EA049630B1 - MULTISPECIFIC BINDING PROTEINS THAT BIND CD33, NKG2D, AND CD16 AND METHODS OF ADMINISTRATION - Google Patents

Abstract

Multispecific binding proteins that bind to and kill human cancer cells expressing CD33 (Siglec-3), as well as pharmaceutical compositions and therapeutic methods applicable to the treatment of cancers expressing CD33, are described. The invention relates to multispecific binding proteins that bind to human cancer cells expressing CD33 and exhibit high potency and maximal target cell lysis compared to monoclonal antibodies to CD33.

Description

Cross reference to related application

This application claims priority to U.S. Provisional Patent Application No. 62/632,756, filed February 20, 2018, which is incorporated herein by reference in its entirety for all purposes, and to U.S. Provisional Patent Application No. 62/677,137, filed May 28, 2018.

List of sequences

The currently pending application contains a sequence listing submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy of the listing, created on February 20, 2019, is named DFY_070WO_SL25.txt and is 571,170 bytes in size.

Field of technology

The invention relates to multispecific binding proteins that bind to CD33 (Siglec-3) and exhibit high efficiency and maximum lysis of target cells compared to monoclonal antibodies to CD33.

State of the art

Cancer continues to be a major health problem despite significant research developments and advances reported in the literature on the treatment of this disease. Some of the most commonly diagnosed cancers in adults include prostate cancer, breast cancer, and lung cancer. Hematologic malignancies, although less common than solid cancers, have poor survival rates. Current treatment options for these cancers are not effective for all patients and/or may have serious side effects. Other cancers also remain difficult to treat with existing therapeutic options.

Cancer immunotherapy is desirable because it is highly specific and can facilitate the destruction of cancer cells by the patient's own immune system. Fusion proteins such as bispecific T cell activators are types of cancer immunotherapy described in the literature that bind to tumor cells and T cells to facilitate tumor cell destruction. T cells are the primary effectors of the adaptive immune system that attack foreign cells as well as host cells that are mutated or misexpressed peptides. Cells targeted by T cells may be infected with a virus, thus expressing foreign proteins, or malignant, in which case they may express mutated proteins. T cells recognize target cells through their T cell receptor (TCR), interacting with intracellular peptides presented by major histocompatibility complex proteins on target cells. Individual T cells typically recognize target cells bearing specific MHC-peptide complexes, but new agents have been developed that usurp and enhance this natural process to achieve therapeutic effect. Bispecific T cell activators link the antigen-binding site(s) of tumor-associated antigens to the antigen-binding site(s) of TCR complex components to redirect T cell activity to desired target cells independent of recognition of the native peptide-MHC complex. For example, Blincyto is an FDA-approved T cell activator that targets CD19 on malignant B cells.

T cells can also be engineered to express chimeric antigen receptors (CARs), which give them the ability to recognize their CAR targets. CARs contain antigen-binding sites for tumor-associated antigens linked to T cell activation domains. These CAR-T cells can also be used to target malignant cells, and some have been approved by the FDA for use against B-cell malignancies.

Antibodies that bind to specific tumor-associated antigens and specific immune cells have been described in the literature (See, for example, WO 2016/134371 and WO 2015/095412). Antibody-drug conjugates or immunocytokines, using antigen-binding sites, target tumor-associated antigens to deliver toxic agents or immunomodulatory cytokines to specific target cells.

Natural killer (NK) cells are a component of the innate immune system and comprise approximately 15% of circulating lymphocytes. NK cells infiltrate virtually all tissues and were originally characterized by their ability to effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells, i.e., via cytolytic granules containing perforin and granzymes, and via death receptor pathways. Activated NK cells also secrete inflammatory cytokines such as IFN-gamma and chemokines that promote the recruitment of other leukocytes to the target tissue.

NK cells respond to signals through a variety of activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy self-cells, their activity is inhibited by activation of immunoglobulin-like receptors on killer cells

- 1 049630 (KIR). Alternatively, when NK cells encounter foreign cells or cancer cells, they are activated via their activating receptors (e.g. NKG2D, NCR, DNAM1). NK cells are also activated by the constant region of some immunoglobulins via CD16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the combined effects of stimulatory and inhibitory signals.

CD33 is a member of the sialic acid-binding immunoglobulin-like lectins. As a transmembrane receptor expressed primarily on myeloid lineage cells, CD33 modulates inflammatory and immune responses through suppressive effects on tyrosine kinase-mediated signaling pathways. For example, CD33 has been shown to consistently suppress the production of proinflammatory cytokines such as IL-1e, TNF-α, and IL-8 by human monocytes.

CD33 is associated with hematopoietic malignancies. It is widely expressed in blasts of nearly all acute myeloid leukemias (AML). Furthermore, stem and/or progenitor cells in hematopoietic malignancies have been found to be CD33+, implying that CD33-targeted therapies could potentially eradicate malignant stem and/or progenitor cells in such cases while sparing normal hematopoietic stem cells. In addition to its expression in AML, CD33 is found on other myeloid neoplasms (e.g., myelodysplastic syndromes and myeloproliferative neoplasms) and on subsets of B-cell and T-cell acute lymphoblastic leukemia (ALL)/lymphoblastic lymphomas. This expression pattern has led to the use of CD33-targeted therapeutics in patients with malignancies including AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis chronic myeloid leukemia, and ALL.

The essence of the invention

The invention provides multispecific binding proteins that bind to CD33 on a cancer cell and to the NKG2D receptor and CD16 receptor on natural killer cells. Such proteins can engage more than one type of NK activating receptors and can block the binding of natural ligands to NKG2D. In some embodiments, the proteins can agonize NK cells in humans and in other species, such as rodents and cynomolgus macaques. Various aspects and embodiments of the invention are described in more detail below.

In some embodiments, the present invention provides a protein that comprises a human CD33 antigen-binding site comprising a heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300 or 302, and further comprises a second antigen-binding site, the same as or different from the antigen-binding site that binds to human CD33.

In certain embodiments, the present invention provides an antigen-binding site in a protein (e.g., a multispecific binding protein) that binds to CD33 on a cancer cell, as well as an NKG2D receptor and a CD16 receptor on natural killer cells to activate the natural killer cell. The binding protein (e.g., a multispecific binding protein) is used in the pharmaceutical compositions and therapeutic methods described herein. Binding of a protein comprising an antigen-binding site that binds to CD33, as well as an NKG2D receptor and a CD16 receptor on a natural killer cell, enhances the activity of the natural killer cell to destroy the cancer cell. Binding of a protein comprising an antigen-binding site that binds to CD33 (e.g., a multispecific binding protein) on a cancer cell brings the cancer cell into proximity of a natural killer cell, facilitating direct and indirect destruction of the cancer cell by the natural killer cell. Further description of exemplary multispecific binding proteins is provided below.

The first component of the multispecific binding proteins of the present disclosure binds to CD33-expressing cells, which may include, but are not limited to, AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, and ALL.

The second component of the multispecific binding proteins of the present disclosure binds to cells expressing the NKG2D receptor, which may include, but are not limited to, NK cells, γδ T cells, and CD8+ αβ T cells. Upon binding to NKG2D, the multispecific binding proteins can block natural ligands, such as ULBP6 and MICA, from binding to NKG2D and activating NKG2D receptors. The NKG2D antigen binding site may include, for example:

(1) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)

- 2 049630 identical to SEQ ID NO: 81, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 82 [ADI-29379];

(2) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 84 [ADI-29463];

(3) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 86 [ADI-27744];

(4) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 87 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 88 [ADI-27749];

(5) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 191 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 88 [A49MI]; or (6) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 89 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 90 [ADI-29378].

The third component for the multispecific binding proteins of the present disclosure binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

In certain embodiments, any of the above isolated antibodies has a KD of 1 nM or less, 5 nM or less, or 12 nM or less for the extracellular domain of human CD33, as measured by surface plasmon resonance (SPR) (e.g., using the Biacore method described in Example 1 below) or by bilayer interferometry (BLI) (e.g., using the Octet method described in Example 1 below) and/or binds CD33 from a subject's body fluid, tissue, and/or cell. In certain embodiments, any of the above isolated antibodies has a Kd (i.e., off-rate, also referred to as K _off ) equal to or less than 1x10'5, ^1x10'4 , ^1x10'3 , ^5x10'3 , 0.01, 0.02, or 0.05 1/s, as measured by SPR (e.g., using the Biacore method described in Example 1 below) or BLI (e.g., using the Octet method described in Example 1 below).

Some proteins of the present disclosure bind to NKG2D with a KD of 10 nM or weaker affinity.

In another aspect, the invention provides one or more isolated nucleic acids comprising sequences encoding the variable region of an immunoglobulin heavy chain and/or an immunoglobulin light chain of any of the above antibodies. The invention provides one or more expression vectors that express the variable region of an immunoglobulin heavy chain and/or an immunoglobulin light chain of any of the above antibodies. Similarly, the invention provides host cells comprising one or more of the above expression vectors and/or isolated nucleic acids.

Also provided are compositions comprising any of the proteins that comprise the CD33 binding domain described herein, and methods for enhancing tumor cell death using these proteins and/or compositions.

In another aspect, the invention provides a method of treating cancer, such as a CD33-associated cancer, in a subject. The method comprises administering to the subject an effective amount of a protein comprising any CD33-binding domain described herein, such as a multispecific protein comprising a CD33-binding domain, an NKG2D-binding domain, and a CD16-binding domain, to treat cancer in the subject.

In another aspect, the invention provides a method of inhibiting cancer growth, such as the growth of a CD33-associated cancer, in a subject. The method comprises exposing the subject to an effective amount of an antibody comprising any CD33-binding domain described herein,

- 3 049630 example, a multispecific protein comprising a CD33 binding domain, an NKG2D binding domain and a CD16 binding domain, for inhibiting cancer growth in a subject.

In another aspect, the invention provides a method for treating cancer in a patient. The method includes administering to a patient in need thereof a therapeutically effective amount of the multispecific binding protein described herein. Examples of cancer for treatment using the multispecific binding protein include, for example, a cancer selected from the group consisting of AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, and ALL.

In another aspect of the invention, there is provided a protein comprising:

(a) a first antigen-binding site containing a Fab fragment that binds NKG2D;

(b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33 comprising a heavy chain variable domain and a light chain variable domain; and (c) an Fc domain of an antibody or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16, wherein the scFv is linked to the Fc domain of an antibody or a portion thereof sufficient to bind CD16 or a third antigen-binding site that binds CD16, through a hinge comprising Ala-Ser or GlyAla-Ser.

In certain embodiments, the scFv is linked to the Fc domain of the antibody. In certain embodiments, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv. In certain embodiments, the disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain.

In certain embodiments, the scFv is linked to the Fc domain of an antibody, wherein the light chain variable domain of the scFv is located at the N-terminus of the heavy chain variable domain of the scFv and is linked to the heavy chain variable domain of the scFv via a flexible linker (Gl yGl yGl yGl ySer)4 ((G4S)4), and the Fab is linked to the Fc domain of the antibody.

In certain embodiments, the present invention provides a protein as described herein, wherein the heavy chain variable domain of an scFv is linked to the light chain variable domain of an scFv via a flexible linker. In certain embodiments, the flexible linker comprises (GlyGlyGlyGlySer)4 ((G4S)4).

In certain embodiments, the present invention provides a protein as described herein, wherein the heavy chain variable domain of the scFv is located at the N-terminus or C-terminus of the light chain variable domain of the scFv. In certain embodiments, the light chain variable domain of the scFv is located at the N-terminus of the heavy chain variable domain of the scFv.

In certain embodiments, the present invention provides a protein as described herein, wherein the Fab fragment is linked to an Fc domain of an antibody, or a portion thereof sufficient to bind CD16 or a third antigen-binding site that binds CD16. In certain embodiments, the heavy chain portion of the Fab fragment comprises a heavy chain variable domain and a CH1 domain, wherein the heavy chain variable domain is linked to the CH1 domain. In certain embodiments, the Fab fragment is linked to an Fc domain of an antibody.

In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) sequences represented by the amino acid sequences of SEQ ID NOs: 93, 94, and 95, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 96, 97, and 98, respectively. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 99, 100, and 101, respectively; and a light chain variable domain comprising a CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 102, 103, and 104, respectively. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising a CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 105, 106, and 107, respectively; and a light chain variable domain comprising a CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 108, 109, and 110, respectively. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising a CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 192, 112, and 193, respectively; and a light chain variable domain comprising the CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences SEQ ID NO: 114, 115 and 116, respectively. In certain

- 4,049,630 embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 192, 112, and 195, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 114, 115, and 116, respectively. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 117, 118, and 119, respectively; and a light chain variable domain comprising the CDR1, CDR2 and CDR3 sequences represented by the amino acid sequences SEQ ID NO: 120, 121 and 122, respectively.

In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 81 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 82. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 84. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 84. or 100%) identical to SEQ ID NO: 85, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 86. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 87, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 88. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 191 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 100%) identical to SEQ ID NO: 88. In certain embodiments, the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 89, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 90.

In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 181, 46, and 182, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 183, 34, and 184, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 36, 185, and 38, respectively.

In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%,

- 5 049630

96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy ... the site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 3, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 15 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 16. In certain embodiments, the second an antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 18. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 18. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 266, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 267. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 268, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 269. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 270 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: NO: 271. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 272, and a light chain variable domain comprising

- 6,049,630 an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 273. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 274 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 274. 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 275. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 276 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 277. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 278 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 279. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 280, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 281. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 281. or 100%) identical to SEQ ID NO: 282, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 283. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 284, and a light chain variable domain comprising an amino acid sequence that is at least 9θ% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 285. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 286, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 286. or 100%) identical to SEQ ID NO: 287. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 288 and a light chain variable domain comprising an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 289. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain, comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 290, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 291. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 292, and a light chain variable domain comprising an amino acid sequence that is at least 9θ% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 293. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 294, and a a light chain domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 295. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,

- 7 049630

97%, 98%, 99%, or 100%) identical to SEQ ID NO: 296, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 297. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 298, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 298. chain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 299. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 300 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 300. 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 301. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 302 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 303. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy ... the site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 418, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 419. In certain embodiments, the second antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 419. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 420, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 421.

In certain embodiments, the present invention provides a protein as described herein comprising a sequence selected from SEQ ID NO: 188, SEQ ID NO:

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

206, SEQ ID NO

212, SEQ ID NO

218, SEQ ID NO

447, SEQ ID NO

453, SEQ ID NO

459, SEQ ID NO

465, SEQ ID NO

471, SEQ ID NO

477, SEQ ID NO

207, SEQ ID NO

213, SEQ ID NO

219, SEQ ID NO

448, SEQ ID NO

454, SEQ ID NO

460, SEQ ID NO

466, SEQ ID NO

472, SEQ ID NO

478, SEQ ID NO

208, SEQ ID NO

214, SEQ ID NO

220, SEQ ID NO

449, SEQ ID NO

455, SEQ ID NO

461, SEQ ID NO

467, SEQ ID NO

473, SEQ ID NO

479, SEQ ID NO

209, SEQ ID NO

215, SEQ ID NO

221, SEQ ID NO

450, SEQ ID NO

456, SEQ ID NO

462, SEQ ID NO

468, SEQ ID NO

474, SEQ ID NO

480, SEQ ID NO

210, SEQ ID NO

216, SEQ ID NO

222, SEQ ID NO

451, SEQ ID NO

457, SEQ ID NO

463, SEQ ID NO

469, SEQ ID NO

475, SEQ ID NO

481, SEQ ID NO

198,

211,

217,

223,

452,

458,

464,

470,

476,

482,

SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487 and SEQ ID NO: 488.

In certain embodiments, the present invention provides a multispecific binding protein as described herein comprising an scFv linked to an Fc domain of an antibody, wherein the scFv linked to the Fc domain of the antibody is a sequence selected from

SEQ ID NO: 187, SEQ ID NO: 197, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227,

SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233,

SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239,

SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243.

In certain embodiments, the present invention provides a protein as described herein comprising a sequence selected from SEQ ID NO: 189, SEQ ID NO: 196, SEQ ID NO: 244, and SEQ ID NO: 245.

In certain embodiments, the present invention provides a protein as described herein comprising a sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 188, SEQ ID NO: 198, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO:

208, SEQ ID NO

214, SEQ ID NO

220, SEQ ID NO

449, SEQ ID NO

455, SEQ ID NO

461, SEQ ID NO

467, SEQ ID NO

209, SEQ ID NO

215, SEQ ID NO

221, SEQ ID NO

450, SEQ ID NO

456, SEQ ID NO

462, SEQ ID NO

468, SEQ ID NO

210, SEQ ID NO

216, SEQ ID NO

222, SEQ ID NO

451, SEQ ID NO

457, SEQ ID NO

463, SEQ ID NO

469, SEQ ID NO

211, SEQ ID NO

217, SEQ ID NO

223, SEQ ID NO

452, SEQ ID NO

458, SEQ ID NO

464, SEQ ID NO

470, SEQ ID NO

212, SEQ ID NO

218, SEQ ID NO

447, SEQ ID NO

453, SEQ ID NO

459, SEQ ID NO

465, SEQ ID NO

471, SEQ ID NO

213, SEQ ID NO

219, SEQ ID NO

448, SEQ ID NO

454, SEQ ID NO

460, SEQ ID NO

466, SEQ ID NO

472, SEQ ID NO

- 8 049630

473, SEQ ID NO: 474, SEQ ID NO: 475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO:

479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, and SEQ ID NO: 484. In certain embodiments, the present invention provides a protein as described herein comprising a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 188, SEQ ID NO: 198, SEQ ID NO: 206, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248,

NO

NO

NO

NO

NO

NO

NO

NO

NO

208, SEQ ID NO

214, SEQ ID NO

220, SEQ ID NO

449, SEQ ID NO

455, SEQ ID NO

461, SEQ ID NO

467, SEQ ID NO

473, SEQ ID NO

209, SEQ ID NO

215, SEQ ID NO

221, SEQ ID NO

450, SEQ ID NO

456, SEQ ID NO

462, SEQ ID NO

468, SEQ ID NO

474, SEQ ID NO

210, SEQ ID NO

216, SEQ ID NO

222, SEQ ID NO

451, SEQ ID NO

457, SEQ ID NO

463, SEQ ID NO

469, SEQ ID NO

475, SEQ ID NO

211, SEQ ID NO

217, SEQ ID NO

223, SEQ ID NO

452, SEQ ID NO

458, SEQ ID NO

464, SEQ ID NO

470, SEQ ID NO

476, SEQ ID NO

212, SEQ ID NO

218, SEQ ID NO

447, SEQ ID NO

453, SEQ ID NO

459, SEQ ID NO

465, SEQ ID NO

471, SEQ ID NO

477, SEQ ID NO

479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483 and SEQ ID NO

207, SEQ ID

213, SEQ ID

219, SEQ ID

448, SEQ ID

454, SEQ ID

460, SEQ ID

466, SEQ ID

472, SEQ ID

478, SEQ ID

484. In certain embodiments, the present invention provides a protein as described herein comprising a sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NO: 188, SEQ ID NO: 198, SEQ ID NO: 206, SEQ ID NO:

NO

NO

NO

NO

NO

NO

NO

NO

NO

208, SEQ ID NO

214, SEQ ID NO

220, SEQ ID NO

449, SEQ ID NO

455, SEQ ID NO

461, SEQ ID NO

467, SEQ ID NO

473, SEQ ID NO

209, SEQ ID NO

215, SEQ ID NO

221, SEQ ID NO

450, SEQ ID NO

456, SEQ ID NO

462, SEQ ID NO

468, SEQ ID NO

474, SEQ ID NO

210, SEQ ID NO

216, SEQ ID NO

222, SEQ ID NO

451, SEQ ID NO

457, SEQ ID NO

463, SEQ ID NO

469, SEQ ID NO

475, SEQ ID NO

211, SEQ ID NO

217, SEQ ID NO

223, SEQ ID NO

452, SEQ ID NO

458, SEQ ID NO

464, SEQ ID NO

470, SEQ ID NO

476, SEQ ID NO

212, SEQ ID NO

218, SEQ ID NO

447, SEQ ID NO

453, SEQ ID NO

459, SEQ ID NO

465, SEQ ID NO

471, SEQ ID NO

477, SEQ ID NO

207, SEQ ID

213, SEQ ID

219, SEQ ID

448, SEQ ID

454, SEQ ID

460, SEQ ID

466, SEQ ID

472, SEQ ID

478, SEQ ID no.

479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, SEQ ID NO: 483 and SEQ ID NO: 484.

In certain embodiments, the present invention provides a protein described herein comprising at least 90% (e.g., 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence selected from SEQ ID NO: 187, SEQ ID NO: 197, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, and SEQ ID NO: 243. In certain embodiments, the present invention provides a protein as described herein comprising a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO: 187, SEQ ID NO: 197, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, and SEQ ID NO: 243. In certain embodiments, the present invention provides a protein as described herein comprising a sequence that is at least 99% identical to an amino acid sequence selected from SEQ ID NO: 187, SEQ ID NO: 197, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ

ID NO:

ID NO:

ID NO:

IN

227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242 and SEQ ID NO: 243.

In another aspect, the present invention provides a composition comprising the protein described herein and a pharmaceutically acceptable carrier.

In another aspect of the present invention, there is provided a method of treating a cancer expressing CD33, the method comprising administering a therapeutically effective amount of a protein or composition thereof described herein to a subject in need thereof.

In certain embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), myeloproliferative neoplasms (MPN), lymphoma, non-Hodgkin lymphomas, and classical Hodgkin lymphoma. In certain embodiments, the AML is selected from undifferentiated acute myeloid leukemia, acute myeloid leukemia with minimal maturation, acute myeloid leukemia with maturation, acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, acute myelomonocytic leukemia with eosinophilia, acute monocytic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia (AMCL), acute basophilic leukemia, acute panmyelosis with fibrosis, and blastic plasmacytoid dendritic cell neoplasia (BPDCN). In some embodiments, the AML is characterized by expression of CLL-1 on leukemia stem cells (LSC) of the AML. In some embodiments, the LSCs further express a membrane marker selected from CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

- 9 049630

In certain embodiments, the AML is minimal residual disease (MRD). In some embodiments, the MRD is characterized by the presence or absence of a mutation selected from FLT3-ITD ((Fms-like tyrosine kinase 3)-internal tandem duplications (ITD)), NPM1 (nucleophosmin 1), DNMT3A (DNMT3A DNA methyltransferase gene), and IDH (isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2)). In certain embodiments, the MDS is selected from MDS with multilineage dysplasia (MDS-MLD), MDS with unilineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS), MDS with excess blasts (MDS-EB), MDS with distinct del(5q), and MDS unclassified (MDS-U). In certain embodiments, the MDS is primary MDS or secondary MDS.

In certain embodiments, ALL is selected from B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL). In certain embodiments, MPN is selected from polycythemia vera, essential thrombocythemia (ET), and myelofibrosis. In certain embodiments, non-Hodgkin lymphoma is selected from B-cell lymphoma and T-cell lymphoma. In certain embodiments, lymphoma is selected from chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL), diffuse large B cell lymphoma (DLBCL), Burkitt lymphoma (BL), primary mediastinal large B cell lymphoma (PMBL), follicular lymphoma. lymphoma, mantle cell lymphoma, hairy cell leukemia, plasma cell myeloma (PCM) or multiple myeloma (MM), mature T/NK cell neoplasms and histiocytic neoplasms.

These and other aspects and advantages of the invention are illustrated by the following figures, detailed description and claims.

Description of graphic materials

The invention can be understood more fully by referring to the following graphic materials.

Figure 1 shows a structural representation of the extracellular domain of human CD33 extracellular domain (ECD). CD33 ECD contains two distinct domains: a distal V domain and a membrane proximal C domain. The ligand binding interface is located in the V domain. The function of the C domain is unknown. CD33 ECD is heavily glycosylated: 2 N-linked glycosylation sites are located in the V domain and 3 N-linked glycosylation sites are located in the C domain. Human CD33 ECD contains several SNPs with the most significant mutation being R69G, which is found in 42% of patients. The R69G SNP is located in the V domain.

Figure 2 shows the alignment of the primary sequences of full-length human and cynomolgus monkey CD33 (SEQ ID NO: 598 and SEQ ID NO: 599, respectively). The V domain is underlined in blue, the C domain is underlined in green. Sequence differences are highlighted in red.

Figures 3A–3K show the SPR profiles of Fab fragments of anti-CD33 monoclonal antibodies binding to the human CD33 ECD measured by Biacore at 37°C. Each Fab fragment includes a CD33-binding clone described herein. Figure 3A shows the Biacore profile of ADI-10159; Figure 3B shows the Biacore profile of ADI-10177; Figure 3C shows the profile

Biacore ADI-11776; Fig. 3D shows the profile of Biacore ADI-11801; Fig. 3E shows the profile

Biacore ADI-11807; Fig. 3F shows the profile of Biacore ADI-11809; Fig. 3G shows the profile

Biacore ADI-11815; Fig. 3H shows the profile of Biacore ADI-11819; Fig. 3I shows the profile of

Biacore ADI-11830; Fig. 3J shows the profile of Biacore ADI-11835 and Fig. 3K shows the profile of the Biacore Fab fragment of lintuzumab.

Figures 4A-4H show the SPR profiles of anti-CD33 mAb Fab fragments binding to the cynomolgus monkey CD33 ECD measured by Biacore at 37°C. Each Fab fragment comprises a CD33-binding clone described herein. Figure 4A shows the Biacore profile of ADI-10159; Figure 4B shows the Biacore profile of ADI-10177; Figure 4C shows the Biacore profile of ADI-11776; Figure 4D shows the Biacore profile of ADI-11807; Figure 4E shows the Biacore profile of ADI-11809; Figure 4F shows the Biacore profile of ADI-11819; Figure 4G shows the Biacore profile of ADI-11830; and Figure 4H shows the Biacore profile of ADI-11835.

Figures 5A-5T show the SPR profiles of anti-CD33 monoclonal antibody Fab fragments binding to the V domain and C domain of human CD33, measured at 37°C. Each Fab fragment comprises a CD33-binding clone described herein. Figures 5A-5J show binding to the V domain; panels K-T show binding to the C domain. Figures 5A and 5K show profiles of Biacore ADI-10159; Figures 5B and 5L show profiles of Biacore ADI-10177; Figures 5C and 5M show profiles of Biacore ADI-11776; Figures 5D and 5N show profiles of Biacore ADI-11801; Figure 5E and 5O show profiles of Biacore ADI-11807; Figs. 5F and 5P show profiles of Biacore ADI11809; Figs. 5G and 5Q show profiles of Biacore ADI-11815; Figs. 5H and 5R show profiles of Biacore ADI-11819; Figs. 5I and 5S show profiles of Biacore ADI-11830; and Figs. 5J and 5T show profiles of Biacore ADI-11835.

Figures 6A-6D show the SPR profiles of a Fab fragment that includes binding of ADI-11815 to various domains of human CD33 and human CD33 bearing the R69G point mutation. Figure 6A

- 10 049630 Fab binding to human CD33 ECD; Fig. 6B Fab binding to the V domain; Fig. 6C Fab binding to the C domain; Fig. 6D Fab binding to human CD33 bearing R69G.

Figures 7A-7D show the SPR profiles of a Fab fragment that includes binding of ADI-11801 to various domains of human CD33 and human CD33 bearing the R69G point mutation. Figure 7A, human CD33 ECD; Figure 7B, V domain; Figure 7C, C domain: Figure 7D, human CD33 bearing R69G.

Figure 8 shows histograms showing the binding of monoclonal antibodies containing CD33-binding clones to CD33 expressed on human AML Molm-13 cells. The anti-CD33 antibody lintuzumab was also tested and the mean fluorescence intensity (MFI) was plotted. Five of the six antibodies showed a higher binding signal to CD33 than lintuzumab.

Figure 9 shows histograms showing the internalization of anti-CD33 antibodies on Molm-13 cells after 24 h. All anti-CD33 antibodies showed similar internalization after 24 h. Lintuzumab showed slightly higher internalization compared to other anti-CD33 antibodies.

Figures 10A-10B show the binding of CD33-targeted TriNKETs to human NKG2D expressed on EL4-hNKG2D and KHYG-1 cells. Figure 10A shows the binding of CD33-targeted TriNKETs to human NKG2D recombinantly expressed on EL4 cells. Figure 10B shows the binding of CD33-targeted TriNKETs to human NKG2D expressed on KHYG-1 cells. For each TriNKET, the fold over background (FOB) value was similar in both EL4-hNKG2D and KHYG-1 cells, and the level of binding was also maintained in both cell lines.

Figure 11 shows the binding of CD33-targeted TriNKETs to CD33 expressed on human AML Molm-13 cells. Four different CD33-binding clones were used with five NKG2D-binding clones to generate a total of 20 different TriNKETs. The NKG2D-binding domains of TriNKETs did not affect the binding of the CD33-binding clones to CD33.

Figure 12 is a graph showing that resting human NK cells are activated by CD33-targeted TriNKETs in co-culture with CD33-expressing THP-1 AML cells.

Figure 13 shows a histogram demonstrating that CD33-targeted TriNKETs induce resting NK cell-mediated killing of Molm-13 AML cells.

Figure 14 shows a histogram demonstrating that CD33-targeted TriNKETs induce resting NK cell-mediated killing of THP-1 cells.

Figure 15A shows line graphs showing that TriNKETs mediate KHYG-1 cell killing of Molm-13 AML cells. Figure 15B shows line graphs showing that TriNKETs mediate resting NK cell killing of Molm-13 AML cells.

Figure 16 shows line graphs demonstrating that TriNKETs mediate the killing of KHYG-1 cells by EOL-1 AML cells.

Fig. 17A shows line graphs showing that TriNKETs mediate the killing of THP-1 AML cells by KHYG-1 cells. Fig. 17B shows line graphs showing that TriNKETs mediate the killing of THP-1 AML cells by resting NK cells.

Fig. 18 shows an image of a multispecific binding protein that contains an NKG2D-binding domain (right arm), a CD33-binding domain (left arm), and an Fc domain or portion thereof that binds to CD16.

Fig. 19 is an illustration of a multispecific binding protein that comprises an NKG2D binding domain or a CD33 binding domain, either of which may be in scFv format, and an Fc domain or portion thereof that binds CD16.

Figure 20 shows an image of TriNKET in the form of Triomab, which is a trifunctional bispecific antibody that retains an IgG-like shape. This chimera consists of two half-antibodies, each with one light and one heavy chain, which are derived from two parent antibodies.

Figure 21 shows a representation of a TriNKET in the form of a common light chain (LC) KiH, which uses knob-in-hole (KIH) technology. KiH is a heterodimer containing 2 Fabs binding to targets 1 and 2 and an Fc stabilized by heterodimerization mutations. A TriNKET in the KiH format can be a heterodimeric construct with 2 Fabs binding to target 1 and target 2, containing two different heavy chains and a common light chain that binds to both heavy chains.

Fig. 22 shows a TriNKET in the form of dual variable domain immunoglobulin (DVD-Ig™), which combines the target binding domains of two monoclonal antibodies through flexible natural linkers to produce a tetravalent IgG-like molecule. DVD-Ig™

- 11 049630 is a homodimeric construct in which the variable domain-targeting antigen 2 is fused to the N-terminus of the variable domain of the Fab-targeting antigen 1. The construct contains normal Fc.

Figure 23 shows a representation of TriNKET in the form of an orthogonal Fab interface (ortho-Fab), which is a heterodimeric construct that contains two Fabs, target 1 and target 2, fused to an Fc. LC-HC pairing is mediated by the orthogonal interface. Heterodimerization is mediated by mutations in the Fc.

Fig. 24 shows an image of TrinKET in Ig 2-in-1 format.

Figure 25 shows an image of TrinKET in the ES form, which is a heterodimeric construct containing two different Fabs binding to target 1 and target 2 fused to an Fc. Heterodimerization is mediated by mutations in the electrostatic interaction in the Fc.

Figure 26 shows a representation of TriNKET in the Fab exchange form: antibodies that exchange Fab fragments by replacing the heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, resulting in bispecific antibodies. The Fab exchange form (cFae) is a heterodimer containing 2 Fabs binding to target 1 and 2 and an Fc stabilized by heterodimerization mutations.

Figure 27 shows an image of TriNKET in the form of a SEED Body, which is a heterodimer containing two Fab fragments binding to targets 1 and 2, and an Fc stabilized by heterodimerization mutations.

Figure 28 shows an image of TriNKET in the LuZ-Y form, in which the leucine zipper is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs, target binding 1 and 2, fused to an Fc.

Fig. 29 shows an image of TriNKET in the form of Cov-X-Body.

Figures 30A-30B show an illustration of TriNKET in λ-Body forms, which are heterodimeric constructs with two different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1-targeted antigen 1 contains LC kappa, while a second Fab-targeted antigen 2 contains LC lambda. Figure 30A shows an illustration of one λ-Body form; Figure 30B shows an illustration of another λ-Body form.

Figure 31 shows a heterodimeric Oasc-Fab construct that includes target 1 Fab binding and target 2 scFab binding fused to Fc. Heterodimerization is mediated by mutations in Fc.

Figure 32 shows DuetMab, which is a heterodimeric construct containing two different Fabs binding to antigens 1 and 2 and an Fc stabilized by heterodimerization mutations. Fabs 1 and 2 contain differential S-S bridges that ensure proper pairing of the light chain (LC) and heavy chain (HC).

Figure 33 shows CrossmAb, which is a heterodimeric construct with two different Fabs binding to targets 1 and 2, fused to a heterodimerization-stabilized Fc. The CL and CH1 domains and the VH and VL domains are swapped, e.g., CH1 is fused in line with VL, and CL is fused in line with VH.

Figure 34 shows Fit-Igs, which are homodimeric constructs in which the Fab that binds to antigen 2 is fused to the N-terminus of the HC Fab that binds to antigen 1. The construct contains wild-type Fc.

Fig. 35 is a graph showing the binding of A49-F3'-TriNKET-107 and 107-F405L mAbs to human cell surface NKG2D expressed on EL4 cells.

Figures 36A-36B are graphs showing the binding of A49-F3'-TriNKET-107 and 107-F405L mAbs to the human CD33+ AML cell lines Mv4-11 (Figure 36A) and Molm-13 (Figure 36B).

Figures 37A-37B are graphs showing the internalization of A49-F3'-TriNKET-107 and 107-F405L mAbs after incubation with EOL-1 cells (Figure 37A) and Molm-13 cells (Figure 37B).

Figures 38A-38D are graphs showing specific lysis of Molm-13 (Figure 38A), EOL-1 (Figure 38B), and THP-1 (Figures 38C and 38D) human AML cells by resting human NK cells in the presence of A49-F3'-TriNKET-107 and anti-CD33 mAbs.

Figure 39 shows a series of flow cytograms demonstrating the expression level of CD3, CD8, NKG2D and CD16 on isolated primary CD8+ T cells.

Figures 40A-40B are graphs showing specific lysis of Molm-13 cells by isolated primary CD8+ T cells in the presence of A49-F3'-TriNKET-107, A49-F3'-TriNKETH76, non-targeted TriNKET, or 107-F405L rnAb (labeled 107 in the figures). The primary CD8+ T cells in Figure 40A were isolated from PBMCs of Donor 1, and the primary CD8+ T cells in Figure 40B were isolated from PBMCs of Donor 2. The dotted lines indicate specific lysis of Molm-13 cells by CD8+ T cells in the absence of TriNKET or antibody.

Figures 41A-41E are histograms showing the binding of A49-F3'-TriNKET-107 to NK cells (Figure 41A), CD8+ T cells (Figure 41B), CD4+ T cells (Figure 41C), B cells (Figure 41D) and

- 12 049630 monocytes (Fig. 41E) in human whole blood. Unfilled dashed lines represent binding of A49-F3'-TriNKET-107 to cells; filled solid lines represent binding of human IgG1 isotype control to cells.

Figures 42A-42B are graphs showing CD33 expression on monocytes. Figure 42A shows CD33 expression on monocytes from four healthy donors (dark gray) and Molm-13 (light gray). The bottom five rows represent signals from cell samples stained with CD33 antibody; the top five rows represent signals from the same samples stained with isotype antibody. Figure 42B shows CD33 expression on monocytes from the same donor before (light gray) and after (dark gray) negative monocyte selection.

Figures 43A-43B are graphs showing the long-term cytotoxicity of NK cells against Molm-13 AML cells and primary human monocytes in the presence of A49-F3'-TriNKET107. Target cell proliferation is plotted against co-culture time with NK cells in the presence of A49-F3'-TriNKET-107 or PMA+ionomycin. Figure 43A shows the results of an experiment using NK cells from one donor, and Figure 43B shows the results of another experiment using NK cells from a different donor.

Fig. 44 illustrates a trispecific antibody (TriNKET) that comprises a CD33-binding scFv targeting NKG2D Fab and a heterodimerized antibody constant domain that binds CD16. The antibody format is referred to herein as F3'-TriNKET.

Detailed description of the invention

The invention provides multispecific binding proteins that bind CD33 on a cancer cell and the NKG2D receptor and the CD16 receptor on natural killer cells to activate the natural killer cell, pharmaceutical compositions comprising such multispecific binding proteins, and therapeutic methods using such multispecific binding proteins and pharmaceutical compositions, including for the treatment of cancer. Various aspects of the invention are set forth in the sections provided below; however, aspects of the invention described in one particular section should not be limited to any particular section.

In one aspect, the invention provides a multispecific binding protein comprising an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and/or cynomolgus/rhesus macaque CD33. In one aspect, the present invention provides an antigen-binding site that recognizes and binds one or more conformational epitopes on the extracellular domain of human CD33 and/or cynomolgus/rhesus macaque CD33. In one aspect, the present invention provides an antigen-binding site that recognizes and binds one or more conformational epitopes on the extracellular domain of human CD33, but does not recognize and/or bind one or more conformational epitopes of the extracellular domain of cynomolgus CD33. In one, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33. In one aspect, the present invention provides an antigen-binding site that binds wild-type human CD33 but not the R69G allele of human CD33. In one aspect, the present invention provides an antigen-binding site that binds to an epitope on human CD33 that includes R69. In one, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33. In one aspect, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not the S128N allele of human CD33. In one aspect, the present invention provides an antigen-binding site that binds to an epitope on human CD33 that includes S128. In one aspect, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds to an extracellular domain in human CD33 and/or cynomolgus monkey CD33, regardless of the glycosylation profile of the target CD33.

In certain embodiments, the present invention provides a multispecific binding protein comprising an antigen-binding site that binds to an extracellular domain in human CD33 and/or cynomolgus monkey CD33 such that the epitopes are unique compared to epitopes targeted by one or more anti-CD33 antibodies known in the art. In some embodiments, the present invention provides an antigen-binding site that binds to an extracellular domain in human CD33 and/or cynomolgus monkey CD33 and exhibits cross-reactivity with human or cynomolgus/rhesus monkey CD33 and high binding affinity for target CD33.

To facilitate understanding of the present invention, a definition of a number of terms and phrases is provided below.

Unless the context clearly dictates otherwise, the use of the singular forms herein includes a plural reference and means one or more.

As used herein, the term antigen-binding site refers to the portion of an immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen-binding site is formed by amino acid residues of the N-terminal variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent regions within the V regions of the heavy and light chains are called hypervariable regions, which are located between more conserved flanking regions known as framework regions or FRs. Thus, the term FRs refers to the amino acid sequences that naturally occur between and adjacent to the hypervariable regions of immunoglobulins. In a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called complementarity determining regions or CDRs. In some animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, providing a single-domain antibody. Antigen-binding sites may exist in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv using a peptide linker to join the variable domain of the heavy chain to the variable domain of the light chain in a single polypeptide. All amino acid positions in the variable regions of the heavy or light chains described herein are numbered according to Kabat numbering.

The CDRs of an antigen-binding site can be defined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). CDRs defined according to these definitions typically include overlapping or subsets of amino acid residues when compared to one another. In some embodiments, the term CDR is a CDR as defined in MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some embodiments, the term CDR is a CDR as defined in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, the CDRs of the heavy chain and the CDRs of the light chain of an antibody are defined using different principles. For example, in certain embodiments, the CDRs of the heavy chain are defined by MacCallum (see above) and the CDRs of the light chain are defined by Kabat (see above). CDRH1, CDRH2, and CDRH3 represent the CDRs of the heavy chain, and CDRL1, CDRL2, and CDRL3 represent the CDRs of the light chain.

As used herein, the terms subject and patient refer to an organism that is to be treated with the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, dogs, cats, etc.) and more preferably include humans.

As used herein, the term effective amount refers to an amount of a compound (e.g., a compound of the present invention) sufficient to achieve beneficial or desired results. An effective amount may be administered in one or more administrations, uses, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term treatment includes any effect, such as reduction, reduction, modulation, alleviation, or elimination, that results in an improvement in a condition, disease, disorder, etc., or in the alleviation of symptoms thereof.

In the context of this document, the term pharmaceutical composition refers to a combination of an active agent with a carrier, inert or active, which makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

As used herein, the term pharmaceutically acceptable carrier refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil/water or water/oil emulsions), and various types of wetting agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975].

As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., an acid or a base) of a compound of the present invention that, when administered to a subject, is capable of providing the compound of the present invention or an active metabolite or residue thereof. As known to those skilled in the art, salts of the compounds of the present invention may be derivatives of inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, chloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-para-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, although not pharmaceutically acceptable in themselves, may be used in the preparation of salts used as

- 14 049630 intermediate compounds in the preparation of compounds according to the invention and their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal hydroxides (e.g., sodium), alkaline earth metal hydroxides (e.g., magnesium), ammonia, and compounds of the formula NW ₄ ⁺ , where W is C1_ ₄ alkyl, and the like.

Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention combined with a suitable cation such as Na ⁺ , NH4 ⁺ , and NW4 ⁺ (where W is a _C1.4 - _alkyl group) and the like.

For therapeutic use, salts of the compounds of the present invention are considered to be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

Throughout this specification, where compositions are described as having, including, or containing particular components, or where processes and methods are described as having, including, or containing particular steps, it is intended that there are additional compositions of the present invention that consist essentially of or consist of the recited components, and that there are processes and methods of the present invention that consist essentially of or consist of the recited processing steps.

Generally, compositions that specify percentages refer to percentages by weight unless otherwise stated. In addition, if a variable is not accompanied by a definition, the previous definition of the variable is used.

Various features and aspects of the invention are described in more detail below.

Antigen binding site

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 21, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 22, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 23. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain of an antibody with an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 comprises a sequence CDR1 represented by the amino acid sequence of SEQ ID NO: 434, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 22, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 435. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 24, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 25, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 26.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. In certain embodiments, the antigen-binding site

- 15 049630 an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 27, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 28, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 29. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 181, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 28, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 436. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 4 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 30, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 31 and the CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 32.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 33, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 34, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 35. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5 comprises a sequence CDR1 represented by the amino acid sequence of SEQ ID NO: 183, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 34, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 184. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 6 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 36, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 37, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 38. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody having an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 6, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 36, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 185, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the antigen-binding site comprises an amino acid sequence that is at least 90% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 188. In certain embodiments, the antigen-binding site comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 188. In certain embodiments, the antigen-binding site comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 188. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 188.

In certain embodiments, the present invention provides an antigen-binding site that comprises a heavy chain variable domain of an antibody that comprises at least 90% (e.g., at least 91%, 92%, 93%,

- 16 049630

94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: sequence of SEQ ID NO: 7, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 39, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 40, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 41. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 437, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 40, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 438. In In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody having an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 8, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 42, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 43, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 44.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 45, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 46, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 47. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9 comprises a sequence CDR1 represented by the amino acid sequence of SEQ ID NO: 181, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 46, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 182. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 48, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 49, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 50. In certain embodiments, the antigen-binding site comprises an amino acid a sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 198. In certain embodiments, the antigen-binding site comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 198. In certain embodiments, the antigen-binding site comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 198. In certain embodiments, the antigen-binding site comprises the amino acid sequence of SEQ ID NO: 198.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11, and an antibody light chain variable domain that comprises an amino acid sequence

- 17 049630 an antibody having at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NO: 12. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 51, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 52, and CDR3 represented by the amino acid sequence of SEQ ID NO: 53. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain having an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 181, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 52, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 439. In certain embodiments, an antigen-binding site that comprises an antibody light chain variable domain having an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 12, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 54, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 55 and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 56.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 57, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 58, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 59. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 440, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 58, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 441. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 60, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 61, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 62.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 16. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 63, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 64, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 65. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (e.g.,

- 18 049 630 at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 442, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 64, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 443. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15, sequence SEQ ID NO: 16, includes a CDR1 sequence represented by the amino acid sequence SEQ ID NO: 66, a CDR2 sequence represented by the amino acid sequence SEQ ID NO: 67, and a CDR3 sequence represented by the amino acid sequence SEQ ID NO: 68.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 18. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 69, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 70, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 71. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 181, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 70, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 444. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 72, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 73, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 74.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 75, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 76, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 77. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 445, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 76, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 446. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 78, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 79, and

- 19 049630 CDR3 sequence represented by the amino acid sequence SEQ ID NO: 80.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 267. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 304, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 305, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 306. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 528, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 305, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 529. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 307, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 308, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 309.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 269. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 310, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 311, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 312. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 530, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 311, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 531. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 269 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 313, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 314, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 315.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 271. In certain embodiments, the

- 20 049 630 an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 316, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 317, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 318. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 532, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 317, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 533. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 319, a CDR2 sequence represented by the amino acid sequence SEQ ID NO: 320, and a CDR3 sequence represented by the amino acid sequence SEQ ID NO: 321.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 273. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 322, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 323, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 324. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 534, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 323, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 535. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 325, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 326, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 327.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 275. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 328, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 329, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 330. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90%

- 21 049630 (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 536, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 329, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 537. In certain embodiments, an antigen-binding site that comprises an antibody light chain variable domain with an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274, identical to the amino acid sequence of SEQ ID NO: 275, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 331, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 332, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 333.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 277. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 334, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 335, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 336. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 538, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 335, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 539. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 337, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 338, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 339.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 279. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 340, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 341, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 342. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 540, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 341, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 541. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:

- 22 049630

279, comprises a CDR1 sequence represented by the amino acid sequence SEQ ID NO: 343, a CDR2 sequence represented by the amino acid sequence SEQ ID NO: 344, and a CDR3 sequence represented by the amino acid sequence SEQ ID NO: 345.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 281. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 346, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 347, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 348. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 542, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 347, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 543. In certain embodiments, an antigen-binding site that comprises an antibody light chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 349, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 350, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 351.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 283. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 352, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 353, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 354. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 544, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 353, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 545. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 355, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 356, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 357.

In certain embodiments, the present invention provides an antigen-binding site that comprises a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID

- 23 049630

NO: 284, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 285. In certain embodiments, the antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 358, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 359 and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 360. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 546, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 359, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 547. In certain embodiments, an antigen-binding site that comprises an antibody light ...2 sequence (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 361, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 362, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 363.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 287. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 364, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 365, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 366. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 548, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 365, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 549. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 367, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 368, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 369.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 289. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 370, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:

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371b and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 372. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain having an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 550, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 371, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 551. In certain embodiments, an antigen-binding site that comprises an antibody light chain variable domain having an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 289, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 373, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 374, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 375.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 291. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 376, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 377, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 378. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 552, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 377, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 553. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 379, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 380, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 381.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 293. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 382, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 383, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 384. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 554, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 383, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 555. In certain embodiments, the

- 25 049630 an antigen-binding site that comprises a variable domain of a light chain of an antibody with an amino acid sequence that is at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 293, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 385, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 386 and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 387.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 295. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence of at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 388, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 389, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 390. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 556, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 389, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 557. In certain embodiments, an antigen-binding site that comprises an antibody light chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 295 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 391, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 392, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 393.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 297. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 394, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 395, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 396. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 558, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 395, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 559. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 397, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 398, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 399.

In certain embodiments, the present invention provides an antigen-binding

- 26 049630 an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298, and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 299. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 299. at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 400, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 401, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 402. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 560, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 401, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 561. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 403, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 404, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 405.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 301. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 406, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 407, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 408. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 562, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 407, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 563. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 409, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 410, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 411.

In certain embodiments, the present invention provides an antigen-binding site that comprises an antibody heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 and an antibody light chain variable domain that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 303. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%,

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96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302, comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 412, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 413, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 414. In certain embodiments, an antigen-binding site that comprises an antibody heavy chain variable domain with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 564, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 565, a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 566, a CDR4 sequence represented by the amino acid sequence of SEQ ID NO: 567, a CDR5 sequence represented by the amino acid sequence of SEQ ID NO: 568, a CDR6 sequence represented by the amino acid sequence of SEQ ID NO: 569, a CDR7 sequence represented by the amino acid sequence of SEQ ID NO: 570, a CDR8 sequence represented by the amino acid sequence of SEQ ID NO: 571, a CDR9 sequence represented by the amino acid sequence of SEQ ID NO: 572, a CDR10 sequence represented by the amino acid sequence of SEQ ID NO: 573, a CDR11 sequence represented by the amino acid sequence of SEQ ID NO: 574, a CDR12 sequence represented by the amino acid sequence of SEQ ID NO: 575, a CDR13 sequence represented by CDR2 represented by the amino acid sequence of SEQ ID NO: 413, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 565. In certain embodiments, an antigen-binding site that comprises a light chain variable domain of an antibody with an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303 comprises a CDR1 sequence represented by the amino acid sequence of SEQ ID NO: 415, a CDR2 sequence represented by the amino acid sequence of SEQ ID NO: 416, and a CDR3 sequence represented by the amino acid sequence of SEQ ID NO: 417.

In each of the above embodiments, it is contemplated herein that the immunoglobulin heavy chain variable region sequences and/or the light chain variable region sequences that together bind CD33 may contain amino acid changes (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the heavy and/or light chain variable regions without significantly affecting their ability to bind CD33.

Table 1 lists the peptide sequences of the heavy-chain variable domains and the light-chain variable domains that in combination (either as a Fab fragment or as a single-chain variable fragment (scFv)) can bind to CD33. Unless otherwise noted, the CDR sequences shown in Table 1 are as defined by Kabat. CD33-binding domains may differ in their binding affinity for CD33. Table 1 also lists the scFv forms of the CD33-binding variable domains of the heavy and light chains. The exemplary nucleic acid sequences shown in Table 1 are the predicted possible nucleic acid sequences from which the corresponding peptide sequences indicated were derived, which were generated using the EMBL-EBI protein sequence reverse translation program.

Table 1 VH VL ADI-10159 [АЫ] (G59) EVQLVESGGGLVQPGGSLRLSCA ASGFTFSSYGMSWVRQAPGKGLE WVANIKQDGSEKYYVDSVKGRF TISRDNAKNSLYLQMNSLRAEDT AVYYCAREGGPYYDSSGYFVYY GMDVWGQGTTVTVSS [SEQ ID NO:1] CDR1: FTFSSYGMS [SEQ ID NO:21] (not by Kabat numbering) or SYGMS [SEQ ID NO:434] CDR2: NIKQDGSEKYYVDSVKG [SEQ ID NO:22] CDR3: AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:23] (not no Kabat) or EGGPYYDSSGYFVYYGMDV [SEQ ID NO:435] DIQMTQSPSTLSASVGDRVTITCR ASQSISSWLAWYQQKPGKAPKLLI YDASSLESGVPSRFSGSGSGTEFTL TISSLQPDDFATYYCQQYESFPTFG GGTKVEIK [SEQ ID NO:2] CDR1: RASQSISSWLA [SEQ ID NO:24] CDR2: DASSLES [SEQ ID NO:25] CDR3: QQYESFPT [SEQ ID NO:26] scFv АЫ DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIY DASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFGCG TKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS

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CAASGFTFSSYGMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGM DVWGQGTTVTVSS [SEQ ID NO:206] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKCLEWV ANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AREGGPYYDSSGYFVYYGMDVWGQGTTVTVSSGGGGSGGGGSGGG GSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPG KAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYE SFPTFGCGTKVEIK [SEQ ID NO:207] Illustrative nucleotide sequence of scFv Abi GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGACGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAG CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGAGA GCTTCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGG CGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCA CCTTCAGCAGCTACGGCATGAGCTGGGTGAGGCAGGCCCCCGGCAA GTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAG TACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACA ACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTAC GACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCC AGGGCACCACGTGACCGTGAGCAGC [SEQ ID NO:246] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACGGCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGG

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CTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGCACCACG TGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAG AGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCA CCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCA GCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGC AGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCG GCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTC GCCACCTACTACTGCCAGCAGTACGAGAGCTTCCCCACCTTCGGCTG CGGCACCAAGGTGGAGATCAAG [SEQ ID NO:247] ADI-10177 [Ab2] EVQLVESGGGLVQPGGSLRLSCA ASGFTFSSYWMSWVRQAPGKGL EWVANIKQDGSEKYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCARPLNAGELDVWGQGT MVTVSS [SEQ ID NOG] CDR1: FTFSSYWMS [SEQ ID NO:27] (not no Kabat) or SYWMS [SEQ ID NO:181] CDR2: NIKQDGSEKYYVDSVKG [SEQ ID NO:28] CDR3: ARPLNAGELDV [SEQ ID NO:29] (not by Kabat numbering) or PLNAGELDV [SEQ ID NO:436] DIQMTQSPSTLSASVGDRVTITCR ASQSISSWLAWYQQKPGKAPKLLI YEASSLESGVPSRFSGSGSGTEFTL TISSLQPDDFATYYCQQLESYPLTF GGGTKVEIK [SEQ ID NO:4] CDR1: RASQSISSWLA [SEQ ID NO:30] CDR2: EASSLES [SEQ ID NO:31] CDR3: QQLESYPLT [SEQ ID NO:32] scFvAb2 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYE ASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGCG TKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS CAASGFTFSSYWMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVT VSS [SEQ ID NO:208] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEW VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY

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CARPLNAGELDVWGOGTMVTVSSGGGGSGGGGSGGGGSGGGGSDI QMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEA SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGCGT KVEIK [SEQ ID NO:209] Illustrative nucleotide sequence of scFv AЬ2 GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAG CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGCTGGAGA GCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCT TCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGA AGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCG AGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO:248] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGG GGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG ACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGG CGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGC TGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGC TGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTT

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CAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGC CTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGCTGGAGAG CTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:249] ADI-11776 [АЬЗ] (H76) EVQLLESGGGLVQPGGSLRLSCA ASGFTFSKYTMSWVRQAPGKGL EWVSAIVGSGESTYFADSVKGRF TISRDNSKNTLYLQMNSLRAEDT AVYYCAREGGPYYDSSGYFVYY GMDVWGQGTTVTVSS [SEQ ID NO:5] CDR1: FTFSKYTMS [SEQ ID NO:33] (not no Kabat) or KYTMS [SEQ ID NO:183] CDR2: AIVGSGESTYFADSVKG [SEQ ID NO:34] CDR3: AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:35] (not no Kabat) or EGGPYYDSSGYFVYYGMDV [SEQ ID NO:184] DIQMTQSPSTLSASVGDRVTITCR ASQSISSWLAWYQQKPGKAPKLLI YKASSLESGVPSRFSGSGSGTEFTL TISSLQPDDFATYYCQQYDDLPTF GGGTKVEIK [SEQ ID NO:6] CDR1: RASQSISSWLA [SEQ ID NO:36] CDR2: KASSLES [SEQ ID NO:37] or KASSLE [SEQ ID NO: 185] (not no Kabat) CDR3: QQYDDLPT [SEQ ID NO:38] scFv АЬЗ DIOMTQSPSTLSASVGDRVTITCRASOSISSWLAWYOQKPGKAPKLLIY KASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOQYDDLPTFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGFTFSKYTMSWVRQAPGKCLEWVSAIVGSGESTYFADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGM DVWGQGTTVTVSS [SEQ ID NO: 198] EVQLLESGGGLVQPGGSLRLSCAASGTFFSKYTMSWVRQAPGKCLEWV SAIVGSGESTYFADSVKGRFTISRDNSKNTLYLOMNSLRAEDTAVYYCA REGGPYYDSSGYFVYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGG SGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKA

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PKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDL PTFGCGTKVEIK [SEQ ID NO:210] Illustrative nucleotide sequence of scFv AЬ3 GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACAAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAG CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGACG ACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGG CGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTG CAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCA CCTTCAGCAAGTACACCATGAGCTGGGTGAGGCAGGCCCCCGGCAA GTGCCTGGAGTGGGTGAGCGCCATCGTGGGCAGCGGCGAGAGCACC TACTTCGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACA ACAGCAAGACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTAC GACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCC AGGGCACCACGTGACCGTGAGCAGC [SEQ ID NO:250] GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAAG TACACCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGAGCGCCATCGTGGGCAGCGGCGAGAGCACCTACTTCGCCGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAAC ACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGG CTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACG TGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAG AGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCA CCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCA GCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGC AGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCG GCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTC GCCACCTACTACTGCCAGCAGTACGACGACCTGCCCACCTTCGGCTG

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CGGCACCAAGGTGGAGATCAAG [SEQ ID NO:251] ADI-11801 [Ab4] QVQLVQSGAEVKKPGASVKVSC KASGYTFSDYYMHWVRQAPGQG LEWMGMINPSWGSTSYAQKFQG RVTMTRDTSTSTVYMELSSSLRSE DTAVYYCAREAADGFVGERYFD LWGRGTLVTVSS [SEQ ID NO:7] CDR1: YTFSDYYMH [SEQ ID NO:39] (not by Kabat numbering) or DYYMH [SEQ ID NO:437] CDR2: MINPSWGSTSYAQKFQG [SEQ ID NO:40] CDR3: AREAADGFVGERYFDL [SEQ ID NO:41] (not by Kabat numbering) or EAADGFVGERYFDL [SEQ ID NO:438] DIVMTQSPLSLPVTPGEPASISCRSS QSLLYSNGYNYLDWYLQKPGQSP QLLIYLGSNRASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCMQD VALPITFGGGTKVEIK [SEQ ID NO:8] CDR1: RSSQSLLYSNGYNYLD [SEQ ID NO:42] CDR2: LGSNRAS [SEQ ID NO:43] CDR3: MQDVALPIT [SEQ ID NO:44] scFv Ab4 DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVAL PITFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSOVQLVQSGAEVKKP GASVKVSCKASGYTFSDYYMHWVRQAPGQCLEWMGMINPSWGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGER YFDLWGRGTLVTVSS [SEQ ID NO:211] QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQCLE WMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCAREAADGFVGERYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSG GGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKP GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QDVALPITFGCGTKVEIK [SEQ ID NO:212] Illustrative nucleotide GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGG CGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCTGTAC AGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCCCGGCC

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scFv sequence АЬ4 AGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGGCCAGCGG CGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACT GCATGCAGGACGTGGCCCTGCCCATCACCTTCGGCTGCGGCACCAAG GTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGA GCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCTGAAGGTGAGCTG CAAGGCCAGCGGCTACACCTTCAGCGACTACTACATGCACTGGGTGA GGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATGATCAACCC CAGCTGGGGCAGCACCAGCTACGCCCAGAAGTTCCAGGGCAGGGTG ACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGA GGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGC AGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO:252] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG CCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCGA CTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGT GGATGGGCATGATCAACCCCAGCTGGGGCAGCACCAGCTACGCCCA GAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGC ACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAG GTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCC TGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAG CCAGAGCCTGCTGTACAGCAACGGCTACAACTACCTGGACTGGTACC TGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAG CAACAGGGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGC GGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAGGCCGAGGACG TGGGCGTGTACTACTGCATGCAGGACGTGGCCCTGCCCATCACCTTC GGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:253] ADI-11807 [АЬ5] EVQLVESGGGLVQPGGSLRLSCA ASGFTFGSYWMSWVRQAPGKGL EWVATIKQDGSEKSYVDSVKGRF TISRDNAKNSLYLQMNSLRAEDT DIQMTQSPSTLSASVGDRVTITCR ASQSISSWLAWYQQKPGKAPKLLI YEASSLESGVPSRFSGSSGTEFTL TISSLQPDDFATYYCQQSQSYPPIT

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(107) AVYYCARPLNAGELDVWGQGT MVTVSS [SEQ GO NO:9] CDR1: FTFGSYWMS [SEQ ID NO:45] (not no Kabat) or SYWMS [SEQ ID NO:181] CDR2: TIKQDGSEKSYVDSVKG [SEQ ID NO:46] CDR3: ARPLNAGELDV [SEQ ID NO:47] (not no Kabat) or RPLNAGELDV [SEQ ID NO: 182] FGGGTKVEIK [SEQ ID NO: 10] CDR1: RASQSISSWLA [SEQ ID NO:48] CDR2: EASSLES [SEQ ID NO:49] CDR3: QQSQSYPPIT [SEQ ID NO:50] scFv Ab5 107 scFv DIQMTQSPSTLSASVGDRVTTITCRASOSISSWLAWYQQKPGKAPKLLIYE ASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOOSQSYPPITFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR LSCAASGFTFGSYWMSWVROAPGKCLEWVATIKQDGSEKSYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGOGTM VTVSS [SEQ ID NO: 188] EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKCLEW VATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARPLNAGELDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDI QMTQSPSTLSASVGDRVTITCRASOSISSWLAWYQQKPGKAPKLLIYEA SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOOSQSYPPITFGCGT KVEIK [SEQ ID NO:213] Illustrative nucleotide sequence of scFv AЬ5 GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAG CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGA GCTACCCCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCC

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TGGTGCAGCCCGGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGG CTTCACCTTCGGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCG GCAAGTGCCTGGAGTGGGTGGCCACCATCAAGCAGGACGGCAGCGA GAAGAGCTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAACGCCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGG CCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGG CGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO:254] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCACCATCAAGCAGGACGGCAGCGAGAAGAGCTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGG GGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG ACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGG CGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGC TGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGC TGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGC CTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGAG CTACCCCCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:255] ADI-11809 [Ab6] EVQLVESGGGLVQPGGSLRLSCA ASGFFPSYWMSWVRQAPGKGL EWVATIKRDGSEKGYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCARPLNAGELDVWGQGT MVTVSS [SEQ ID NO: 11] CDR1: FTFPSYWMS [SEQ ID NO:51] (not no Kabat) or SYWMS DIQMTQSPSTLSASVGDRVTITCR ASQSISSWLAWYQQKPGKAPKLLI YEASSLESGVPSRFSGSGSGTEFTL TISSLQPDDFATYYCQQSQSYPPIT FGGGTKVEIK [SEQ ID NO: 12] CDR1: RASQSISSWLA [SEQ ID NO:54] CDR2: EASSLES [SEQ ID NO:55]

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[SEQ ID NO:181] CDR2: TIKRDGSEKGYVDSVKG [SEQ ID NO:52] CDR3: ARPLNAGELDV [SEQ ID NO:53] (not by Kabat numbering) or PLNAGELDV [SEQ ID NO:439] CDR3: QQSQSYPPIT [SEQ ID NO:56] scFvAb6 DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYE ASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR LSCAASGFTFPSYWMSWVRQAPGKCLEWVATIKRDGSEKGYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTM VTVSS [SEQ ID NO:214] EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKCLEW VATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARPLNAGELDVWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDI QMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEA SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPrrFGCGT KVEIK [SEQ ID NO:215] Illustrative nucleotide sequence of scFv AB6 GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAG CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGA GCTACCCCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCC TGGTGCAGCCCGGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGG CTTCACCTTCCCCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCG GCAAGTGCCTGGAGTGGGTGGCCACCATCAAGAGGGACGGCAGCGA GAAGGGCTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGG GACAACGCCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGG

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CCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGG CGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO:256] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCCCCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCACCATCAAGAGGGACGGCAGCGAGAAGGGCTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGCCCCTGAACGCCGGCGAGCTGGACGTGTGG GGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG ACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGG CGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGC TGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGC TGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGC CTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGAG CTACCCCCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:257] ADI-11815 [Ab7] QVQLVQSGAEVKKPGASVKVSC KASGYTFGTYYMHWVRQAPGQ GLEWMGIINPSRGSTVYAQKFQG RVTMTRDTSTSTVYMELSSSLRSE DTAVYYCARGAGYDDEDMDVW GKGTTVTVSS [SEQ ID NO: 13] CDR1: YTFGTYYMH [SEQ ID NO:57] (not by Kabat numbering) or TYYMH [SEQ ID NO:440] CDR2: IINPSRGSTVYAQKFQG [SEQ ID NO:58] CDR3: ARGAGYDDEDMDV [SEQ ID NO :59] (not by Kabat numbering) DIQMTQSPSSVSASVGDRVTITCR ASQGIDSWLAWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQAHSYPLT FGGGTKVEIK [SEQ ID NO: 14] CDR1:RASQGIDSWLA [SEQ ID NO:60] CDR2: AASSLQS [SEQ ID NO:61] CDR3: QQAHSYPLT [SEQ ID NO:62]

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or GAGYDDEDMDV [SEQ ID NO:441] scFv Ab7 DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFGTYYMHWVRQAPGQCLEWMGIINPSRGSTVYAQKFQ GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWG KGTTVTVSS [SEQ ID NO:216] QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQCLE WMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCARGAGYDDEDMDVWGKGTTVTVSSGGGGSGGGGSGGGGSGGG GSDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTF GCGTKVEIK [SEQ ID NO:217] Illustrative nucleotide sequence of scFv AЬ7 GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCGACAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTGCCAGCAGGCCCACAG CTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA AGAAGCCCGGCGCCAGCTGAAGGTGAGCTGCAAGGCCAGCGGCTA CACCTTCGGCACCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCC AGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCAGGGGCAGCAC CGTGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGAC ACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCG AGGACACCGCCGTGTACTACTGCGCCAGGGGCGCCGGCTACGACGA CGAGGACATGGACGTGTGGGGCAAGGGCACCACGTGACCGTGAGC AGC [SEQ ID NO:258] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG CCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCGGCAC

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CTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGT GGATGGGCATCAACCCCAGCAGGGGCAGCACCGTGTACGCCCA GAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGC ACCTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCGCCAGGGGCGCCGGCTACGACGACGAGGACATGGA CGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGC GGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGC GGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCA GCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCAT CGACAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCA GCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCAT CAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGG CCCACAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATC AAG [SEQ ID NO: 259] ADI-11819 [Ab8] EVQLVESGGGLVKPGGSLRLSCA ASGFTFSSYAMSWVRQAPGKGLE WVSSISSSSEGIYYADSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAV YYCAREGGPYYDSSGYFVYYGM DVWGQGTTVTVSS [SEQ ID NO:15] CDR1: FTFSSYAMS [SEQ ID NO:63] (not by Kabat numbering) or SYAMS [SEQ ID NO:442] CDR2: SISSSSEGIYYADSVKG [SEQ ID NO:64] CDR3: AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:65] (not no Kabat) or EGGPYYDSSGYFVYYGMDV [SEQ ID NO:443] DIQMTQSPSTLSASVGDRVTITCR ASNSISSWLAWYQQKPGKAPKLLI YEASSTKSGVPSRFSGSGSGTEFTL TISSLQPDDFATYYCQQYDDLPTF GGGTKVEIK [SEQ ID NO: 16] CDR1: RASNSISSWLA [SEQ ID NO:66] CDR2: EASSTKS [SEQ ID NO:67] CDR3: QQYDDLPT [SEQ ID NO:68]

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scFvAb8 DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYE ASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGCG TKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLS CAASGFTFSSYAMSWVRQAPGKCLEWVSSISSSSEGIYYADSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDV WGQGTTVTVSS [SEQ ID NO:218] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWV SSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR [SEQ ID NO:219] Illustrative nucleotide sequence of scFv AB8 GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCAACAGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGAGGCCAGCAGCACCAAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAG CCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGACG ACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGG CGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTG AAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCA CCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAA GTGCCTGGAGTGGGTGAGCAGCATCAGCAGCAGCAGCGAGGGCATC TACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACA ACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGA GGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTAC GACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCC AGGGCACCACGTGACCGTGAGCAGC [SEQ ID NO:260] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGAGCAGCATCAGCAGCAGCAGCGAGGGCATCTACTACGCCGA

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CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGG CTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCG TGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAG AGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCA CCTGCAGGGCCAGCAACAGCATCAGCAGCTGGCTGGCCTGGTACCA GCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAGGCCAGC AGCACCAAGGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCG GCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTC GCCACCTACTACTGCCAGCAGTACGACGACCTGCCCACCTTCGGCTG CGGCACCAAGGTGGAGATCAAG [SEQ ID NO:261] ADI-11830 [Ab9] EVQLVESGGGLVQPGGSLRLSCA ASGFTFSSYWMSWVRQAPGKGL EWVANINTDGSEVYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCARDVGPGIAYQGHFDY WGQGTLVTVSS [SEQ ID NO: 17] CDR1: FTFSSYWMS [SEQ ID NO:69] (not no Kabat) or SYWMS [SEQ ID NO:181] CDR2: NINTDGSEVYYVDSVKG [SEQ ID NO:70] CDR3: ARDVGPGJAYQGHFDY [SEQ ID NO:71] (not by Kabat numbering) or DVGPGIAYQGHFDY [SEQ ID NO:444] DIQMTQSPSSSLSASVGDRVTITCRA SQVIYSYLNWYQQKPGKAPKLLIY AASSLKSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQVYDTPLTF GGGTKVEIK [SEQ ID NO: 18] CDR1: RASQVIYSYLN [SEQ ID NO:72] CDR2: AASSLKS [SEQ ID NO:73] CDR3: QQVYDTPLT [SEQ ID NO:74] scFvAb9 DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIY AASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFTFSSYWMSWVRQAPGKCLEWVANINTDGSEVYYVDSVK

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GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDY WGQGTLVTVSS [SEQ ID NO:220] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEW VANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARDVGPGIAYQGHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGG GGSDIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKL LIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLT FGCGTKVEIK [SEQ ID NO:221] Illustrative nucleotide sequence of scFv AЬ9 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGTGATCTACAG CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAGCCTGAAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACG ACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCT TCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAACACCGACGGCAGCGAGG TGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCA TCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTG ACCGTGAGCAGC [SEQ ID NO:262] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAACACCGACGGCAGCGAGGTGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGC CACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCG

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GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCT GAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGC CAGGTGATCTACAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCA AGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGAAGAGCGG CGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTG CCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGG TGGAGATCAAG [SEQ ID NO:263] ADI-11835 [Ab 10] (135) QLQLQESGPGLVKPSETLSLTCTV SGGSISSTDYYWGWIRQPPGKGL EWIGSIGYSGTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVY YCARETAHDVHGMDVWGQGTT VTVSS [SEQ ID NO: 19] CDR1: GSJSSTDYYWG [SEQ ID NO:75] (not by Kabat numbering) or STDYYWG [SEQ ID NO:445] CDR2: SIGYSGTYYNPSLKS [SEQ ID NO:76] CDR3: ARETAHDVHGMDV [SEQ ID NO:77] (not by Kabat numbering) or ETAHDVHGMDV [SEQ ID NO:446] EIVLTQSPATLSLSPGERATLSCRA SHSVYSYLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQYDNLPTF GGGTKVEIK [SEQ ID NO:20] CDR1: RASHSVYSYLA [SEQ ID NO:78] CDR2: DASNRAT [SEQ ID NO:79] CDR3: QQYDNLPT [SEQ ID NO:80] scFv Ab 10 EIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGCG TKVEIKGGGGSGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLT CTVSGGSISSTDYYWGWIRQPPGKCLEWIGSIGYSGTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVT VSS [SEQ ID NO:222] QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKCLEWI GSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARET

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AHDVHGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVLT QSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNR ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGCGTKVEI TO [SEQ GO NO:223] Illustrative nucleotide sequence of scFv Ab 10 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCACAGCGTGTACAG CTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTG CTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGACAA CCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCCAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGA AGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAG CATCAGCAGCACCGACTACTACTGGGGCTGGATCAGGCAGCCCCCC GGCAAGTGCCTGGAGTGGATCGGCAGCATCGGCTACAGCGGCACCT ACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACAC CAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCC GACACCGCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGC ACGGCATGGACGTGTGGGGCCAGGGCACCACGTGACCGTGAGCAG C [SEQ ID NO:264] CAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCG AGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAG CACCGACTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGC CTGGAGTGGATCGGCAGCATCGGCTACAGCGGCACCTACTACAACC CCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAA CCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCC GTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGCACGCATGG ACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGG CGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGG CGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG AGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCACAGCG TGTACAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCC AGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCG

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CCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATC AGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTA CGACAACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:265] ADI-10154 [Abll] EVQLLESGGGLVQPGGSLRLSCA ASGFTFSSYAMSWVRQAPGKGLE WVSAISASGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTA VYYCARPRAYYDSSGFKVNYGM DVWGQGTTVTVSS [SEQ ID NO:266] CDR1: SYAMS [SEQ ID NO:304] or FTFSSYAMS [SEQ ID NO:528] (not no Kabat) CDR2: AISASGGSTYYADSVKG [SEQ ID NO:305] CDR3: PRAYYDSSGFKVNYGMDV [SEQ ID NO:306] or ARPRAYYDSSGFKVNYGMDV [SEQ ID NO:529] (not by Kabat numbering) EIVLTQSPGTLSLSPGERATLSCRA SQSVSSSFLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQASSSPPTF GGGTKVEIK [SEQ ID NO:267] CDR1: RASQSVSSSFLA [SEQ ID NO:307] CDR2: GASSRAT [SEQ ID NO:308] CDR3: QQASSSPPT [SEQ ID NO:309] scFv Abll EIVLTOSPGTLSLSPGERATLSCRASQSVSSSFLAWYOQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQASSSPPTFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGFTFSSYAMSWVRQAPGKCLEWVSAISASGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARPRAYYDSSGFKVNYGMD VWGQGTTVTVSS [SEQ ID NO:447] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWV SAISASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA RPRAYYDSSGFKVNYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS GGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQA

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PRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQASSS PPTFGCGTKVEIK [SEQ ID NO:448] Illustrative nucleotide sequence of scFv Ab 11 GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGG CTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAG CAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGCC AGCAGCAGCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCA AGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCA GCGGCGGCGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGG CCTGGTGCAGCCCGGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGC GGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCC CGGCAAGTGCCTGGAGTGGGTGAGCGCCATCAGCGCCAGCGGCGGC AGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCA GGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCAGGGCCTAC TACGACAGCAGCGGCTTCAAGGTGAACTACGGCATGGACGTGTGGG GCCAGGGCACCACGTGACCGTGAGCAGC [SEQ ID NO:489] GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGAGCGCCATCAGCGCCAGCGGCGGCAGCACCTACTACGCCGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAAC ACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGCCCAGGGCCTACTACGACAGCAGCGGCTTC AAGGTGAACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGA CCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAG CCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGC TGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTTCCTGGCCTGGTACC AGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAG CAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGC GGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTT

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CGCCGTGTACTACTGCCAGCAGGCCAGCAGCAGCCCCCCCACCTTCG GCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:490] ADI-10155 [Ab 12] EVQLLESGGGLVQPGGSLRLSCA ASGFTFSSYAMSWVRQAPGKGLE WVSGISGSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTA VYYCAREGHSSSYYDHAFDIWG QGTMVTVSS [SEQ ID NO:268] CDR1: SYAMS [SEQ ID NO:310] or FTFSSYAMS [SEQ ID NO:530] (not no Kabat) CDR2: GISSGGSTYYADSVKG [SEQ ID NO:311] CDR3: EGHSSSYYDHAFDI [SEQ ID NO:312] or AREGHSSSYYDHAFDI [SEQ ID NO:531] (not no Kabat) EIVLTQSPGTLSLSPGERATLSCRA SQSVSSDYLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQHSSAPPTF GGGTKVEIK [SEQ ID NO:269] CDR1: RASQSVSSDYLA [SEQ ID NO:313] CDR2: GASSRAT [SEQ ID NO:314] CDR3: QQHSSAPPT [SEQ ID NO:315] EIVLTQSPGTLLSLSPGERATLSCRASQSVSSDYLAWYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHSSAPPTFGC scFv Abl2 GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRL SCAASGFTFSSYAMSWVRQAPGKCLEWVSGISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAREGHSSSYYDHAFDIWGQG TMVTVSS [SEQ ID NO:449] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLEWV SGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA REGHSSSYYDHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGS EIVLTQSPGTLSLSPGERATLSCRASOSVSSDYLAWYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHSSAPPTFGC GTKVEIK [SEQ ID NO:450] Illustrative GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG

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nucleotide sequence of scFv Ab 12 CGACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGG CTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAG CAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCAC AGCAGCGCCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCA AGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCA GCGGCGGCGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGG CCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGC GGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCC CGGCAAGTGCCTGGAGTGGGTGAGCGGCATCAGCGGCAGCGGCGGC AGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCA GGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAG GGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCCACAGC AGCAGCTACTACGACCACGCCTTCGACATCTGGGGCCAGGGCACCAT GGTGACCGTGAGCAGC [SEQ ID NO:491] GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGAGCGGCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAAC ACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCACAGCAGCAGCTACTACGACCA CGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCC TGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAG CCAGAGCGTGAGCAGCGACTACCTGGCCTGGTACCAGCAGAAGCCC GGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCA CCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTT CACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACT ACTGCCAGCAGCACAGCAGCGCCCCCCCCACCTTCGGCTGCGGCACC AAGGTGGAGATCAAG [SEQ ID NO:492] ADI-10157 QVQLQESGPGLVKPSETLSLTCTV SGGSISSYYWSWIRQPPGKGLEWI GSIYYSGSTNYNPSLKSRVTISVD EIVMTQSPATLSVSPGERATLSCCR ASQSVSSNLAWYQQKPGQAPRLLI YGASTRATGIPARFSGSGSGTEFTL

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[AYZ] TSKNQFSLKLSSVTAADTAVYYC ARVGGVYSTIETYGMDVWGQGT TVTVSS [SEQ GO NO:270] CDR1: SYYWS [SEQ GO NO:316] or GSISSYYWS [SEQ GO NO:532] (not by Kabat) CDR2: SIYYSGSTNYNPSLKS [SEQ ID NO:317] CDR3: VGGVYSTIETYGMDV [SEQ ID NO:318] or ARVGGVYSTIETYGMDV [SEQ ID NO:533] (not no Kabat) TISSLQSEDFAVYYCQQYTVYPPT FGGGTKVEIK [SEQ ID NO:271] CDR1: RASQSVSSNLA [SEQ ID NO:319] CDR2: GASTRAT [SEQ ID NO:320] CDR3: QQYTVYPPT [SEQ ID NO:321] scFv AЫЗ EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIY GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTVYPPTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLS LTCTVSGGSISSYYWSWIRQPPGKCLEWIGSIYYSGSTNYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARVGGVYSTIETYGMDVWGQGT TVTVSS [SEQ ID NO:451] QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKCLEWIGS IYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVG GVYSTIETYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEI VMTQSPATLSVSPGERTLSCRASQSVSSNLAWYQQKPGQAPRLLIYGA STRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTVYPPTFGCGT KVEIK [SEQ ID NO:452] Illustrative nucleotide sequence of scFv AC3 GAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCGTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTG CTGATCTACGGCGCCAGCACCAGGGCCACCGGCATCCCCGCCAGGTT CAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGC CTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACACCGT GTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC

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GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGG TGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGG CAGCATCAGCAGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGC AAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCA ACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACAC CAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCC GACACCGCCGTGTACTACTGCGCCAGGGTGGGCGGCGTGTACAGCA CCATCGAGACCTACGGCATGGACGTGTGGGGCCAGGGCACCACCGT GACCGTGAGCAGC [SEQ ID NO:493] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCG AGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAG CTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGT GGATCGGCAGCATCTACTACAGCGGCCAGCACCAACTACAACCCCAG CCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAG TTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTA CTACTGCGCCAGGGTGGGCGGCGTGTACAGCACCATCGAGACCTAC GGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGAGATCGTGATGACCCAGAGCCCCGCCACCCT GAGCGTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC CAGACCGTGAGCAGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCC AGGCCCCCAGGCTGCTGATCTACGGCGCCAGCACCAGGGCCACCGG CATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACC CTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTG CCAGCAGTACACCGTGTACCCCCCCACCTTTCGGCTGCGGCACCAAGG TGGAGATCAAG [SEQ ID NO:494] ADI-10158 [Ab 14] QVQLQQWGAGLLKPSETLSLTCA VYGGSFSGYYWSWIRQPPGKGLE WIGEIDHSGSTNYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVY YCARQGIHGLRYFDLWGRGTLV TVSS [SEQ ID NO:272] CDR1: GYYWS [SEQ ID NO:322] EIVMTQSPATLSLSPGERATLSCRA SQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQDHNFPYTF GGGTKVEIK [SEQ ID NO:273] CDR1: RASQSVSSYLA [SEQ ID

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or GSFSGYYWS [SEQ Ш NO:534] (not by Kabat) CDR2: EIDHSGSTNYNPSLKS [SEQ ID NO:323] CDR3: QGIHGLRYFDL [SEQ ID NO:324] or ARQGIHGLRYFDL [SEQ ID NO:535] (not by Kabat numbering) NO:325] CDR2: DASNRAT [SEQ ID NO:326] CDR3: QQDHNFPYT [SEQ ID NO:327] scFv Ab 14 EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQDHNFPYTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQQWGAGLLKPSETL SLTCAVYGGSFSGYYWSWIRQPPGKCLEWIGEIDHSGSTNYNPSLKSRV TISVDTSKNQFSLKLSSVTAADTAVYYCARQGIHGLRYFDLWGRGTLVT VSS [SEQ ID NO:453] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKCLEWI GEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR QGIHGLRYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVM TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQDHNFPYTFGCGTK VEIK [SEQ ID NO:454] Illustrative nucleotide sequence of scFv Ab 14 GAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTG CTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACCACA ACTTCCCCTACACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGC TGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGG CAGCTTCAGCGGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCA AGTGCCTGGAGTGGATCGGCGAGATCGACCACAGCGGCAGCACCAA

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CTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACC AGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCG ACACCGCCGTGTACTACTGCGCCAGGCAGGGCATCCACGGCCTGAG GTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO:495] CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCG AGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGC TACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGT GGATCGGCGAGATCGACCACAGCGGCAGCACCAACTACAACCCCAG CCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAG TTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTA CTACTGCGCCAGGCAGGCATCCACGGCCTGAGGTACTTCGACCTGT GGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAG CGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAG CGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC GGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCA GCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCT GCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGG TTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCA GCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACCAC AACTTCCCCTACACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:496] ADI-10160 [Ab 15] EVQLVESGGGLVQPGGSLRLSCA AS GFTFS S YWMS W VRQAPGKGL EWVANINQDGSEKYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCAREANYYGNVGDDYW GQGTLVTVSS [SEQ ID NO:274] CDR1: SYWMS [SEQ ID NO:328] or FTFSSYWMS [SEQ ID NO:536] (not by Kabat numbering) CDR2: NINQDGSEKYYVDSVKG [SEQ ID NO:329] DIQMTQSPSSLSSASVGDRVTITCRA SQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQQYVTPITFG GGTKVEIK [SEQ ID NO:275] CDR1: RASQSISSYLN [SEQ ID NO:331] CDR2: AASSLQS [SEQ ID NO:332] CDR3: QQQYVTPIT [SEQ ID NO:333]

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CDR3: EANYYGNVGDDY [SEQ ID NO:330] or AREANYYGNVGDDY [SEQ ID NO:537] (not no Kabat) DIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQYVTPITFGCG scFv Ab 15 TKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS CAASGFFSSYWMSWVRQAPGKCLEWVANINQDGSEKYYVDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQG TLVTVSS [SEQ ID NO:455] EVOLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVROAPGKCLEW VANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CAREANYYGNVGDDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGG SDIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQYyTPITFGC GTKVEIK [SEQ ID NO:456] Illustrative nucleotide sequence of scFv Ab 15 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCAGTACGT GACCCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGG TGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTT CACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGA AGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACG GCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGT GAGCAGC [SEQ ID NO:497]

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GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAACCAGGACGGCAGCGAGAAGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGA CGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCG CCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAG CATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCC CCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCC CAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCCTGACC ATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCA GCAGTACGTGACCCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAG ATCAAG [SEQ ID NO:498] ADI-10161 [Ab 16] EVQLVESGGGLVQPGGSLRLSCA ASGFTFSSYWMSWVRQAPGKGL EWVANINQDGSEKYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCAREGGDSWYHAFDIWG QGTMVTVSS [SEQ ID NO:276] CDR1: SYWMS [SEQ ID NO:334] or FTFSSYWMS [SEQ ID NO:538] (not by Kabat numbering) CDR2: NINQDGSEKYYVDSVKG [SEQ ID NO:335] CDR3: EGGDSWYHAFDI [SEQ ID NO:336] or AREGGDSWYHAFDI [SEQ ID NO:539] (not no Kabat) DIQMTQSPSSVSASVGDRVTITCR ASQGISSWLAWYQQKPGKAPKLLI YAASNLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQKLSLPLT FGGGTKVEIK [SEQ ID NO:277] CDR1: RASQGISSWLA [SEQ ID NO:337] CDR2: AASNLQS [SEQ ID NO:338] CDR3: QQKLSLPLT [SEQ ID NO:339]

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scFv Ab 16 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIY AASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKLSLPLTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL RLSCAASGFTFSSYWMSWVRQAPGKCLEWVANINQDGSEKYYVDSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGDSWYHAFDIWG QGTMVTVSS [SEQ ID NO:457] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEW VANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CAREGGDSWYHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGG SDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLI YAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKLSLPLTF GCGTKVEIK [SEQ ID NO:458] Illustrative nucleotide sequence of scFv Ab 16 GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAACCTGCAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTGCCAGCAGAAGCTGAG CCTGCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGG TGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTT CACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGA AGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCGACAGCT GGTACCACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTG AGCAGC [SEQ ID NO:499] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAACCAGGACGGCAGCGAGAAGTACTACGTGGA

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CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGGCGGCGACAGCTGGTACCACGCCTTC GACATCTGGGGCCAGGCACCATGGTGACCGTGAGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGC CAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGC ATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCC CCAAGCTGCTGATCTACGCCGCCAGCAACCTGCAGAGCGGCGTGCCC AGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCA TCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAG AAGCTGAGCCTGCCCCTGACCTTTCGGCTGCGGCACCAAGGTGGAGAT CAAG [SEQ ID N0:500] ADI-10163 [Ab 17] QVQLQESGPGLVKPSQTLSLTCT VSGGSISSGGYYWSWIRQHPGKG LEWIGSIYYSGSTYYNPSLKSRVT ISVDTSKNQFSLKLSSVTAADTAV YYCARDRLDYSYNYGMDVWGQ GTTVTVSS [SEQ ID NO:278] CDR1: SGGYYWS [SEQ ID NO:340] or GSISSGGYYWS [SEQ ID NO: 540] (not by Kabat numbering) CDR2: SIYYSGSTYYNPSLKS [SEQ ID NO:341] CDR3: DRLDYSYNYGMDV [SEQ ID NO:342] or ARDRLDYSYNYGMDV [SEQ ID NO:541] (not no Kabat) DIQMTQSPSSLSSASVGDRVTITCRA SQSISSYLNWYQQKPGKAPKLLIY GASSLQSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQVYSAPFTFG GGTKVEIK [SEQ ID NO:279] CDR1: RASQSISSYLN [SEQ ID NO:343] CDR2: GASSLQS [SEQ ID NO:344] CDR3: QQVYSAPFT [SEQ ID NO:345] scFv Ab 17 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYG ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSAPFTFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSL

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TCTVSGGSISSGGYYWSWIRQHPGKCLEWIGSIYYSGSTYYNPSLKSRVT ISVDTSKNQFSLKLSSVTAADTAVYYCARDRLDYSYNYGMDVWGQGT TVTVSS [SEQ ID NO:459] OVQLQESGPGLVKPSOTLSLTCTVSGGSISSGGYYWSWIRQHPGKCLEW IGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR DRLDYSYNYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSD IQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSAPFTFGCGT KVEIK [SEQ ID NO:460] Illustrative nucleotide sequence of scFv Ab 17 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGGCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CCTGCAGCCCGAGGACTTCGCCACCTACTGCCAGCAGGTGTACA GCGCCCCCTTCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGG TGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGG CAGCATCAGCAGCGGCGGCTACTACTGGAGCTGGATCAGGCAGCAC CCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCA GCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGT GGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACC GCCGCCGACACCGCCGTGTACTACTGCGCCAGGGACAGGCTGGACT ACAGCTACAACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGT GACCGTGAGCAGC [SEQ ID NO:501] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCC AGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAG CGGCGGCTACTACTGGAGCTGGATCAGGCAGCACCCCGGCAAGTGC CTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCTACTACA ACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAA GAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACC GCCGTGTACTACTGCGCCAGGGACAGGCTGGACTACAGCTACAACT

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ACGGCATGGACGTGTGGGGCCAGGCACCACCGTGACCGTGAGCAG CGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAG CGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGC CTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCA GCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGG CAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCAGCCTGCAGAGC GGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCA CCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTAC TGCCAGCAGGTGTACAGCGCCCCCTTCACCTTCGGCTGCGGCACCAA GGTGGAGATCAAG [SEQ ID NO:502] ADI-10164 [Ab 18] QVQLQESGPGLVKPSETLSLTCA VSGYSISSGYYWGWIRQPPGKGL EWIGSIYHSGSTNYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVY YCARLPPWFGFSYFDLWGRGTLV TVSS [SEQ ID NO:280] CDR1: SGYYWG [SEQ ID NO:346] or YSISSGYYWG [SEQ ID NO:542] (not by Kabat numbering) CDR2: SIYHSGSTNYNPSLKS [SEQ ID NO:347] CDR3: LPPWFGFSYFDL [SEQ ID NO:348] or ARLPPWFGFSYFDL [SEQ ID NO:543] (not by Kabat numbering) EIVLTQSPATLSLSPGERATLSCRA SQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQVDNYPPTF GGGTKVEIK [SEQ ID NO:281] CDR1: RASQSVSSYLA [SEQ ID NO:349] CDR2: DASNRAT [SEQ ID NQ:350] CDR3: QQVDNYPPT [SEQ ID NO:351] scFv Ab 18 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQVDNYPPTFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSL TCAVSGYSISSGYYWGWIRQPPGKCLEWIGSIYHSGSTNYNPSLKSRVTI SVDTSKNQFSLKLSSVTAADTAVYYCARLPPWFGFSYFDLWGRGTLVT VSS [SEQ ID NO:461] QVQLQESGPGLVKPSETLSLTCA VSGYSISSGYYWGWIRQPPGKCLEWI

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GSIYHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARL PPWFGFSYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLT QSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQVDNYPPTFGCGTKV EIK [SEQ ID NO:462] Illustrative nucleotide sequence of scFv Ab 18 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTG CTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGTGGACA ACTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTG GTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGAGCGGCT ACAGCATCAGCAGCGGCTACTACTGGGGCTGGATCAGGCAGCCCCC CGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACCACAGCGGCAGC ACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGG ACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGC CGCCGACACCGCCGTGTACTACTGCGCCAGGCTGCCCCCCTGGTTCG GCTTCAGCTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTG AGCAGC [SEQ ID NO:503] CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCG AGACCCTGAGCCTGACCTGCGCCGTGAGCGGCTACAGCATCAGCAG CGGCTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTG GAGTGGATCGGCAGCATCTACCACAGCGGCAGCACCAACTACAACC CCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAA CCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCC GTGTACTACTGCGCCAGGCTGCCCCCCTGGTTCGGCTTCAGCTACTTC GACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCT GAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGC GTGAGCAGCTACCTGGCCCTGGTACCAGCAGAAGCCCGGCCAGCCC

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CCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCC CGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCCTGACC ATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCA GGTGGACAACTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAG ATCAAG [SEQ ID NO:504] ADI-10165 [Ab 19] EVQLVESGGGLVQPGGSLRLSCA ASGFTFSSYWMSWVRQAPGKGL EWVANIKQDGSEKYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCARDVGPGIAYQGHFDY WGQGTLVTVSS [SEQ ID NO:282] CDR1: SYWMS [SEQ ID NO:352] or FTFSSYWMS [SEQ ID NO:544] (not by Kabat numbering) CDR2: NIKQDGSEKYYVDSVKG [SEQ ID NO:353] CDR3: DVGPGIAYQGHFDY [SEQ ID NO:354] or ARDVGPGIAYQGHFDY [SEQ ID NO:545] (not by Kabat numbering) DIQMTQSPSSLSSASVGDRVTITCRA SQSISSYLNWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQVYDTPLTF GGGTKVEIK [SEQ ID NO:283] CDR1: RASQSISSYLN [SEQ ID NO:355] CDR2: AASSLQS [SEQ ID NO:356] CDR3: QQVYDTPLT [SEQ ID NO:357] scFv Ab 19 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR LSCAASGFTFSSYWMSWVRQAPGKCLEWVANIKQDGSEKYYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYW GQGTLVTVSS [SEQ ID NO:463] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEW VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARDVGPGIAYQGHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGG GGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLT

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FGCGTKVEIK [SEQ ID NO:464] Illustrative nucleotide sequence of scFv Ab 19 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAG CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGA CACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGG TGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTT CACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGA AGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCA TCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTG ACCGTGAGCAGC [SEQ ID NO:505] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGC CACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCT GAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGC CAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCA AGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGG CGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTG CCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGG

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TGGAGATCAAG [SEQ ID NO:506] ADI-10167 [Ab20] QLQLQESGPGLVKPSETLSLTCTV SGGSISSSSYYWGWIRQPPGKGLE WIGSIYYSGSTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVY YCARETAHDVHGMDVWGQGTT VTVSS [SEQ ID NO:284] CDR1: SSSYYWG [SEQ ID NO:358] or GSISSSSYYWG [SEQ ID NO:546] (not by Kabat numbering) CDR2: SIYYSGSTYYNPSLKS [SEQ ID NO:359] CDR3: ETAHDVHGMDV [SEQ ID NO:360] or ARETAHDVHGMDV [SEQ ID NO:547] (not by Kabat numbering) EIVLTQSPATLSLSPGERATLSCRA SQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQYDNLPTFG GGTKVEIK [SEQ ID NO:285] CDR1: RASQSVSSYLA [SEQ ID NO:361] CDR2: DASNRAT [SEQ ID NO:362] CDR3: QQYDNLPT [SEQ ID NO:363] scFv Ab20 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASNRATGIPARFSGSGSGTDFTISSLEPEDFAVYYCQQYDNLPTFGCG TKVEIKGGGGSGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLT CTVSGGSISSSSYYWGWIRQPPGKCLEWIGSIYYSGSTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVT VSS [SEQ ID NO:465] OLOLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIROPPGKCLEWI GSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARE TAHDVHGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIVL TOSPATLSLSPGERATLSCRASOSVSSYLAWYOQKPGOAPRLLIYDASN RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGCGTKV EIK [SEQ ID NO:466] Illustrative nucleotide GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTG

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scFv sequence АЬ20 CTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGACAA CCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCCAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGA AGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAG CATCAGCAGCAGCAGCTACTACTGGGGCTGGATCAGGCAGCCCCCC GGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCA CCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGA CACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCC GCCGACACCGCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACG TGCACGGCATGGACGTGTGGGGCCAGGGCACCACGTGACCGTGAG CAGC [SEQ GO NO:507] CAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCG AGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAG CAGCAGCTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGC CTGGAGTGGATCGGCAGCATCTACTACAGCGGGCAGCACCTACTACA ACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAA GAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACC GCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGCACGGCA TGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGG CGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGG CGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGC CTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGA GCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGC CCCCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATC CCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAG CAGTACGACAACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGA TCAAG [SEQ GO NO:508] ADI-10168 [АЬ21] QVQLVQSGAEVKKPGSSVKVSC KASGGTFSSYAISWVRQAPGQGL EWMGSIIPIFGTANYAQKFQGRV TITADESTSTAYMELSSLRSEDTA EIVLTQSPATLSLSPGERATLSCRA SQSVSSYLAWYQQKPGQAPRLLIY DASKRATGIPARFSGSGSGTDFTLT ISSLEPEDFAVYYCQQSSNHPSTFG

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VYYCAREVGYGWYTKIAFDIWG QGTMVTVSS [SEQ ID NO:286] CDR1: SYAIS [SEQ ID NO:364] or GTFSSYAIS [SEQ ID NO:548] (not no Kabat) CDR2: SIIPIFGTANYAQKFQG [SEQ ID NO:365] CDR3: EVGYGWYTKIAFDI [SEQ ID NO:366] or AREVGYGWYTKIAFDI [SEQ ID NO:549] (not no Kabat) GGTKVEIK [SEQ ID NO:287] CDR1: RASQSVSSYLA [SEQ ID NO:367] CDR2: DASKRAT [SEQ ID NO:368] CDR3: QQSSNHPST [SEQ ID NO:369] scFv Ab21 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYD ASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNHPSTFGCG TKVEIKGGGGSGGGGSGGGGSGGGGSQVOLVQSGAEVKKPGSSVKV SCKASGGTFSSYAISWVRQAPGOCLEWMGSIIPIFGTANYAQKFOGRVTI TADESTSTAYMELSSLRSEDTAVYYCAREVGYGWYTKIAFDIWGQGTM VTVSS [SEQ ID NO:467] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQCLEW MGSIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCA REVGYGWYTKIAFDIWGOGTMVTVSSGGGGSGGGGSGGGGSGGGG SEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNHPSTFGC GTKVEIK [SEQ ID NO:468] Illustrative nucleotide sequence of scFv AB21 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTG CTGATCTACGACGCCAGCAAGAGGGCCACCGGCATCCCCGCCAGGT TCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGC AACCACCCCAGCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG

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GCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGT GAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGC GGCACCTTCAGCAGCTACGCCATCAGCTGGGTGAGGCAGGCCCCCG GCCAGTGCCTGGAGTGGATGGGCAGCATCATCCCCATCTTCGGCACC GCCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCACCGCCG ACGAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAG CGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGTGGGCTACGGC TGGTACACCAAGATCGCCTTCGACATCTGGGGCCAGGGCACCATGGT GACCGTGAGCAGC [SEQ ID NO:509] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCA GCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGCAG CTACGCCATCAGCTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAG TGGATGGGCAGCATCATCCCCATCTTCGGCACCGCCAACTACGCCCA GAAGTTCCAGGGCAGGGTGACCATCACCGCCGACGAGAGCACCAGC ACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGTGGGCTACGGCTGGTACACCAAGAT CGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCC TGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAG CCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGC CAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAAGAGGGCCACCG GCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCAC CCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACT GCCAGCAGAGCAGCAACCACCCCAGCACCTTTCGGCTGCGGCACCAA GGTGGAGATCAAG [SEQ ID NO:510] ADI-10173 [Ab22] QVQLVQSGAEVKKPGASVKVSC KASGYTFTSYYMHWVRQAPGQG LEWMGIINPSGGSTTYAQKFQGR VTMTRDTSTSTVYMELSSLRSED TAVYYCAREAADGFVGERYFDL WGRGTLVTVSS [SEQ ID NO:288] CDR1: SYYMH [SEQ ID NO:370] or YTFTSYYMH [SEQ ID DIVMTQSPLSLPVTPGEPASISCRSS QSLLHSNGYNYLDWYLQKPGQSP QLLIYLGSNRASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCMQA LGVPLTFGGGTKVEIK [SEQ ID NO:289] CDR1: RSSQSLLHSNGYNYLD

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NO:550] (not by Kabat numbering) CDR2: IINPSGGSTTYAQKFQG [SEQ ID NO:371] CDR3:EAADGFVGERYFDL[SEQ ID NO:372] or AREAADGFVGERYFDL [SEQ ID NO :551] (not by Kabat numbering) [SEQ ID NO:373] CDR2: LGSNRAS [SEQ ID NO:374] CDR3: MQALGVPLT [SEQ ID NO:375] scFv Ab22 DIVMTQSPLSLPVTPGEPASISCRSSOSLLHSNGYNYLDWYLQKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMOALGV PLTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKK PGASVKVSCKASGYTFTSYYMHWVRQAPGQCLEWMGIINPSGGSTTYA OKFOGRVTMTRDTSTVYMELSSLRSEDTAVYYCAREAADGFVGERY FDLWGRGTLVTVSS [SEQ ID NO:469] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQCLE WMGIINPSGGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YCAREAADGFVGERYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSGG GGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ ALGVPLTFGCGTKVEIK [SEQ ID NO:470] Illustrative nucleotide sequence of scFv AЬ22 GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGG CGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCTGCAC AGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCCCGGCC AGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGGCCAGCGG CGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACT GCATGCAGGCCCTGGGCGTGCCCCTGACCTTCGGCTGCGGCACCAAG GTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGA GCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCTGAAGGTGAGCTG CAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGA GGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCC CAGCGGCGGCAGCACCACCTACGCCCAGAAGTTCCAGGGCAGGGTG

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ACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGA GGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGC AGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO:511] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG CCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAG CTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGT GGATGGGCATCATCAACCCCAGCGGCGGCAGCACCACCTACGCCCA GAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGC ACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAG GTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCC TGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAG CCAGAGCCTGCTGCACAGCAACGGCTACAACTACCTGGACTGGTACC TGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAG CAACAGGGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGC GGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAGGCCGAGGACG TGGGCGTGTACTACTGCATGCAGGCCCTGGGCGTGCCCCTGACCTTC GGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:512] ADI-11802 [Ab23] QVQLVQSGAEVKKPGASVKVSC KASGYTFSGYYMHWVRQAPGQG LEWMGMINPYGGSTRYAQKFQG RVTMTRDTSTSTVYMELSSSLRSE DTAVYYCAREAADGFVGERYFD LWGRGTLVTVSS [SEQ ID NO:290] CDR1: GYYMH [SEQ ID NO:376] or YTFSGYYMH [SEQ ID NO:552] (not no Kabat) CDR2: MINPYGGSTRYAQKFQG [SEQ ID NO:377] DIVMTQSPLSLPVTPGEPASISCRSS QSLLYSNGYNYLDWYLQKPGQSP QLLIYLGSNRASGVPDRFSGSGSG TDFTLKISRVEAEDVGVYYCMQD VALPITFGGGTKVEIK [SEQ ID NO:291] CDR1: RSSQSLLYSNGYNYLD [SEQ ID NO:379] CDR2: LGSNRAS [SEQ ID NO:380] CDR3: MQDVALPIT [SEQ ID NO:381]

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CDR3:EAADGFVGERYFDL[SEQ ID NO:378] or AREAADGFVGERYFDL [SEQ ID NO:553] (not by Kabat numbering) scFv Ab23 DIVMTQSPLSLPVTPGEPASISCRSSOSLLYSNGYNYLDWYLQKPGQSPQ LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMODVAL PITFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSOVQLVQSGAEVKKP GASVKVSCKASGYTFSGYYMHWVRQAPGQCLEWMGMINPYGGSTRY AOKFOGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGER YFDLWGRGTLVTVSS [SEQ ID NO:471] QVOLVQSGAEVKKPGASVKVSCKASGYTFSGYYMHWVROAPGQCLE WMGMINPYGGSTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCAREAADGFVGERYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSG GGGSDIVMTOSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKP GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM QDVALPITFGCGTKVEIK [SEQ ID NO:472] Illustrative nucleotide sequence of scFv AB23 GACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGG CGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCTGTAC AGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCCCGGCC AGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGGCCAGCGG CGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACT GCATGCAGGACGTGGCCCTGCCCATCACCTTCGGCTGCGGCACCAAG GTGGAGATCAAGGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGA GCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCTGAAGGTGAGCTG CAAGGCCAGCGCTACACCTTCAGCGGCTACTACATGCACTGGGTGA GGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATGATCAACCC CTACGGCGGCAGCACCAGGTACGCCCAGAAGTTCCAGGGCAGGGTG ACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGA GGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGC AGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO:513]

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CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG CCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCGG CTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGT GGATGGGCATGATCAACCCCTACGGCGGCAGCACCAGGTACGCCCA GAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGC ACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAG GTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCC TGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAG CCAGAGCCTGCTGTACAGCAACGGCTACAACTACCTGGACTGGTACC TGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAG CAACAGGGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGC GGCACCGACTTCACCCCTGAAGATCAGCAGGGTGGAGGCCGAGGACG TGGGCGTGTACTACTGCATGCAGGACGTGGCCCTGCCCATCACCTTC GGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:514] ADI-11812 [Ab24] QVQLVQSGAEVKKPGASVKVSC KASGYTFEIYYMHWVRQAPGQG LEWMGIINPSSGSTVYAQKFQGR VTMTRDTSTSTVYMELSSLRSED TAVYYCARGAGYDDEDMDVWG KGTTVTVSS [SEQ ID NO:292] CDR1: IYYMH [SEQ ID NO:382] or YTFEIYYMH [SEQ ID NO:554] (not by Kabat numbering) CDR2: IINPSSGSTVYAQKFQG [SEQ ID NO:383] CDR3: GAGYDDEDMDV [SEQ ID NO:384] or ARGAGYDDEDMDV [SEQ ID NO:555] (not no Kabat) DIQMTQSPSSVSASVGDRVTITCR ASQGIDSWLAWYQQKPGKAPKLL IYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQAHSYPLT FGGGTKVEIK [SEQ ID NO:293] CDR1: RASQGIDSWLA [SEQ ID NO:385] CDR2: AASSLQS [SEQ ID NO:386] CDR3: QQAHSYPLT [SEQ ID NO:387] scFv Ab24 DIOMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY

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AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASV KVSCKASGYTFEIYYMHWVRQAPGQCLEWMGIINPSSGSTVYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGK GTTVTVSS [SEQ ID NO:473] QVQLVQSGAEVKKPGASVKVSCKASGYTFEIYYMHWVRQAPGQCLEW MGIINPSSGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC ARGAGYDDEDMDVWGKGTTVTVSSGGGGSGGGGSGGGGSGGGGS DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFG CGTKVEIK [SEQ ID NO:474] Illustrative nucleotide sequence of scFv AЬ24 GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCGACAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTGCCAGCAGGCCCACAG CTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGA AGAAGCCCGGCGCCAGCTGAAGGTGAGCTGCAAGGCCAGCGGCTA CACCTTCGAGATCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCC AGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCAGCGGCAGCAC CGTGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGAC ACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCG AGGACACCGCCGTGTACTACTGCGCCAGGGGCGCCGGCTACGACGA CGAGGACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGC AGC [SEQ ID NO:515] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG CCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCGAGAT CTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGT GGATGGGCATCATCAACCCCAGCAGCGGCAGCACCGTGTACGCCCA GAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGC

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ACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCCAGGGGCGCCGGCTACGACGACGAGGACATGGA CGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGCGGGCGGCGGC GGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGC GGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCA GCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCAT CGACAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGCAAGGCCCCC AAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCA GCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCAT CAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGG CCCACAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATC AAG [SEQ ID NO:516] ADI-11825 [Ab25] EVQLVESGGGLVQPGGSLRLSCA ASGFTFGGYWMSWVRQAPGKGL EWVANINQDGSEEYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCAREANYYGNVGDDYW GQGTLVTVSS [SEQ ID NO:294] CDR1: GYWMS [SEQ ID NO:388] or FTFGGYWMS [SEQ ID NO:556] (not by Kabat numbering) CDR2: NINQDGSEEYYVDSVKG [SEQ ID NO:389] CDR3: EANYYGNVGDDY [SEQ ID NO:390] or AREANYYGNVGDDY [SEQ ID NO:557] (not no Kabat) DIQMTQSPSSSLSASVGDRVTITCRA SQSIYNYLNWYQQKPGKAPKLLIY AASNLHSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQAFHVPITF GGGTKVEIK [SEQ ID NO:295] CDR1: RASQSIYNYLN [SEQ ID NO:391] CDR2: AASNLHS [SEQ ID NO:392] CDR3: QQAFHVPIT [SEQ ID NO:393] scFv Ab25 DIOMTQSPSSLSASVGDRVTITCRASQSIYNYLNWYQQKPGKAPKLLIY AASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFHVPITFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVOLVESGGGLVQPGGSLR LSCAASGFTFGGYWMSWVRQAPGKCLEWVANINQDGSEEYYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQ

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GTLVTVSS [SEQ ID NO:475] EVQLVESGGGLVQPGGSLRLSCAASGFTFGGYWMSWVRQAPGKCLEW VANINQDGSEEYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CAREANYYGNVGDDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG SDIQMTQSPSSLSSASVGDRVTITCRASQSIYNYLNWYQQKPGKAPKLLIY AASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFHVPITFGC GTKVEIK [SEQ ID NO:476] Illustrative nucleotide sequence of scFv AЬ25 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCTACAA CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAACCTGCACAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTGCCAGCAGGCCTTCCA CGTGCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGG TGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTT CACCTTCGGCGGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGG AGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACG GCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGT GAGCAGC [SEQ ID NO:517] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGGCGGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAACCAGGACGGCAGCGAGGAGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGA CGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC

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GGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCG CCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAG CATCTACAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCC CCCAAGCTGCTGATCTACGCCGCCAGCAACCTGCACAGCGGCGTGCC CAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACC ATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCA GGCCTTCCACGTGCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAGA TCAAG [SEQ ID NO:518] ADI-11826 [Ab26] EVQLVESGGGLVQPGGSLRLSCA ASGFTFPGYWMSWVRQAPGKGL EWVANINQDGSEVYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCAREANYYGNVGDDYW GQGTLVTVSS [SEQ ID NO:296] CDR1: GYWMS [SEQ ID NO:394] or FTFPGYWMS [SEQ ID NO:558] (not by Kabat numbering) CDR2: NINQDGSEVYYVDSVKG [SEQ ID NO:395] CDR3: EANYYGNVGDDY [SEQ ID NO:396] or AREANYYGNVGDDY [SEQ ID NO:559] (not no Kabat) DIQMTQSPSSLSSASVGDRVTITCRA SQSIYNYLNWYQQKPGKAPKLLIY AASSTQSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQAFHVPITFG GGTKVEIK [SEQ ID NO:297] CDR1: RASQSIYNYLN [SEQ ID NO:397] CDR2: AASSTQS [SEQ ID NO:398] CDR3: QQAFHVPIT [SEQ ID NO:399] scFv Ab26 DIQMTQSPSSLSASVGDRVTITCRASOSIYNYLNWYQQKPGKAPKLLIY AASSTQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCOOAFHVPITFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR LSCAASGFTFPGYWMSWVROAPGKCLEWVANINQDGSEVYYVDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQ GTLVTVSS [SEQ ID NO:477] EVQLVESGGGLVQPGGSLRLSCAASGFTFPGYWMSWVRQAPGKCLEW VANINODGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CAREANYYGNVGDDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGG

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SDIQMTQSPSSLSSASVGDRVTITCRASQSIYNYLNWYQQKPGKAPKLLIY AASSTQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFHVPITFGC GTKVEIK [SEQ ID NO:478] Illustrative nucleotide sequence of scFv AЬ26 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCTACAA CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCAGCACCCAGAGCGGCGTGCCCAGCAGGT TCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG CCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCC ACGTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCT TCACCTTCCCCGGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGG TGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACG GCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGT GAGCAGC [SEQ ID NO:519] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCCCCGGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAACCAGGACGGCAGCGAGGTGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGAGGCCAACTACGGCAACGTGGGCGA CGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGC GGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGC GGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCG CCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAG CATCTACAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCC CCCAAGCTGCTGATCTACGCCGCCAGCAGCACCCAGAGCGGCGTGC CCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAC

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CATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGC AGGCCTTCCACGTGCCCATCACCTTTCGGCTGCGGCACCAAGGTGGAG ATCAAG [SEQ ID NO:520] ADI-11828 [Ab27] EVQLVESGGGLVQPGGSLRLSCA ASGFTFSSYWMSWVRQAPGKGL EWVANINQDGSEVYYVDSVKGR FTISRDNAKNSLYLQMNSLRAED TAVYYCARDVGPGIAYQGHFDY WGQGTLVTVSS [SEQ ID NO:298] CDR1: SYWMS [SEQ ID NO:400] or FTFSSYWMS [SEQ ID NO:560] (not by Kabat numbering) CDR2: NINQDGSEVYYVDSVKG [SEQ ID NO:401] CDR3: DVGPGIAYQGHFDY [SEQ ID NO:402] or ARDVGPGIAYQGHFDY [SEQ ID NO :561] (not by Kabat numbering) DIQMTQSPSSLSSASVGDRVTITCRA SQSIYYYLNWYQQKPGKAPKLLIY AASSRQSGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQVYDTPLTF GGGTKVEIK [SEQ ID NO:299] CDR1: RASQSIYYYLN [SEQ ID NO:403] CDR2: AASSRQS [SEQ ID NO:404] CDR3: QQVYDTPLT [SEQ ID NO:405] scFv Ab27 DIQMTOSPSSLSASVGDRVTITCRASOSIYYYLNWYQQKPGKAPKLLIY AASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCOOVYDTPLTFG CGTKVEIKGGGGSGGGGSGGGGSGGGGSEVOLVESGGGLVOPGGSL RLSCAASGFTFSSYWMSWVRQAPGKCLEWVANINQDGSEVYYVDSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDY WGQGTLVTVSS [SEQ ID NO:479] EVOLVESGGGLVQPGGSLRLSCAASGFFSSYWMSWVROAPGKCLEW VANINQDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYY CARDVGPGIAYQGHFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGG GGSDIOMTQSPSSLSASVGDRVTITCRASOSIYYYLNWYQQKPGKAPKL LIYAASSROSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCOOVYDTPLT FGCGTKVEIK [SEQ ID NO:480] Illustrator GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGG

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willow GCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCTACTA nucleotide CTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG naya CTGATCTACGCCGCCAGCAGCAGGCAGAGCGGCGTGCCCAGCAGGT consistency TCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAG efficiency CCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACG scFv АЬ27 ACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTG GTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCT TCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGC AAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGG TGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGA CAACGCCAAGACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCC GAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCA TCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTG ACCGTGAGCAGC [SEQ ID NO:521] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCG GCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGC TACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGT GGGTGGCCAACATCAACCAGGACGGCAGCGAGGTGTACTACGTGGA CAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAAC AGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCG TGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGC CACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCT GAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGC CAGAGCATCTACTACCTGAACTGGTACCAGCAGAAGCCCGGCA AGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCAGGCAGAGCGG CGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTG CCAGCAGGTGTACGACACCCCTGACCTTCGGCTGCGGCACCAAGG TGGAGATCAAG [SEQ ID NO:522] ADI-11839 QVQLVQSGAEVKKPGASVKVSC DIQMTQSPSSVSASVGDRVTITCE KASGYTFSNYYMHWVRQAPGQG ASKGISSWLAWYQQKPGKAPKLLI

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[Ab28] LEWMGWINPFSGGTRYAQKFQG RVTMTRDTSTSTVYMELSSSLRSE DTAVYYCARDVGSSAYYYMDV WGKGTTVTVSS [SEQ ID NO:300] CDR1: NYYMH [SEQ ID NO:406] or YTFSNYYMH [SEQ ID NO:562] (not by Kabat numbering) CDR2:WINPFSGGTRYAQKFQG [SEQ ID NO:407] CDR3: DVGSSAYYYMDV [SEQ ID NO:408] or ARDVGSSAYYYMDV [SEQ ID NO :563] (not no Kabat) YAASDLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQAFLFPPT FGGGTKVEIK [SEQ ID NO:301] CDR1: EASKGISSWLA [SEQ ID NO:409] CDR2: AASDLQS [SEQ ID NO:410] CDR3: QQAFLPPT [SEQ ID NO:411] scFv Ab28 DIQMTQSPSSVSASVGDRVTITCEASKGISSWLAWYQQKPGKAPKLLIY AASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFLFPPTFGC GTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYTFSNYYMHWVRQAPGQCLEWMGWINPFSGGTRYAOKFO GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDVGSSAYYYMDVWG KGTTVTVSS [SEQ ID NO:481] QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMHWVRQAPGQCLE WMGWINPFSGGTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAV YYCARDVGSSAYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSGG GGSDIQMTQSPSSVSASVGDRVTITCEASKGISSWLAWYQQKPGKAPKL LIYAASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFLFPPT FGCGTKVEIK [SEQ ID NO:482] Illustrative nucleotide sequence GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGG GCGACAGGGTGACCATCACCTGCGAGGCCAGCAAGGGCATCAGCAG CTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGCCGCCAGCGACCTGCAGAGCGGCGTGCCCAGCAGGTT CAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC CTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCT

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scFv Ab28 GTTCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCG GCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCG GCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAA GAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTAC ACCTTCAGCAACTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCA GTGCCTGGAGTGGATGGGCTGGATCAACCCCTTCAGCGGCGGCACC AGGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACA CCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGA GGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCAGCAGCGCC TACTACTACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGA GCAGC [SEQ ID NO:523] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG CCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCAA CTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGT GGATGGGCTGGATCAACCCCTTCAGCGGCGGCACCAGGTACGCCCA GAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGC ACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCG TGTACTACTGCGCCAGGGACGTGGGCAGCAGCGCCTACTACTACATG GACGTGTGGGGCAAGGGCACCACGTGACCGTGAGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGC CAGCGTGGGCGACAGGGTGACCATCACCTGCGAGGCCAGCAAGGGC ATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGCAAGGCCC CCAAGCTGCTGATCTACGCCGCCAGCGACCTGCAGAGCGGCGTGCCC AGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCA TCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAG GCCTTCCTGTTCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGAT CAAG [SEQ ID NO:524] ADI-10152 [Ab29] EVQLVQSGAEVKKPGESLKISCK GSGYSFTSYWIGWVRQMPGKGL EWMGSIYPGDSDTRYSPSFQGQV TISADKSISTAYLQWSSLKASDTA MYYCARELAYGDYKGGVDYWG QGTLVTVSS [SEQ ID NO:302] EIVLTQSPGTLSLSPGERATLSCRA SQSVSSSFLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTL TISRLEPEDFAVYYCQQLDSPPPTF GGGTKVEIK [SEQ ID NO:303] CDR1: RASQSVSSSFLA [SEQ ID

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CDR1: SYWIG [SEQ ID NO:412] or YSFTSYWIG [SEQ ID NO:564] (not by Kabat numbering) CDR2: SIYPGDSDTRYSPSFQG [SEQ ID NO:413] CDR3: ELAYGDYKGGVDY [SEQ ID NO:414] or ARELAYGDYKGGVDY [SEQ ID NO:565] (not by Kabat numbering) NO:415] CDR2: GASSRAT [SEQ ID NO:416] CDR3: QQLDSPPPT [SEQ ID NO:417] scFv Ab29 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQLDSPPPTFGC GTKVEIKGGGGSGGGGSGGGGSC SCKGSGYSFTSYWIGWVRQMPGKl TISADKSISTAYLQWSSLKASDTAM TLVTVSS [SEQ ID NO:483] EVQLVQSGAEVKKPGESLKISCKG5 MGSIYPGDSDTRYSPSFQGQVTISA ARELAYGDYKGGVDYWGQGTLVI SEIVLTQSPGTLSLSPGERATLSCRA GASSRATGIPDRFSGSGSGTDFTLTI 1GGGSEVQLVQSGAEVKKPGESLKI XEWMGSIYPGDSDTRYSPSFQGQV [YYCARELAYGDYKGGVDYWGQG JGYSFTSYWIGWVRQMPGKCLEW DKSISTAYLQWSSLKASDTAMYYC rVSSGGGGSGGGGSGGGGSGGGG SQSVSSSFLAWYQQKPGQAPRLLIY SRLEPEDFAVYYCQQLDSPPPTFGC GTKVEIK [SEQ ID NO:484] Illustrative nucleotide sequence of scFv AЬ29 GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCG GCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAG CAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGG CTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACA GGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAG CAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCTGG ACAGCCCCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAA GGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAG CGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGCAGAGCGGCGCCGAG GTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCG GCTACAGCTTCACCAGCTACTGGATCGGCTGGGTGAGGCAGATGCCC

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GGCAAGTGCCTGGAGTGGATGGGCAGCATCTACCCCGGCGACAGCG ACACCAGGTACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGC CGACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAG GCCAGCGACACCGCCATGTACTACTGCGCCAGGGAGCTGGCCTACG GCGACTACAAGGGCGGCGTGGACTACTGGGGCCAGGGCACCCTGGT GACCGTGAGCAGC [SEQ ID NO:525] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCG AGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCACCAG CTACTGGATCGGCTGGGTGAGGCAGATGCCCGGCAAGTGCCTGGAG TGGATGGGCAGCATCTACCCCGGCGACAGCGACACCAGGTACAGCC CCAGCTTCCAGGGCCAGGTGACCATCAGCGCCGACAAGAGCATCAG CACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCC ATGTACTACTGCGCCAGGGAGCTGGCCTACGGCGACTACAAGGGCG GCGTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGG CGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGG CGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTG AGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCC AGAGCGTGAGCAGCAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGG CCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACC GGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCA CCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTAC TGCCAGCAGCTGGACAGCCCCCCCCACCTTCGGCTGCGGCACCAA GGTGGAGATCAAG [SEQ ID NO:526] Clone 28031-01 (mutation) WO201204 5752 QVQLVQSGAEVKKPGSSVKVSC KASGGTFSDYAISWVRQAPGQGL EWMGRIIPILGVADYAQKFQGRV TITADKSTRTAYMELSSLRSEDTA VYYCARNWADAFDIWGQGTMV TVSS [SEQ ID NO:418] CDR1: DYAIS [SEQ ID NO:422] CDR2: RIIPILGVADYAQKFQG [SEQ ID NO:423] CDR3: NWADAFDI [SEQ ID DIQLTQSPSSLSASVGDRVTITCRA SQGISSVLAWYQQKPGKAPKLLIY DASSLESGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQFDSSrrFGQ GTKLEIK [SEQ ID NO:419] CDR1: RASQGISSVLA [SEQ ID NO:425] CDR2: DASSLES [SEQ ID NO:426] CDR3: QQFDSSIT [SEQ ID NO:427]

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NO:424] scFv clone 280-31-01 (mutation) WO201204 5752 DIQLTQSPSSLSSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYD ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFDSSITFGCGT KLEIKGGGGSGGGGSGGGGSGGGGSQVOLVQSGAEVKKPGSSVKVS CKASGGTFSDYAISWVRQAPGQCLEWMGRIIPILGVADYAQKFQGRVTI TADKSTRTAYMELSSLRSEDTAVYYCARNWADAFDIWGQGTMVTVSS [SEQ ID NO:485] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQAPGQCLEW MGRIIPILGVADYAQKFQGRVTITADKSTRTAYMELSSLRSEDTAVYYC ARNWADAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQL TOSPSLSASVGDRVTITCRASQGISSVLAWYOQKPGKAPKLLIYDASSL ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFDSSITFGCGTKLEIK [SEQ ID NO:486] lintuzuma b QVQLVQSGAEVKKPGSSVKVSC KASGYTFTDYNMHWVRQAPGQ GLEWIGYIYPYNGGTGYNQKFKS KATITADESTNTAYMELSSLRSED TAVYYCARGRPAMDYWGQGTL VTVSS [SEQ ID NO:420] CDR1: DYNMH [SEQ ID NO:428] CDR2: YIYPYNGGTGYNQKFKS [SEQ ID NO:429] CDR3: GRPAMDY [SEQ ID NO:430] DIQMTQSPSSLSSASVGDRVTITCRA SESVDNYGISFMNWFQQKPGKAP KLLIYAASNQGSGVPSRFSGSGSG TDFTLTISSLQPDDFATYYCQQSKE VPWTFGQGTKVEIK [SEQ ID NO:421] CDR1: RASESVDNYGISFMN [SEQ IDNO:431] CDR2: AASNQGS [SEQ ID NO:432] CDR3: QQSKEVPWT [SEQ ID NO:433] scFv lintuzuma ba DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPK LLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVP WTFGCGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKP GSSVKVSCKASGYTFTDYNMHWVRQAPGQCLEWIGYIYPYNGGTGYN QKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQ GTLVTVSS [SEQ ID NO:487] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQCLE WIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYY CARGRPAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQM TOSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYA ASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGC GTKVEIK [SEQ ID NO:488]

An antigen-binding site that binds an epitope on the extracellular domain of human CD33 and/or cynomolgus/rhesus macaque CD33

In one aspect, the invention provides an antigen-binding site comprising a heavy chain variable domain that binds an epitope on the extracellular domain of human CD33 and/or cynomolgus/rhesus macaque CD33.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVANIKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGM

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DVWGQGTTVTVSS [SEQ ID NO: 1]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 1. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYGMS [SEQ ID NO: 21] as the first complementarity determining region 1 (CDR1), NKQDGSEKYYVDSVKG [SEQ ID NO: 22] as the second CDR (CDR2), and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 23] as the third CDR (CDR3) of SEQ ID NO: 1. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYGMS [SEQ ID NO: 434] as the first complementarity determining region 1 (CDR1), NIKQDGSEKYYVDSVKG [SEQ ID NO: 22] as the second CDR (CDR2) and EGGPYYDSSGYFVYYGMDV [SEQ ID NO: 435] as the third CDR (CDR3) of SEQ ID NO: 1. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFGGGTKVEIK [SEQ ID NO: 2]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 may be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, which comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 24] as CDR1, DASSLES [SEQ ID NO: 25] as CDR2, and QQYESFPT [SEQ ID NO: 26] as CDR3 of SEQ ID NO: 2.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSE KYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 3]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYWMS [SEQ ID NO: 27] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO: 28] as CDR2, and ARPLNAGELDV [SEQ ID NO: 29] as CDR3 of SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYWMS [SEQ ID NO: 181] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO: 28] as CDR2, and PLNAGELDV [SEQ ID NO: 436] as CDR3 of SEQ ID NO: 3. In certain embodiments, the heavy chain variable domain of the antibody, which comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGGGTKVEIK [SEQ ID NO: 4]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 4 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 30] as CDR1, EASSLES [SEQ ID NO: 31] as CDR2, and QQLESYPLT [SEQ ID NO: 32] as CDR3 of SEQ ID NO: 4.

In certain embodiments, the present invention provides an antigen-binding

- 84 049630 an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus macaque CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGKGLEWVSAIVGSGEST YFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGM

DVWGQGTTVTVSS [SEQ ID NO: 5]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSKYTMS [SEQ ID NO: 33] as CDR1, AIVGSGESTYFADSVKG [SEQ ID NO: 34] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 35] as CDR3 of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises the amino acid sequences KYTMS [SEQ ID NO: 183] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO: 34] as CDR2, and EGGPYYDSSGYFVYYGMDV [SEQ ID NO: 184] as CDR3 of SEQ ID NO: 5. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO: 6]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 6 that includes the amino acid sequences RASQSISSWLA [SEQ ID NO: 36] as CDR1, KASSLES [SEQ ID NO: 37], or KASSLE [SEQ ID NO: 185] as CDR2 and QQLESYPLT [SEQ ID NO: 38] as CDR3 of SEQ ID NO: 6.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWG RGTLVTVSS [SEQ ID NO: 7]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFSDYYMH [SEQ ID NO: 39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO: 40] as CDR2, and AREAADGFVGERYFDL [SEQ ID NO: 41] as CDR3 of SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequences DYYMH [SEQ ID NO: 437] as CDR1, AIVGSGESTYFADSVKG [SEQ ID NO: 34] as CDR2, and EAADGFVGERYFDL [SEQ ID NO: 438] as CDR3 of SEQ ID NO: 7. In certain embodiments, the variable domain a heavy chain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO: 8]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,

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98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 8, which comprises the amino acid sequences RSSQSLLYSNGYNYLD [SEQ ID NO: 42] as CDR1, LGSNRAS [SEQ ID NO: 43] as CDR2 and MQDVALPIT [SEQ ID NO: 44] as CDR3 of SEQ ID NO: 8.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 9]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFGSYWMS [SEQ ID NO: 45] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO: 46] as CDR2, and ARPLNAGELDV [SEQ ID NO: 47] as CDR3 of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYWMS [SEQ ID NO: 181] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO: 46] as CDR2, and RPLNAGELDV [SEQ ID NO: 182] as CDR3 of SEQ ID NO: 9. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO: 10]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 48] as CDR1, EASSLES [SEQ ID NO: 49] as CDR2, and QQSQSYPPIT [SEQ ID NO: 50] as CDR3 of SEQ ID NO: 10.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKGLEWVATIKRDGSEK GYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVT VSS [SEQ ID NO: 11]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFPSYWMS [SEQ ID NO: 51] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO: 52] as CDR2, and ARPLNAGELDV [SEQ ID NO: 53] as CDR3 of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYWMS [SEQ ID NO: 181] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO: 52] as CDR2, and PLNAGELDV [SEQ ID NO: 439] as CDR3 of SEQ ID NO: 11. In certain embodiments, the heavy chain variable domain an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO: 12]. In certain embodiments, the heavy chain variable domain of an antibody is at least

- 86 049630 re 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11, may be paired with an antibody light chain variable domain that is at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12, which comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 54] as CDR1, EASSLES [SEQ ID NO: 55] as CDR2, and QQSQSYPPIT [SEQ ID NO: 56] as CDR3 of SEQ ID NO: 12.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKG TTVTVSS [SEQ ID NO: 13]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 13. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFGTYYMH [SEQ ID NO: 57] as CDR1, IINPSRGSTVYAQKFQG [SEQ ID NO: 58] as CDR2, and ARGAGYDDEDMDV [SEQ ID NO: 59] as CDR3 of SEQ ID NO: 13. In some embodiments, the heavy chain variable domain comprises the amino acid sequences TYYMH [SEQ ID NO: 440] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO: 52] as CDR2, and GAGYDDEDMDV [SEQ ID NO: 441] as CDR3 of SEQ ID NO: 13. In certain embodiments, the variable domain a heavy chain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO: 14]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14 that comprises the amino acid sequences RASQGIDSWLA [SEQ ID NO: 60] as CDR1, AAsSlQS [SEQ ID NO: 61] as CDR2, and QQAHSYPLT [SEQ ID NO: 62] as CDR3 of SEQ ID NO: 14.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMD VWGQGTTVTVSS [SEQ ID NO: 15]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 15. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYAMS [SEQ ID NO: 63] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO: 64] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 65] as CDR3 of SEQ ID NO: 15. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYAMS [SEQ ID NO: 442] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO: 64] as CDR2, and EGGPYYDSSGYFVYYGMDV [SEQ ID NO: 443] as CDR3 of SEQ ID NO: 15. In certain In some embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO: 15.

- 87 049630 or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO: 16]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 16 that comprises the amino acid sequences RASNSISSWLA [SEQ ID NO: 66] as CDR1, EASSTKS [SEQ ID NO: 67] as CDR2, and QQYDDLPT [SEQ ID NO: 68] as CDR3 of SEQ ID NO: 16.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS [SEQ ID NO: 17]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 17. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYWMS [SEQ ID NO: 69] as CDR1, NINTDGSEVYYVDSVKG [SEQ ID NO: 70] as CDR2, and ARDVGPGIAYQGHFDY [SEQ ID NO: 71] as CDR3 of SEQ ID NO: 17. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYWMS [SEQ ID NO: 181] as CDR1, NINTDGSEVYYVDSVKG [SEQ ID NO: 70] as CDR2, and DVGPGIAYQGHFDY [SEQ ID NO: 444] as CDR3 of SEQ ID NO: 17. In certain embodiments a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFGGGTKVEIK [SEQ ID NO: 18]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18 that comprises the amino acid sequences RASQVIYSYLN [SEQ ID NO: 72] as CDR1, AASSLKS [SEQ ID NO: 73] as CDR2, and QQVYDTPLT [SEQ ID NO: 74] as CDR3 of SEQ ID NO: 18.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKGLEWIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS [SEQ ID NO: 19]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 19. In some embodiments, the heavy chain variable domain comprises the amino acid sequences GSISSTDYYWG [SEQ ID NO: 75] as CDR1, SIGYSGTYYNPSLKS [SEQ ID NO: 76] as CDR2, and ARETAHDVHGMDV [SEQ ID NO: 77] as CDR3 of SEQ ID NO: 19. In some embodiments, the heavy chain variable domain comprises the amino acid sequences STDYYWG [SEQ ID NO: 445] as CDR1, SIGYSGTYYNPSLKS [SEQ ID NO: 76] as CDR2, and ETAHDVHGMDV [SEQ ID NO: 446] as CDR3 of SEQ ID NO: 19. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19, is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, the variable domain of a light chain

- 88 049630 a yellow chain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of EIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGGGTKVEIK [SEQ ID NO: 20]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20 that comprises the amino acid sequences RASHSVYSYLA [SEQ ID NO: 78] as CDR1, DASNRAT [SEQ ID NO: 79] as CDR2, and QQYDNLPT [SEQ ID NO: 80] as CDR3 of SEQ ID NO: 20.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 304 as CDR1, SEQ ID NO: 305 as CDR2, and SEQ ID NO: 306 as CDR3 of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 528 as CDR1, SEQ ID NO: 305 as CDR2 and SEQ ID NO: 529 as CDR3 of SEQ ID NO: 266. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267, which comprises the amino acid sequences of SEQ ID NO: 307 as CDR1, SEQ ID NO: 308 as CDR2, and SEQ ID NO: 309 as CDR3 of SEQ ID NO: 267.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 268. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 310 as CDR1, SEQ ID NO: 311 as CDR2, and SEQ ID NO: 312 as CDR3 of SEQ ID NO: 268. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 530 as CDR1, SEQ ID NO: 311 as CDR2 and SEQ ID NO: 531 as CDR3 of SEQ ID NO: 268. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 269. In certain embodiments, an antibody heavy chain variable domain that is at least

- 89 049630 which is 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268, can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 269, which comprises the amino acid sequences of SEQ ID NO: 313 as CDR1, SEQ ID NO: 314 as CDR2, and SEQ ID NO: 315 as CDR3 of SEQ ID NO: 269.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 316 as CDR1, SEQ ID NO: 317 as CDR2, and SEQ ID NO: 318 as CDR3 of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 532 as CDR1, SEQ ID NO: 317 as CDR2 and SEQ ID NO: 533 as CDR3 of SEQ ID NO: 270. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271, which comprises the amino acid sequences of SEQ ID NO: 319 as CDR1, SEQ ID NO: 320 as CDR2, and SEQ ID NO: 321 as CDR3 of SEQ ID NO: 271.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 322 as CDR1, SEQ ID NO: 323 as CDR2, and SEQ ID NO: 324 as CDR3 of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 534 as CDR1, SEQ ID NO: 323 as CDR2 and SEQ ID NO: 535 as CDR3 of SEQ ID NO: 272. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273, which comprises the amino acid sequences of SEQ ID NO: 325 as CDR1, SEQ ID NO: 326 as CDR2, and SEQ ID NO: 327 as CDR3 of SEQ ID NO: 273.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site

- 90 049630 comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 274. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 328 as CDR1, SEQ ID NO: 329 as CDR2, and SEQ ID NO: 330 as CDR3 of SEQ ID NO: 274. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 536 as CDR1, SEQ ID NO: 329 as CDR2, and SEQ ID NO: 537 as CDR3 of SEQ ID NO: 274. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 275. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 275, which comprises the amino acid sequences of SEQ ID NO: 331 as CDR1, SEQ ID NO: 332 as CDR2, and SEQ ID NO: 333 as CDR3 of SEQ ID NO: 275.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 276. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 334 as CDR1, SEQ ID NO: 335 as CDR2, and SEQ ID NO: 336 as CDR3 of SEQ ID NO: 276. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 538 as CDR1, SEQ ID NO: 335 as CDR2 and SEQ ID NO: 539 as CDR3 of SEQ ID NO: 276. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody. at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 can be paired to an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277, which includes the amino acid sequences of SEQ ID NO: 337 as CDR1, SEQ ID NO: 338 as CDR2, and SEQ ID NO: 339 as CDR3 of SEQ ID NO: 277.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 340 as CDR1, SEQ ID NO: 341 as CDR2, and SEQ ID NO: 342 as CDR3 of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 540 as CDR1, SEQ

- 91 049630

ID NO: 341 as CDR2 and SEQ ID NO: 541 as CDR3 of SEQ ID NO: 278. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279, which comprises the amino acid sequences of SEQ ID NO: 343 as CDR1, SEQ ID NO: 344 as CDR2, and SEQ ID NO: 345 as CDR3 of SEQ ID NO: 279.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 346 as CDR1, SEQ ID NO: 347 as CDR2, and SEQ ID NO: 348 as CDR3 of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 542 as CDR1, SEQ ID NO: 347 as CDR2 and SEQ ID NO: 543 as CDR3 of SEQ ID NO: 280. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281, which comprises the amino acid sequences of SEQ ID NO: 349 as CDR1, SEQ ID NO: 350 as CDR2, and SEQ ID NO: 351 as CDR3 of SEQ ID NO: 281.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 352 as CDR1, SEQ ID NO: 353 as CDR2, and SEQ ID NO: 354 as CDR3 of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 544 as CDR1, SEQ ID NO: 353 as CDR2 and SEQ ID NO: 545 as CDR3 of SEQ ID NO: 282. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with a light chain variable domain of an antibody.

- 92 049630 an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282. 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 283, which comprises the amino acid sequences of SEQ ID NO: 355 as CDR1, SEQ ID NO: 356 as CDR2 and SEQ ID NO: 357 as CDR3 of SEQ ID NO: 283.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 358 as CDR1, SEQ ID NO: 359 as CDR2, and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 546 as CDR1, SEQ ID NO: 359 as CDR2 and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 547. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285, which comprises the amino acid sequences of SEQ ID NO: 361 as CDR1, SEQ ID NO: 362 as CDR2, and SEQ ID NO: 363 as CDR3 of SEQ ID NO: 285.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 364 as CDR1, SEQ ID NO: 365 as CDR2, and SEQ ID NO: 366 as CDR3 of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 548 as CDR1, SEQ ID NO: 365 as CDR2 and SEQ ID NO: 549 as CDR3 of SEQ ID NO: 286. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 may be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287, which comprises the amino acid sequences of SEQ ID NO: 367 as CDR1, SEQ

- 93 049630

ID NO: 368 as CDR2 and SEQ ID NO: 369 as CDR3 of SEQ ID NO: 287.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 370 as CDR1, SEQ ID NO: 371 as CDR2, and SEQ ID NO: 372 as CDR3 of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 550 as CDR1, SEQ ID NO: 371 as CDR2 and SEQ ID NO: 551 as CDR3 of SEQ ID NO: 288. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289, which comprises the amino acid sequences of SEQ ID NO: 373 as CDR1, SEQ ID NO: 374 as CDR2, and SEQ ID NO: 375 as CDR3 of SEQ ID NO: 289.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 376 as CDR1, SEQ ID NO: 377 as CDR2, and SEQ ID NO: 378 as CDR3 of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 552 as CDR1, SEQ ID NO: 377 as CDR2 and SEQ ID NO: 553 as CDR3 of SEQ ID NO: 290. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291, which comprises the amino acid sequences of SEQ ID NO: 379 as CDR1, SEQ ID NO: 380 as CDR2, and SEQ ID NO: 381 as CDR3 of SEQ ID NO: 291.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292. In some embodiments, the heavy chain variable domain of an antibody is at least 95% identical to SEQ ID NO: 292. In some embodiments, the heavy chain variable domain comprises an amino acid

- 94 049630 amino acid sequences of SEQ ID NO: 382 as CDR1, SEQ ID NO: 383 as CDR2, and SEQ ID NO: 384 as CDR3 of SEQ ID NO: 292. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 554 as CDR1, SEQ ID NO: 383 as CDR2, and SEQ ID NO: 555 as CDR3 of SEQ ID NO: 292. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292, is fused with the light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 293. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 293, which comprises the amino acid sequences of SEQ ID NO: 385 as CDR1, SEQ ID NO: 386 as CDR2, and SEQ ID NO: 387 as CDR3 of SEQ ID NO: 293.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 294. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 388 as CDR1, SEQ ID NO: 389 as CDR2, and SEQ ID NO: 390 as CDR3 of SEQ ID NO: 294. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 556 as CDR1, SEQ ID NO: 389 as CDR2 and SEQ ID NO: 557 as CDR3 of SEQ ID NO: 294. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 295. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 may be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 295, which comprises the amino acid sequences of SEQ ID NO: 391 as CDR1, SEQ ID NO: 392 as CDR2, and SEQ ID NO: 393 as CDR3 of SEQ ID NO: 295.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 296. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 394 as CDR1, SEQ ID NO: 395 as CDR2, and SEQ ID NO: 396 as CDR3 of SEQ ID NO: 296. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 558 as CDR1, SEQ ID NO: 395 as CDR2, and SEQ ID NO: 559 as CDR3 of SEQ ID NO: 296. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 is combined with a light chain variable domain to form an antigen-binding site capable of

- 95 049630 contact CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297, which comprises the amino acid sequences of SEQ ID NO: 397 as CDR1, SEQ ID NO: 398 as CDR2, and SEQ ID NO: 399 as CDR3 of SEQ ID NO: 297.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33 and cynomolgus/rhesus macaque CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 298. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 400 as CDR1, SEQ ID NO: 401 as CDR2, and SEQ ID NO: 402 as CDR3 of SEQ ID NO: 298. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 560 as CDR1, SEQ ID NO: 401 as CDR2 and SEQ ID NO: 561 as CDR3 of SEQ ID NO: 298. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299, which comprises the amino acid sequences of SEQ ID NO: 403 as CDR1, SEQ ID NO: 404 as CDR2, and SEQ ID NO: 405 as CDR3 of SEQ ID NO: 299.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 406 as CDR1, SEQ ID NO: 407 as CDR2, and SEQ ID NO: 408 as CDR3 of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 562 as CDR1, SEQ ID NO: 407 as CDR2 and SEQ ID NO: 563 as CDR3 of SEQ ID NO: 300. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) ID

- 96 049630 identical to the amino acid sequence of SEQ ID NO: 300, can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 301, which includes the amino acid sequences of SEQ ID NO: 409 as CDR1, SEQ ID NO: 410 as CDR2, and SEQ ID NO: 411 as CDR3 of SEQ ID NO: 301.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope on the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 412 as CDR1, SEQ ID NO: 413 as CDR2, and SEQ ID NO: 414 as CDR3 of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 564 as CDR1, SEQ ID NO: 413 as CDR2 and SEQ ID NO: 565 as CDR3 of SEQ ID NO: 302. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303, which comprises the amino acid sequences of SEQ ID NO: 415 as CDR1, SEQ ID NO: 416 as CDR2, and SEQ ID NO: 417 as CDR3 of SEQ ID NO: 303.

An antigen-binding site that recognizes and binds a conformational epitope on the extracellular domain of human CD33 and/or cynomolgus/rhesus macaque CD33

In one aspect of the present invention, there is provided an antigen-binding site comprising a heavy chain variable domain that recognizes and binds one or more conformational epitopes on the extracellular domain of human CD33 and/or cynomolgus/rhesus monkey CD33.

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds a conformational epitope located, in part, within the V domain of the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSE KYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 3]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYWMS [SEQ ID NO: 27] as CDR1, NKQDGSEKYYVDSVKG [SEQ ID NO: 28] as CDR2, and ARPLNAGELDV [SEQ ID NO: 29] as CDR3 of SEQ ID NO: 3. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 is combined with a light chain variable domain with formation of an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGGGTKVEIK [SEQ ID NO: 4]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 may be paired with an antibody light chain variable domain that is at least 90%

- 97 049630 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 4, which comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 30] as CDR1, EASSLES [SEQ ID NO: 31] as CDR2, and QQLESYPLT [SEQ ID NO: 32] as CDR3 of SEQ ID NO: 4.

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds a conformational epitope located, in part, within the V domain of the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKGLEWVATIKRDGSEK GYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVT VSS [SEQ ID NO: 11]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFPSYWMS [SEQ ID NO: 51] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO: 52] as CDR2, and ARPLNAGELDV [SEQ ID NO: 53] as CDR3 of SEQ ID NO: 11. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 is combined with a light chain variable domain. chains to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO: 12]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 may be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 54] as CDR1, EASSLES [SEQ ID NO: 55] as CDR2, and QQSQSYPPIT [SEQ ID NO: 56] as CDR3 of SEQ ID NO: 12. In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds a conformational epitope located, in part, within the V domain of the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMD VWGQGTTVTVSS [SEQ ID NO: 15]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 15. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYAMS [SEQ ID NO: 63] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO: 64] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 65] as CDR3 of SEQ ID NO: 15. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 is combined with variable domain of the light chain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO: 16]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 can be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 16, which comprises the amino acid sequences RASNSISSWLA [SEQ ID NO: 66] as CDR1, EASSTKS [SEQ ID NO: 67] as CDR2, and QQYDDLPT [SEQ ID NO: 68] as CDR3. ID NO: 68] as CDR3 of SEQ ID NO: 16.

- 98 049630

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds a conformational epitope located partially within the V domain of the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 316 as CDR1, SEQ ID NO: 317 as CDR2, and SEQ ID NO: 318 as CDR3 of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 532 as CDR1, SEQ ID NO: 317 as CDR2 and SEQ ID NO: 533 as CDR3 of SEQ ID NO: 270. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271, which comprises the amino acid sequences of SEQ ID NO: 319 as CDR1, SEQ ID NO: 320 as CDR2, and SEQ ID NO: 321 as CDR3 of SEQ ID NO: 271.

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds a conformational epitope located partially within the V domain of the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 322 as CDR1, SEQ ID NO: 323 as CDR2, and SEQ ID NO: 324 as CDR3 of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 534 as CDR1, SEQ ID NO: 323 as CDR2 and SEQ ID NO: 535 as CDR3 of SEQ ID NO: 272. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273, which comprises the amino acid sequences of SEQ ID NO: 325 as CDR1, SEQ ID NO: 326 as CDR2, and SEQ ID NO: 327 as CDR3 of SEQ ID NO: 273.

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds a conformational epitope located, in part, within the V domain of the extracellular domain of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280. In some embodiments, the variable domain is

- 99 049 630 the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 346 as CDR1, SEQ ID NO: 347 as CDR2, and SEQ ID NO: 348 as CDR3 of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 542 as CDR1, SEQ ID NO: 347 as CDR2, and SEQ ID NO: 543 as CDR3 of SEQ ID NO: 280. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 280, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281, which comprises the amino acid sequences of SEQ ID NO: 349 as CDR1, SEQ ID NO: 350 as CDR2, and SEQ ID NO: 351 as CDR3 of SEQ ID NO: 281.

An antigen-binding site that recognizes and binds a conformational epitope on the extracellular domain of human CD33 but not a conformational epitope on the extracellular domain of cynomolgus/rhesus macaque

In one aspect of the present invention, there is provided an antigen-binding site that recognizes and binds one or more conformational epitopes on the extracellular domain of human CD33, but does not recognize and/or bind one or more conformational epitopes on the extracellular domain of cynomolgus monkey CD33.

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds one or more conformational epitopes of the extracellular domain of human CD33, but does not recognize and/or bind one or more conformational epitopes of the extracellular domain of cynomolgus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWG RGTLVTVSS [SEQ ID NO: 7]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFSDYYMH [SEQ ID NO: 39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO: 40] as CDR2, and AREAADGFVGERYFDL [SEQ ID NO: 41] as CDR3 of SEQ ID NO: 7. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 is combined with a variable domain light chain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO: 8]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 may be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 8, which comprises the amino acid sequences RSSQSLLYSNGYNYLD [SEQ ID NO: 42] as CDR1, LGSNRAS [SEQ ID NO: 43] as CDR2, and MQDVALPIT [SEQ ID NO: 44] as CDR3 of SEQ ID NO: 8.

In certain embodiments, the present invention provides an antigen-binding site that recognizes and binds one or more conformational epitopes of an extracellular

- 100 049630 of the precise domain of human CD33, but does not recognize and/or bind one or more conformational epitopes of the extracellular domain of cynomolgus monkey CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS [SEQ ID NO: 13]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 13. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFGTYYMH [SEQ ID NO: 57] as CDR1, IINPSRGSTVYAQKFQG [SEQ ID NO: 58] as CDR2, and ARGAGYDDEDMDV [SEQ ID NO: 59] as CDR3 of SEQ ID NO: 13. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 is combined with a variable light chain domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO: 14]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 can be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14 that comprises the amino acid sequences RASQGIDSWLA [SEQ ID NO: 60] as CDR1, AASSLQS [SEQ ID NO: 61] as CDR2, and QQAHSYPLT [SEQ ID NO: 62] as CDR3 of SEQ ID NO: 14.

In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKG TTVTVSS [SEQ ID NO: 13] binds the full-length extracellular domain of human CD33, but does not bind the V domain or the C domain of human CD33 individually and does not cross-block binding to human CD33 by lintuzumab. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 and is paired with a light chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO: 14], binds to the full-length extracellular domain of human CD33, but does not bind the V domain or C domain of human CD33 alone and does not cross-block binding to human CD33 by lintuzumab. In certain embodiments, an antigen-binding site that comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 and is paired with a light chain variable domain of an antibody that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14 that comprises the amino acid sequences RASQGIDSWLA [SEQ ID NO: 60] as CDR1, AASSLQS [SEQ ID NO: 61] as CDR2 and QQAHSYPLT [SEQ ID NO: 62] as CDR3 of SEQ ID NO: 14, binds to the full-length extracellular domain of human CD33, but does not bind the V domain or C domain of human CD33 individually and does not cross-block binding to human CD33 by lintuzumab.

Antigen-binding site that binds to the R69G allele of human CD33

In one embodiment, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33. In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical

- 101 049630 amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGM DVWGQGTTVTVSS [SEQ ID NO: 1]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 1. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYGMS [SEQ ID NO: 21] as the first complementarity determining region 1 (CDR1), NKQDGSEKYYVDSVKG [SEQ ID NO: 22] as the second CDR (CDR2), and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 23] as the third CDR (CDR3) of SEQ ID NO: 1. In certain embodiments, a heavy chain variable domain of an antibody that comprises at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, is combined with the variable domain of the light chain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFGGGTKVEIK [SEQ [ID NO: 2]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 can be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 24] as CDR1, DASSLES [SEQ ID NO: 25] as CDR2, and QQYESFPT [SEQ ID NO: 26] as CDR3 of SEQ ID NO: 2.

In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 3]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYWMS [SEQ ID NO: 27] as CDR1, NKQDGSEKYYVDSVKG [SEQ ID NO: 28] as CDR2, and ARPLNAGELDV [SEQ ID NO: 29] as CDR3 of SEQ ID NO: 3. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 is combined with a light chain variable domain with formation of an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGGGTKVEIK [SEQ ID NO: 3] [ID NO: 4]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 can be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 4 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 30] as CDR1, EASSLES [SEQ ID NO: 31] as CDR2, and QQLESYPLT [SEQ ID NO: 32] as CDR3 of SEQ ID NO: 4.

In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGKGLEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO: 5]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 5. In some embodiments, the

- 102 049630 embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSKYTMS [SEQ ID NO: 33] as CDR1, AIVGSGESTYFADSVKG [SEQ ID NO: 34] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 35] as CDR3 of SEQ ID NO: 5. In some embodiments, the heavy chain variable domain comprises the amino acid sequences KYTMS [SEQ ID NO: 183] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO: 34] as CDR2, and EGGPYYDSSGYFVYYGMDV [SEQ ID NO: 184] as CDR3 of SEQ ID NO: 5. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ [ID NO: 6]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5 may be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 6 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 36] as CDR1, KASSLES [SEQ ID NO: 37] or KASSLE [SEQ ID NO: 185] as CDR2 and QQYDDLPT [SEQ ID NO: 38] as CDR3 of SEQ ID NO: 6.

In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMV TVSS [SEQ ID NO: 9]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFGSYWMS [SEQ ID NO: 45] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO: 46] as CDR2, and ARPLNAGELDV [SEQ ID NO: 47] as CDR3 of SEQ ID NO: 9. In some embodiments, the heavy chain variable domain comprises the amino acid sequences SYWMS [SEQ ID NO: 181] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO: 46] as CDR2, and RPLNAGELDV [SEQ ID NO: 182] as CDR3 of SEQ ID NO: 9. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO: 10]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9 can be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10, which comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 48] as CDR1, EASSLES [SEQ ID NO: 49] as CDR2, and QQSQSYPPIT [SEQ ID NO: ID NO: 50] as CDR3 of SEQ ID NO: 10. In certain embodiments, the present invention provides an antigen-binding site that binds the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKGLEWVATIKRDGSEK GYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVT VSS [SEQ ID NO: 11]. In some embodiments, the heavy chain variable domain of an antibody is at least

- 103 049630 at least 95% identical to SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFPSYWMS [SEQ ID NO: 51] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO: 52] as CDR2, and ARPLNAGELDV [SEQ ID NO: 53] as CDR3 of SEQ ID NO: 11. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 is combined with a light chain variable domain to form antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO: 12]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12 that comprises the amino acid sequences RASQSISSWLA [SEQ ID NO: 54] as CDR1, EASSLES [SEQ ID NO: 55] as CDR2, and QQSQSYPPIT [SEQ ID NO: 56] as CDR3 of SEQ ID NO: 12. In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS [SEQ ID NO: 13]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 13. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFGTYYMH [SEQ ID NO: 57] as CDR1, IINPSRGSTVYAQKFQG [SEQ ID NO: 58] as CDR2, and ARGAGYDDEDMDV [SEQ ID NO: 59] as CDR3 of SEQ ID NO: 13. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 is combined with a variable light chain domain to form an antigen-binding site capable of binding to CD33. For example, the variable domain of the heavy chain of an antibody. at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13, can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO: 14]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13 can be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14 that comprises the amino acid sequences RASQGIDSWLA [SEQ ID NO: 60] as CDR1, AASSLQS [SEQ ID NO: 61] as CDR2, and QQAHSYPLT [SEQ ID NO: 62] as CDR3 of SEQ ID NO: 14.

In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO: 15]. In some embodiments, the heavy chain variable domain of an antibody is at least 95% identical to SEQ ID NO: 15. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYAMS [SEQ ID NO: 63] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO: 64] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 65] as CDR3 of SEQ ID NO: 15. In certain embodiments, a heavy chain variable domain of an antibody that comprises at least 90% (e.g., 91%, 92%, 93%, 94%, 95%,

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96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 15, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO: 15] [ID NO: 16]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 16, which comprises the amino acid sequences RASNSISSWLA [SEQ ID NO: 66] as CDR1, EASSTKS [SEQ ID NO: 67] as CDR2, and QQYDDLPT [SEQ ID NO: 68] as CDR3 of SEQ ID NO: 16.

In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS [SEQ ID NO: 17]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 17. In some embodiments, the heavy chain variable domain comprises the amino acid sequences FTFSSYWMS [SEQ ID NO: 69] as CDR1, NINTDGSEVYYVDSVKG [SEQ ID NO: 70] as CDR2, and ARDVGPGIAYQGHFDY [SEQ ID NO: 71] as CDR3 of SEQ ID NO: 17. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 is combined with a variable light chain domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPS RFSGSGGTDFTLTISSLQPEDFATYYCQQVYDTPLTFGGGTKVEIK [SEQ ID NO: 18]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 may be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18, which comprises the amino acid sequences RASQVIYSYLN [SEQ ID NO: 72] as CDR1, AASSLKS [SEQ ID NO: 73] as CDR2, and QQVYDTPLT [SEQ ID NO: 74] as CDR3. ID NO: 74] as CDR3 of SEQ ID NO: 18.

In certain embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKGLEWIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTV SS [SEQ ID NO: 19]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 19. In some embodiments, the heavy chain variable domain comprises the amino acid sequences GSISSTDYYWG [SEQ ID NO: 75] as CDR1, SIGYSGTYYNPSLKS [SEQ ID NO: 76] as CDR2, and ARETAHDVHGMDV [SEQ ID NO: 77] as CDR3 of SEQ ID NO: 19. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19 is combined with a light chain variable domain. to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19 can be paired with a light chain variable domain of an antibody that is at least 90%

- 105 049630 (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence EIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGGGTKVEIK [SEQ ID NO: 20]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20, which comprises the amino acid sequences RASHSVYSYLA [SEQ ID NO: 78] as CDR1, DASNRAT [SEQ ID NO: 79] as CDR2, and QQYDNLPT [SEQ ID NO: 80] as CDR3 of SEQ ID NO: 20.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33;

the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 304 as CDR1, SEQ ID NO: 305 as CDR2, and SEQ ID NO: 306 as CDR3 of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 528 as CDR1, SEQ ID NO: 305 as CDR2 and SEQ ID NO: 529 as CDR3 of SEQ ID NO: 266. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267, which comprises the amino acid sequences of SEQ ID NO: 307 as CDR1, SEQ ID NO: 308 as CDR2, and SEQ ID NO: 309 as CDR3 of SEQ ID NO: 267.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 268. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 310 as CDR1, SEQ ID NO: 311 as CDR2, and SEQ ID NO: 312 as CDR3 of SEQ ID NO: 268. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 530 as CDR1, SEQ ID NO: 311 as CDR2 and SEQ ID NO: 531 as CDR3 of SEQ ID NO: 268. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 269. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268, may be paired with a light chain variable domain of an antibody by at least 90% (e.g., at least 91%, 92%, 93%,

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94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 269, which includes the amino acid sequences of SEQ ID NO: 313 as CDR1, SEQ ID NO: 314 as CDR2 and SEQ ID NO: 315 as CDR3 of SEQ ID NO: 269.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 316 as CDR1, SEQ ID NO: 317 as CDR2, and SEQ ID NO: 318 as CDR3 of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 532 as CDR1, SEQ ID NO: 317 as CDR2 and SEQ ID NO: 533 as CDR3 of SEQ ID NO: 270. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271, which comprises the amino acid sequences of SEQ ID NO: 319 as CDR1, SEQ ID NO: 320 as CDR2, and SEQ ID NO: 321 as CDR3 of SEQ ID NO: 271.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 322 as CDR1, SEQ ID NO: 323 as CDR2, and SEQ ID NO: 324 as CDR3 of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 534 as CDR1, SEQ ID NO: 323 as CDR2 and SEQ ID NO: 535 as CDR3 of SEQ ID NO: 272. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273, which comprises the amino acid sequences of SEQ ID NO: 325 as CDR1, SEQ ID NO: 326 as CDR2, and SEQ ID NO: 327 as CDR3 of SEQ ID NO: 273.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274. In some embodiments,

- 107 049 630 the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 274. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 328 as CDR1, SEQ ID NO: 329 as CDR2, and SEQ ID NO: 330 as CDR3 of SEQ ID NO: 274. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 536 as CDR1, SEQ ID NO: 329 as CDR2, and SEQ ID NO: 537 as CDR3 of SEQ ID NO: 274. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 274, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 275. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 275, which comprises the amino acid sequences of SEQ ID NO: 331 as CDR1, SEQ ID NO: 332 as CDR2, and SEQ ID NO: 333 as CDR3 of SEQ ID NO: 275.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33;

the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 276. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 334 as CDR1, SEQ ID NO: 335 as CDR2, and SEQ ID NO: 336 as CDR3 of SEQ ID NO: 276. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 538 as CDR1, SEQ ID NO: 335 as CDR2 and SEQ ID NO: 539 as CDR3 of SEQ ID NO: 276. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277, which comprises the amino acid sequences of SEQ ID NO: 337 as CDR1, SEQ ID NO: 338 as CDR2, and SEQ ID NO: 339 as CDR3 of SEQ ID NO: 277.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 340 as CDR1, SEQ ID NO: 341 as CDR2, and SEQ ID NO: 342 as CDR3 of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 540 as CDR1, SEQ ID NO: 341 as CDR2 and SEQ ID NO: 541 as CDR3 of SEQ ID NO: 278. In certain embodiments, a heavy chain variable domain of an antibody that comprises the amino acid sequence

- 108 049630 an amino acid sequence that is at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 278, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279, which comprises the amino acid sequences of SEQ ID NO: 343 as CDR1, SEQ ID NO: 344 as CDR2, and SEQ ID NO: 345 as CDR3 of SEQ ID NO: 279.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 346 as CDR1, SEQ ID NO: 347 as CDR2, and SEQ ID NO: 348 as CDR3 of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 542 as CDR1, SEQ ID NO: 347 as CDR2 and SEQ ID NO: 543 as CDR3 of SEQ ID NO: 280. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281, which comprises the amino acid sequences of SEQ ID NO: 349 as CDR1, SEQ ID NO: 350 as CDR2, and SEQ ID NO: 351 as CDR3 of SEQ ID NO: 281.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 352 as CDR1, SEQ ID NO: 353 as CDR2, and SEQ ID NO: 354 as CDR3 of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 544 as CDR1, SEQ ID NO: 353 as CDR2 and SEQ ID NO: 545 as CDR3 of SEQ ID NO: 282. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283.

- 109 049630 In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283, which comprises the amino acid sequences of SEQ ID NO: 355 as CDR1, SEQ ID NO: 356 as CDR2, and SEQ ID NO: 357 as CDR3 of SEQ ID NO: 283.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 358 as CDR1, SEQ ID NO: 359 as CDR2, and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 546 as CDR1, SEQ ID NO: 359 as CDR2 and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 547. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285, which comprises the amino acid sequences of SEQ ID NO: 361 as CDR1, SEQ ID NO: 362 as CDR2, and SEQ ID NO: 363 as CDR3 of SEQ ID NO: 285.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33;

the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 364 as CDR1, SEQ ID NO: 365 as CDR2, and SEQ ID NO: 366 as CDR3 of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 548 as CDR1, SEQ ID NO: 365 as CDR2 and SEQ ID NO: 549 as CDR3 of SEQ ID NO: 286. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287, which comprises the amino acid sequences of SEQ ID NO: 367 as CDR1, SEQ ID NO: 368 as CDR2, and SEQ ID NO: 369 as CDR3 of SEQ ID NO: 287.

- 110 049630

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 292. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 382 as CDR1, SEQ ID NO: 383 as CDR2, and SEQ ID NO: 384 as CDR3 of SEQ ID NO: 292. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 554 as CDR1, SEQ ID NO: 383 as CDR2 and SEQ ID NO: 555 as CDR3 of SEQ ID NO: 292. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 293. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 293, which comprises the amino acid sequences of SEQ ID NO: 385 as CDR1, SEQ ID NO: 386 as CDR2, and SEQ ID NO: 387 as CDR3 of SEQ ID NO: 293.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 294. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 388 as CDR1, SEQ ID NO: 389 as CDR2, and SEQ ID NO: 390 as CDR3 of SEQ ID NO: 294. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 556 as CDR1, SEQ ID NO: 389 as CDR2 and SEQ ID NO: 557 as CDR3 of SEQ ID NO: 294. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 295. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 may be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 295, which comprises the amino acid sequences of SEQ ID NO: 391 as CDR1, SEQ ID NO: 392 as CDR2, and SEQ ID NO: 393 as CDR3 of SEQ ID NO: 295.

In some embodiments, the present invention provides an antigen-binding site that binds the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 296. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 394 as CDR1, SEQ ID NO: 395 as CDR2, and SEQ ID NO: 396 as

- 111 049630 as CDR3 of SEQ ID NO: 296. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 558 as CDR1, SEQ ID NO: 395 as CDR2, and SEQ ID NO: 559 as CDR3 of SEQ ID NO: 296. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297, which comprises the amino acid sequences of SEQ ID NO: 397 as CDR1, SEQ ID NO: 398 as CDR2, and SEQ ID NO: 399 as CDR3 of SEQ ID NO: 297.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 298. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 400 as CDR1, SEQ ID NO: 401 as CDR2, and SEQ ID NO: 402 as CDR3 of SEQ ID NO: 298. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 560 as CDR1, SEQ ID NO: 401 as CDR2 and SEQ ID NO: 561 as CDR3 of SEQ ID NO: 298. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299, which comprises the amino acid sequences of SEQ ID NO: 403 as CDR1, SEQ ID NO: 404 as CDR2, and SEQ ID NO: 405 as CDR3 of SEQ ID NO: 299.

In some embodiments, the present invention provides an antigen-binding site that binds to the R69G allele of human CD33;

the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 412 as CDR1, SEQ ID NO: 413 as CDR2, and SEQ ID NO: 414 as CDR3 of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 564 as CDR1, SEQ ID NO: 413 as CDR2, and SEQ ID NO: 565 as CDR3 of SEQ ID NO: 302. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, the heavy chain variable domain of an antibody

- 112 049630 an antibody chain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 may be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303, which comprises the amino acid sequences of SEQ ID NO: 415 as CDR1, SEQ ID NO: 416 as CDR2, and SEQ ID NO: 417 as CDR3 of SEQ ID NO: 303.

An antigen-binding site that does not bind to the R69G allele of human CD33

In one aspect, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds wild-type human CD33 but not the R69G allele of human CD33. In certain embodiments, the present invention provides an antigen-binding site that does not bind the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWG RGTLVTVSS [SEQ ID NO: 7]. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFSDYYMH [SEQ ID NO: 39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO: 40] as CDR2, and AREAADGFVGERYFDL [SEQ ID NO: 41] as CDR3 of SEQ ID NO: 7. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 is combined with a variable domain light chain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO: 8]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 may be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 8, which comprises the amino acid sequences RSSQSLLYSNGYNYLD [SEQ ID NO: 42] as CDR1, LGSNRAS [SEQ ID NO: 43] as CDR2, and MQDVALPIT [SEQ ID NO: 44] as CDR3 of SEQ ID NO: 8.

In certain embodiments, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 370 as CDR1, SEQ ID NO: 371 as CDR2, and SEQ ID NO: 372 as CDR3 of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 550 as CDR1, SEQ ID NO: 371 as CDR2 and SEQ ID NO: 551 as CDR3 of SEQ ID NO: 288. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 may be

- 113 049630 is paired with a light chain variable domain of an antibody that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289. In certain embodiments, a heavy chain variable domain of an antibody that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 may be paired with a light chain variable domain of an antibody that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 289, which comprises the amino acid sequences of SEQ ID NO: 373 as CDR1, SEQ ID NO: 374 as CDR2 and SEQ ID NO: 375 as CDR3 of SEQ ID NO: 289.

In certain embodiments, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 376 as CDR1, SEQ ID NO: 377 as CDR2, and SEQ ID NO: 378 as CDR3 of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 552 as CDR1, SEQ ID NO: 377 as CDR2 and SEQ ID NO: 553 as CDR3 of SEQ ID NO: 290. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291, which comprises the amino acid sequences of SEQ ID NO: 379 as CDR1, SEQ ID NO: 380 as CDR2, and SEQ ID NO: 381 as CDR3 of SEQ ID NO: 291.

In certain embodiments, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not to the R69G allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 406 as CDR1, SEQ ID NO: 407 as CDR2, and SEQ ID NO: 408 as CDR3 of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 562 as CDR1, SEQ ID NO: 407 as CDR2 and SEQ ID NO: 563 as CDR3 of SEQ ID NO: 300. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300, may be paired with a light chain variable domain of an antibody by at least 90% (e.g.,

- 114 049630 at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 301, which includes the amino acid sequences of SEQ ID NO: 409 as CDR1, SEQ ID NO: 410 as CDR2 and SEQ ID NO: 411 as CDR3 of SEQ ID NO: 301.

An antigen-binding site that binds to a unique epitope, including R69, on human CD33

In one aspect, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds to a unique epitope on human CD33 that comprises R69. In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWG RGTLVTVSS [SEQ ID NO: 7].

In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 7. In some embodiments, the heavy chain variable domain comprises the amino acid sequences YTFSDYYMH [SEQ ID NO: 39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO: 40] as CDR2, and AREAADGFVGERYFDL [SEQ ID NO: 41] as CDR3 of SEQ ID NO: 7. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 is combined with a variable domain light chain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 can be paired with an antibody light chain variable domain that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO: 8]. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7 may be paired with an antibody light chain variable domain that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 8, which comprises the amino acid sequences RSSQSLLYSNGYNYLD [SEQ ID NO: 42] as CDR1, LGSNRAS [SEQ ID NO: 43] as CDR2, and MQDVALPIT [SEQ ID NO: 44] as CDR3 of SEQ ID NO: 8.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 370 as CDR1, SEQ ID NO: 371 as CDR2, and SEQ ID NO: 372 as CDR3 of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 550 as CDR1, SEQ ID NO: 371 as CDR2 and SEQ ID NO: 551 as CDR3 of SEQ ID NO: 288. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288, may be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical in amino acid sequence after

- 115 049630 of the sequence SEQ ID NO: 289, which includes the amino acid sequences SEQ ID NO: 373 as CDR1, SEQ ID NO: 374 as CDR2 and SEQ ID NO: 375 as CDR3 of SEQ ID NO: 289.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 376 as CDR1, SEQ ID NO: 377 as CDR2, and SEQ ID NO: 378 as CDR3 of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 552 as CDR1, SEQ ID NO: 377 as CDR2 and SEQ ID NO: 553 as CDR3 of SEQ ID NO: 290. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291, which comprises the amino acid sequences of SEQ ID NO: 379 as CDR1, SEQ ID NO: 380 as CDR2, and SEQ ID NO: 381 as CDR3 of SEQ ID NO: 291.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 406 as CDR1, SEQ ID NO: 407 as CDR2, and SEQ ID NO: 408 as CDR3 of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 562 as CDR1, SEQ ID NO: 407 as CDR2 and SEQ ID NO: 563 as CDR3 of SEQ ID NO: 300. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301, which comprises the amino acid sequences of SEQ ID NO: 409 as CDR1, SEQ ID NO: 410 as CDR2, and SEQ ID NO: 411 as CDR3 of SEQ ID NO: 301.

An antigen-binding site that binds to the S128N allele of human CD33.

In one embodiment, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33.

In some embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence of at least

- 116 049630

90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 268. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 310 as CDR1, SEQ ID NO: 311 as CDR2, and SEQ ID NO: 312 as CDR3 of SEQ ID NO: 268. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 530 as CDR1, SEQ ID NO: 311 as CDR2, and SEQ ID NO: 531 as as CDR3 of SEQ ID NO: 268. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 269. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 268 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 269, which comprises the amino acid sequences of SEQ ID NO: 313 as CDR1, SEQ ID NO: 314 as CDR2, and SEQ ID NO: 315 as CDR3 of SEQ ID NO: 269.

In some embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 274. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 328 as CDR1, SEQ ID NO: 329 as CDR2, and SEQ ID NO: 330 as CDR3 of SEQ ID NO: 274. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 536 as CDR1, SEQ ID NO: 329 as CDR2 and SEQ ID NO: 537 as CDR3 of SEQ ID NO: 274. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 275. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 274 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 275, which comprises the amino acid sequences of SEQ ID NO: 331 as CDR1, SEQ ID NO: 332 as CDR2, and SEQ ID NO: 333 as CDR3 of SEQ ID NO: 275.

In certain embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 276. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 334 as CDR1, SEQ ID NO: 335 as CDR2, and SEQ ID NO: 336 as CDR3 of SEQ ID NO: 276. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 538 as CDR1, SEQ ID NO: 335 as CDR2 and SEQ ID NO: 539 as CDR3 SEQ ID NO: 276. In

- 117 049630 in certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 276 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 277, which comprises the amino acid sequences of SEQ ID NO: 337 as CDR1, SEQ ID NO: 338 as CDR2, and SEQ ID NO: 339 as CDR3 of SEQ ID NO: 277.

In certain embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 292. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 382 as CDR1, SEQ ID NO: 383 as CDR2, and SEQ ID NO: 384 as CDR3 of SEQ ID NO: 292. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 554 as CDR1, SEQ ID NO: 383 as CDR2 and SEQ ID NO: 555 as CDR3 of SEQ ID NO: 292. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 293. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 292 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 293, which comprises the amino acid sequences of SEQ ID NO: 385 as CDR1, SEQ ID NO: 386 as CDR2, and SEQ ID NO: 387 as CDR3 of SEQ ID NO: 293.

In certain embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 294. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 388 as CDR1, SEQ ID NO: 389 as CDR2, and SEQ ID NO: 390 as CDR3 of SEQ ID NO: 294. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 556 as CDR1, SEQ ID NO: 389 as CDR2 and SEQ ID NO: 557 as CDR3 of SEQ ID NO: 294. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 can be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least

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91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 295. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 294. 100%) identical to the amino acid sequence of SEQ ID NO: 295, which includes the amino acid sequences of SEQ ID NO: 391 as CDR1, SEQ ID NO: 392 as CDR2 and SEQ ID NO: 393 as CDR3 of SEQ ID NO: 295.

In some embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 296. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 394 as CDR1, SEQ ID NO: 395 as CDR2, and SEQ ID NO: 396 as CDR3 of SEQ ID NO: 296. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 558 as CDR1, SEQ ID NO: 395 as CDR2 and SEQ ID NO: 559 as CDR3 of SEQ ID NO: 296. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 296 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 297, which comprises the amino acid sequences of SEQ ID NO: 397 as CDR1, SEQ ID NO: 398 as CDR2, and SEQ ID NO: 399 as CDR3 of SEQ ID NO: 297.

In certain embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 298. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 400 as CDR1, SEQ ID NO: 401 as CDR2, and SEQ ID NO: 402 as CDR3 of SEQ ID NO: 298. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 560 as CDR1, SEQ ID NO: 401 as CDR2 and SEQ ID NO: 561 as CDR3 of SEQ ID NO: 298. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 298 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 299, which comprises the amino acid sequences of SEQ ID NO: 403 as CDR1, SEQ ID NO: 404 as CDR2, and SEQ ID NO: 405 as CDR3 of SEQ ID NO: 299.

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In certain embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 376 as CDR1, SEQ ID NO: 377 as CDR2, and SEQ ID NO: 378 as CDR3 of SEQ ID NO: 290. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 552 as CDR1, SEQ ID NO: 377 as CDR2 and SEQ ID NO: 553 as CDR3 of SEQ ID NO: 290. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 290 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 291, which comprises the amino acid sequences of SEQ ID NO: 379 as CDR1, SEQ ID NO: 380 as CDR2, and SEQ ID NO: 381 as CDR3 of SEQ ID NO: 291.

In certain embodiments, the present invention provides an antigen-binding site that binds to the S128N allele of human CD33 that comprises R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 406 as CDR1, SEQ ID NO: 407 as CDR2, and SEQ ID NO: 408 as CDR3 of SEQ ID NO: 300. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 562 as CDR1, SEQ ID NO: 407 as CDR2 and SEQ ID NO: 563 as CDR3 of SEQ ID NO: 300. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 300 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 301, which comprises the amino acid sequences of SEQ ID NO: 409 as CDR1, SEQ ID NO: 410 as CDR2, and SEQ ID NO: 411 as CDR3 of SEQ ID NO: 301.

An antigen-binding site that does not bind to the S128N allele of human CD33.

In one aspect, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds to wild-type human CD33 but not to the S128N allele of human CD33.

In some embodiments, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,

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97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 412 as CDR1, SEQ ID NO: 413 as CDR2, and SEQ ID NO: 414 as CDR3 of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 564 as CDR1, SEQ ID NO: 413 as CDR2, and SEQ ID NO: 565 as CDR3 of SEQ ID NO: 302. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303, which comprises the amino acid sequences of SEQ ID NO: 415 as CDR1, SEQ ID NO: 416 as CDR2, and SEQ ID NO: 417 as CDR3 of SEQ ID NO: 303.

In some embodiments, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 304 as CDR1, SEQ ID NO: 305 as CDR2, and SEQ ID NO: 306 as CDR3 of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 528 as CDR1, SEQ ID NO: 305 as CDR2 and SEQ ID NO: 529 as CDR3 of SEQ ID NO: 266. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267, which comprises the amino acid sequences of SEQ ID NO: 307 as CDR1, SEQ ID NO: 308 as CDR2, and SEQ ID NO: 309 as CDR3 of SEQ ID NO: 267.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 316 as CDR1, SEQ ID NO: 317 as CDR2, and SEQ ID NO: 318 as CDR3 of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 532 as CDR1, SEQ ID NO: 317 as CDR2 and SEQ ID NO: 533 as CDR3 of SEQ ID NO: 270. In certain embodiments, the variable domain of the heavy

- 121 049630 antibody chain that comprises an amino acid sequence that is at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 270, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271, which comprises the amino acid sequences of SEQ ID NO: 319 as CDR1, SEQ ID NO: 320 as CDR2, and SEQ ID NO: 321 as CDR3 of SEQ ID NO: 271.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 322 as CDR1, SEQ ID NO: 323 as CDR2, and SEQ ID NO: 324 as CDR3 of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 534 as CDR1, SEQ ID NO: 323 as CDR2 and SEQ ID NO: 535 as CDR3 of SEQ ID NO: 272. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273, which comprises the amino acid sequences of SEQ ID NO: 325 as CDR1, SEQ ID NO: 326 as CDR2, and SEQ ID NO: 327 as CDR3 of SEQ ID NO: 273.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 340 as CDR1, SEQ ID NO: 341 as CDR2, and SEQ ID NO: 342 as CDR3 of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 540 as CDR1, SEQ ID NO: 341 as CDR2 and SEQ ID NO: 541 as CDR3 of SEQ ID NO: 278. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, a heavy chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID

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NO: 278 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278. 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 279, which includes the amino acid sequences of SEQ ID NO: 343 as CDR1, SEQ ID NO: 344 as CDR2 and SEQ ID NO: 345 as CDR3 of SEQ ID NO: 279.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 346 as CDR1, SEQ ID NO: 347 as CDR2, and SEQ ID NO: 348 as CDR3 of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 542 as CDR1, SEQ ID NO: 347 as CDR2 and SEQ ID NO: 543 as CDR3 of SEQ ID NO: 280. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281, which comprises the amino acid sequences of SEQ ID NO: 349 as CDR1, SEQ ID NO: 350 as CDR2, and SEQ ID NO: 351 as CDR3 of SEQ ID NO: 281.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 352 as CDR1, SEQ ID NO: 353 as CDR2, and SEQ ID NO: 354 as CDR3 of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 544 as CDR1, SEQ ID NO: 353 as CDR2 and SEQ ID NO: 545 as CDR3 of SEQ ID NO: 282. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 282, may be paired with a light chain variable domain of an antibody, at least

- 123 049630

90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283, which includes the amino acid sequences of SEQ ID NO: 355 as CDR1, SEQ ID NO: 356 as CDR2, and SEQ ID NO: 357 as CDR3 of SEQ ID NO: 283.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 358 as CDR1, SEQ ID NO: 359 as CDR2, and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 546 as CDR1, SEQ ID NO: 359 as CDR2 and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 547. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285, which comprises the amino acid sequences of SEQ ID NO: 361 as CDR1, SEQ ID NO: 362 as CDR2, and SEQ ID NO: 363 as CDR3 of SEQ ID NO: 285.

In certain embodiments, the present invention provides an antigen binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33 that includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 364 as CDR1, SEQ ID NO: 365 as CDR2, and SEQ ID NO: 366 as CDR3 of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 548 as CDR1, SEQ ID NO: 365 as CDR2 and SEQ ID NO: 549 as CDR3 of SEQ ID NO: 286. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287, which comprises the amino acid sequences of SEQ ID NO: 367 as CDR1, SEQ ID NO: 368 as CDR2, and SEQ ID NO: 369 as CDR3 of SEQ ID NO: 287.

In certain embodiments, the present invention provides an antigen-binding site that binds to wild-type human CD33 but not to the S128N allele of human CD33,

- 124 049630 which includes R69; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 370 as CDR1, SEQ ID NO: 371 as CDR2, and SEQ ID NO: 372 as CDR3 of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 550 as CDR1, SEQ ID NO: 371 as CDR2 and SEQ ID NO: 551 as CDR3 of SEQ ID NO: 288. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289, which comprises the amino acid sequences of SEQ ID NO: 373 as CDR1, SEQ ID NO: 374 as CDR2, and SEQ ID NO: 375 as CDR3 of SEQ ID NO: 289.

An antigen-binding site that binds to a unique epitope, including S128, on human CD33.

In one aspect, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds to a unique epitope on human CD33 that comprises S128.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 412 as CDR1, SEQ ID NO: 413 as CDR2, and SEQ ID NO: 414 as CDR3 of SEQ ID NO: 302. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 564 as CDR1, SEQ ID NO: 413 as CDR2 and SEQ ID NO: 565 as CDR3 of SEQ ID NO: 302. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 302 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 303, which comprises the amino acid sequences of SEQ ID NO: 415 as CDR1, SEQ ID NO: 416 as CDR2, and SEQ ID NO: 417 as CDR3 of SEQ ID NO: 303.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain that comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,

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97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 304 as CDR1, SEQ ID NO: 305 as CDR2, and SEQ ID NO: 306 as CDR3 of SEQ ID NO: 266. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 528 as CDR1, SEQ ID NO: 305 as CDR2, and SEQ ID NO: 529 as CDR3 of SEQ ID NO: 266. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 266 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 267, which comprises the amino acid sequences of SEQ ID NO: 307 as CDR1, SEQ ID NO: 308 as CDR2, and SEQ ID NO: 309 as CDR3 of SEQ ID NO: 267.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 316 as CDR1, SEQ ID NO: 317 as CDR2, and SEQ ID NO: 318 as CDR3 of SEQ ID NO: 270. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 532 as CDR1, SEQ ID NO: 317 as CDR2 and SEQ ID NO: 533 as CDR3 of SEQ ID NO: 270. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 270 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 271, which comprises the amino acid sequences of SEQ ID NO: 319 as CDR1, SEQ ID NO: 320 as CDR2, and SEQ ID NO: 321 as CDR3 of SEQ ID NO: 271.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 322 as CDR1, SEQ ID NO: 323 as CDR2, and SEQ ID NO: 324 as CDR3 of SEQ ID NO: 272. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 534 as CDR1, SEQ ID NO: 323 as CDR2 and SEQ ID NO: 535 as CDR3 of SEQ ID NO: 272. In certain embodiments, a heavy chain variable domain of an antibody that comprises

- 126 049630 an amino acid sequence that is at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of SEQ ID NO: 272, combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 272 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 273, which comprises the amino acid sequences of SEQ ID NO: 325 as CDR1, SEQ ID NO: 326 as CDR2, and SEQ ID NO: 327 as CDR3 of SEQ ID NO: 273.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 340 as CDR1, SEQ ID NO: 341 as CDR2, and SEQ ID NO: 342 as CDR3 of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 540 as CDR1, SEQ ID NO: 341 as CDR2 and SEQ ID NO: 541 as CDR3 of SEQ ID NO: 278. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279, which comprises the amino acid sequences of SEQ ID NO: 343 as CDR1, SEQ ID NO: 344 as CDR2, and SEQ ID NO: 345 as CDR3 of SEQ ID NO: 279.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 346 as CDR1, SEQ ID NO: 347 as CDR2, and SEQ ID NO: 348 as CDR3 of SEQ ID NO: 280. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 542 as CDR1, SEQ ID NO: 347 as CDR2 and SEQ ID NO: 543 as CDR3 of SEQ ID NO: 280. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280.

- 127 049630 of SEQ ID NO: 281. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 280 can be paired with an antibody light chain variable domain that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 281 that comprises the amino acid sequences of SEQ ID NO: 349 as CDR1, SEQ ID NO: 350 as CDR2 and SEQ ID NO: 351 as CDR3 of SEQ ID NO: 281.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 352 as CDR1, SEQ ID NO: 353 as CDR2, and SEQ ID NO: 354 as CDR3 of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 544 as CDR1, SEQ ID NO: 353 as CDR2 and SEQ ID NO: 545 as CDR3 of SEQ ID NO: 282. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283, which comprises the amino acid sequences of SEQ ID NO: 355 as CDR1, SEQ ID NO: 356 as CDR2, and SEQ ID NO: 357 as CDR3 of SEQ ID NO: 283.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 358 as CDR1, SEQ ID NO: 359 as CDR2, and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 546 as CDR1, SEQ ID NO: 359 as CDR2 and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 547. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285, which comprises the amino acid sequences of SEQ ID NO: 361 as CDR1, SEQ ID NO: 362 as CDR2, and SEQ ID NO: 363 as CDR3 of SEQ ID NO: 285.

In some embodiments, the present invention provides an antigen-binding

- 128 049630 site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 364 as CDR1, SEQ ID NO: 365 as CDR2, and SEQ ID NO: 366 as CDR3 of SEQ ID NO: 286. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 548 as CDR1, SEQ ID NO: 365 as CDR2 and SEQ ID NO: 549 as CDR3 of SEQ ID NO: 286. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 286 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 287, which comprises the amino acid sequences of SEQ ID NO: 367 as CDR1, SEQ ID NO: 368 as CDR2, and SEQ ID NO: 369 as CDR3 of SEQ ID NO: 287.

In some embodiments, the present invention provides an antigen-binding site that binds to a unique epitope on human CD33 that includes S128; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 370 as CDR1, SEQ ID NO: 371 as CDR2, and SEQ ID NO: 372 as CDR3 of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 550 as CDR1, SEQ ID NO: 371 as CDR2 and SEQ ID NO: 551 as CDR3 of SEQ ID NO: 288. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289, which comprises the amino acid sequences of SEQ ID NO: 373 as CDR1, SEQ ID NO: 374 as CDR2, and SEQ ID NO: 375 as CDR3 of SEQ ID NO: 289.

The antigen-binding site binds to the V domain of human CD33 in a glycosylation-sensitive manner.

In one aspect of the present invention, there is provided an antigen binding site comprising a heavy chain variable domain that binds to the V domain of human CD33 in a glycosylation sensitive manner, e.g. binds to the V domain of CD33 only when the V domain is deglycosylated.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope in the V domain of human CD33 only if the V domain is deglycosylated; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid

- 129 049630 a sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 340 as CDR1, SEQ ID NO: 341 as CDR2, and SEQ ID NO: 342 as CDR3 of SEQ ID NO: 278. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 540 as CDR1, SEQ ID NO: 341 as CDR2 and SEQ ID NO: 541 as CDR3 of SEQ ID NO: 278. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 278 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 279, which comprises the amino acid sequences of SEQ ID NO: 343 as CDR1, SEQ ID NO: 344 as CDR2, and SEQ ID NO: 345 as CDR3 of SEQ ID NO: 279.

In certain embodiments, the present invention provides an antigen binding site that binds an epitope in the V domain of human CD33 only if the V domain is deglycosylated; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 352 as CDR1, SEQ ID NO: 353 as CDR2, and SEQ ID NO: 354 as CDR3 of SEQ ID NO: 282. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 544 as CDR1, SEQ ID NO: 353 as CDR2 and SEQ ID NO: 545 as CDR3 of SEQ ID NO: 282. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 282 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 283, which comprises the amino acid sequences of SEQ ID NO: 355 as CDR1, SEQ ID NO: 356 as CDR2, and SEQ ID NO: 357 as CDR3 of SEQ ID NO: 283.

In certain embodiments, the present invention provides an antigen-binding site that binds an epitope in the V domain of human CD33 only if the V domain is deglycosylated; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 284. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 358 as CDR1, SEQ ID NO: 359 as CDR2, and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 284. In some embodiments, the

- 130 049630 the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 546 as CDR1, SEQ ID NO: 359 as CDR2, and SEQ ID NO: 360 as CDR3 of SEQ ID NO: 547. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 284 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 285, which comprises the amino acid sequences of SEQ ID NO: 361 as CDR1, SEQ ID NO: 362 as CDR2, and SEQ ID NO: 363 as CDR3 of SEQ ID NO: 285.

In certain embodiments, the present invention provides an antigen binding site that binds an epitope in the V domain of human CD33 only if the V domain is deglycosylated; the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain of the antibody is at least 95% identical to SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 370 as CDR1, SEQ ID NO: 371 as CDR2, and SEQ ID NO: 372 as CDR3 of SEQ ID NO: 288. In some embodiments, the heavy chain variable domain comprises the amino acid sequences of SEQ ID NO: 550 as CDR1, SEQ ID NO: 371 as CDR2 and SEQ ID NO: 551 as CDR3 of SEQ ID NO: 288. In certain embodiments, a heavy chain variable domain of an antibody that comprises an amino acid sequence that is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 is combined with a light chain variable domain to form an antigen-binding site capable of binding to CD33. For example, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 can be paired with an antibody light chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289. In certain embodiments, an antibody heavy chain variable domain that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 288 may be paired with a light chain variable domain of an antibody that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 289, which comprises the amino acid sequences of SEQ ID NO: 373 as CDR1, SEQ ID NO: 374 as CDR2, and SEQ ID NO: 375 as CDR3 of SEQ ID NO: 289.

An antigen-binding site that binds to the extracellular domain of human CD33 and/or cynomolgus monkey CD33, regardless of the glycosylation profile of the target CD33

In one aspect, the present invention provides an antigen-binding site comprising a heavy chain variable domain that comprises an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, or 302, which binds to the extracellular domain of human CD33 regardless of the glycosylation profile of the target CD33.

In certain embodiments, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds to an extracellular domain in human CD33 and/or cynomolgus monkey CD33 such that the epitopes are unique compared to epitopes targeted by one or more anti-CD33 antibodies known in the art. In certain embodiments, the present invention provides an antigen-binding site comprising a heavy chain variable domain that binds to an extracellular domain in human CD33 and/or cynomolgus monkey CD33 that exhibits cross-reactivity

- 131 049630 human CD33 and cynomolgus/rhesus macaque CD33 activity and high binding affinity to target CD33.

The second antigen-binding site is the same as or different from the antigen-binding site that binds human CD33.

In some embodiments, the present invention provides a protein that comprises a human CD33 antigen-binding site comprising a heavy chain variable domain that comprises an amino acid sequence that is at least 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300 or 302, and further comprises a second antigen-binding site, the same as or different from the antigen-binding site that binds human CD33.

Proteins with antigen-binding sites

The heavy chain variable domain of the antibody of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300 and/or 302 may optionally be linked to an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an antibody constant region, such as an IgG constant region, including hinge domains, CH2 and CH3 domains with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human antibody constant region, such as a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the constant region of an antibody of another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be included in a constant region compared to the constant region of human IgG1, such as Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and/or K439. Illustrative substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K36OE, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

In certain embodiments, mutations that can be included in the CH1 constant region of human IgG1 can be at amino acid residues V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In certain embodiments, mutations that can be included in the Ck constant region of human IgG1 can be at amino acid residues E123, F116, S176, V163, S174, and/or T164.

Multispecific binding proteins

In certain embodiments, the present invention provides an antigen-binding site in a protein (e.g., a multispecific binding protein) that binds to CD33 on a cancer cell, as well as the NKG2D receptor and the CD16 receptor on natural killer cells to activate the natural killer cell. As used herein, the term "antibody" encompasses proteins (e.g., multispecific binding proteins) that contain one or more antigen-binding sites (e.g., an antigen-binding site that binds CD33), and is not limited to monospecific antibodies. In certain embodiments, the protein (e.g., a multispecific binding protein) or antibody is a trispecific antibody, also referred to as trispecific NK cell activation therapy (TriNKET). The protein (e.g., a multispecific binding protein) is useful in the pharmaceutical compositions and therapeutic methods described herein. Binding of a protein comprising an antigen-binding site that binds to CD33 and the NKG2D receptor and CD16 receptor on a natural killer cell enhances the natural killer cell's activity in destroying a cancer cell. Binding of a protein comprising an antigen-binding site that binds to CD33 (e.g., a multispecific binding protein) on a cancer cell brings the cancer cell closer to the natural killer cell, facilitating direct and indirect destruction of the cancer cell by the natural killer cell. Further description of exemplary multispecific binding proteins is provided below.

In certain embodiments of the present disclosure, the first component of the multispecific binding proteins binds to CD33-expressing cells, which may include, but are not limited to, AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, and ALL.

In certain embodiments of the present disclosure, the second component of the multi-spec

- 132 049630 multispecific binding proteins bind to cells expressing the NKG2D receptor, which may include, but are not limited to, NK cells, γδ T cells, and CD8+ αβ T cells. Upon binding to NKG2D, multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NKG2D receptors.

In certain embodiments of the present disclosure, the third component for the multispecific binding proteins binds to cells expressing CD16, an Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

In another aspect of the present invention there is provided a protein comprising an antigen-binding site that binds NKG2D, wherein the antigen-binding site comprises a heavy chain variable domain comprising: a CDR1 comprising the amino acid sequence SYSMN [SEQ ID NO: 192]; a CDR2 comprising the amino acid sequence SISSSSSYIYYADSVKG [SEQ ID NO: 112]; and a CDR3 comprising the amino acid sequence GAPXGAAAGWFDP [SEQ ID NO: 527], wherein X is A, V, L, I, P, F, W, G, S, T, C, N, Q or Y; and a light chain variable domain comprising: CDR1 comprising the amino acid sequence RASQGISSWLA [SEQ ID NO: 114], CDR2 comprising the amino acid sequence AASSLQS [SEQ ID NO: 115], and CDR3 comprising the amino acid sequence QQGVSFPRT [SEQ ID NO: 116].

In certain embodiments, X is A, V, L, I, P, F, or W. In certain embodiments, X is V, L, or I. In certain embodiments, the amino acid sequence of a CDR3 in a heavy chain variable domain comprises the sequence of SEQ ID NO: 123. In certain embodiments, the amino acid sequence of a CDR3 in a heavy chain variable domain comprises the sequence of SEQ ID NO: 195, SEQ ID NO: 588, SEQ ID NO: 591, SEQ ID NO: 594, or SEQ ID NO: 597.

In certain embodiments, the antigen-binding site comprises a heavy chain variable domain comprising an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 191; and a light chain variable domain comprising an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 81. In certain embodiments, the antigen-binding site comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 191; and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 81.

In certain embodiments, the antigen-binding site that binds NKG2D is in the form of a Fab fragment. In certain embodiments, the antigen-binding site that binds NKG2D is in the form of an scFv.

In certain embodiments, the present invention provides a protein comprising: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D as described herein; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds a tumor-associated antigen (e.g., CD33); and (c) an Fc domain of an antibody or a portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16.

The multispecific binding proteins described herein can have a variety of formats. For example, one format is a heterodimeric, multispecific antibody that includes a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain, and a second immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc domain (hinge-CH2-CH3), a first heavy chain variable domain, and optionally a first heavy chain CH1 domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen-binding site that binds CD33. The second immunoglobulin heavy chain includes a second Fc domain (hinge-CH2-CH3), a second heavy chain variable domain, and optionally a second heavy chain CH1 domain. The second immunoglobulin light chain includes the second light chain variable domain and the second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms the antigen-binding site that binds NKG2D. The first Fc domain and the second Fc domain together are capable of binding to CD16.

Another exemplary format includes a heterodimeric, multispecific antibody that includes a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain. The first immunoglobulin heavy chain includes a first Fc domain (hinge-CH2-CH3) fused via a linker or antibody hinge to a single-chain variable fragment (scFv) consisting of a heavy and light chain variable domain that connect and bind CD33 or NKG2D. The second immunoglobulin heavy chain includes a second Fc domain (hinge-CH2CH3), a second heavy chain variable domain, and optionally a heavy chain CH1 domain. The light chain

- 133 049630 immunoglobulin includes a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain is linked to the immunoglobulin light chain and binds to NKG2D or CD33. The first Fc domain and the second Fc domain together are capable of binding to CD16.

One or more additional binding motifs may be fused to the C-terminus of the CH3 domain of the constant region, optionally via a linker sequence. In certain embodiments, the antigen-binding site may be a single-chain or disulfide-stabilized variable region (scFv) or may form a tetravalent or trivalent molecule.

In some embodiments, the multispecific binding protein is in the form of Triomab, which is a trifunctional bispecific antibody that retains an IgG-like shape. This chimera consists of two half-antibodies, each with one light and one heavy chain, which are derived from two parent antibodies.

In some embodiments, the multispecific binding protein is in the form of a KiH common light chain (LC) that utilizes knob-in-hole (KIH) technology. KIH involves engineering the _CH3 domains to create a knob or hole in each heavy chain to facilitate heterodimerization. The concept of the Fc knob-in-hole (KiH) technology was to introduce a knob into one CH3 domain (CH3A) by replacing a small residue with a bulkier one (e.g., T366W _CH3A in EU numbering). To accommodate a knob on another CH3 domain (CH3B), an additional hole surface was created by replacing the immediate neighboring residues to the knob with smaller ones (e.g., T366S/L368A/Y407V _CH3B ). The knob mutation was optimized by structured screening of a phage library ((Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J. Mol. Biol. (1997) 270(l):26–35). X-ray crystal structures of Fc variants of KiH (Elliott JM, Ultsch M., Lee J., Tong R., Takeda K., Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2–CH3 hydrophobic interaction. J. Mol. Biol. (2014) 426(9): 1947–57; Mimoto F., Kadono S., Katada H., Igawa T., Kamikawa T., Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs. Mol. Immunol. (2014) 58(1):132-8) demonstrated that heterodimerization is thermodynamically favored by hydrophobic interactions driven by steric complementarity at the main interface between the CH3 domain, whereas knob-knob and hole-hole interfaces do not favor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

In some embodiments, the multispecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the target binding domains of two monoclonal antibodies through flexible natural linkers to produce a tetravalent IgG-like molecule.

In some embodiments, the multispecific binding protein is in the form of an orthogonal Fab interface (ortho-Fab). In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. 2014) 32(2): 191-8), the structural regional design introduces additional mutations at the LC and C boundary of _V h- _CH1 in only one Fab, without any changes in the other Fab.

In some embodiments, the multispecific binding protein is in a 2-to-1 Ig format. In certain embodiments, the multispecific binding protein is in an ES form, which is a heterodimeric construct comprising two different Fabs that bind to target 1 and target 2 fused to an Fc. Heterodimerization is achieved by electrostatic interaction mutations in the Fc.

In some embodiments, the multispecific binding protein is in the form of a λ-body, which is a heterodimeric construct with two different Fabs fused to an Fc stabilized by heterodimerization mutations: Fab1-targeted antigen 1 comprises LC kappa, while a second Fab1-targeted antigen 2 comprises LC lambda. Fig. 30A is an exemplary depiction of one form of a λ-Body; Fig. 30B is an exemplary depiction of another form of a λ-Body.

In some embodiments, the multispecific binding protein is in the form of a Fab exchange (antibodies that exchange Fab fragments by exchanging the heavy chain and attached light chain (half-molecule) for a heavy-light chain pair from another molecule, resulting in bispecific antibodies). In certain embodiments, the multispecific binding protein is in the form of a SEED Body. The chain exchange engineered domain (SEED) platform was developed to create asymmetric and bispecific antibody-like molecules, which expands the therapeutic potential of natural antibodies. This protein-engineered platform is based on the exchange of structurally related

- 134 049630 immunoglobulin sequences in conserved CH3 domains. The SEED design allows for efficient generation of AG/GA heterodimers without promoting homodimerization of the SEED AG and GA CH3 domains. (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)). In certain embodiments, the multispecific binding protein is in the LuZ-Y form, in which the leucine zipper is used to induce heterodimerization of two different HCs. (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

In some embodiments, the multispecific binding protein is in the form of a Cov-X-body. In bispecific CovX-bodies, two different peptides are joined together using a branched azetidinone linker and fused to an antibody scaffold under mild conditions in a site-specific manner. While the pharmacophores are responsible for the functional activity, the antibody scaffold provides a long half-life and an Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to create optimized or unique bispecific antibodies. (Doppalapudi VR et al., PNAS (2010), 107(52); 22611-22616).

In some embodiments, the multispecific binding protein is in a heterodimeric form of Oasc-Fab, which includes Fab binding to target 1 and scFab binding to target 2 fused to Fc. Heterodimerization is achieved by mutations in Fc.

In some embodiments, the multispecific binding protein is in the form of DuetMab, which is a heterodimeric construct comprising two different Fabs that bind to antigens 1 and 2 and an Fc stabilized by heterodimerization mutations. Fabs 1 and 2 contain differential S-S bridges that ensure proper pairing of the LC and HC.

In some embodiments, the multispecific binding protein is in the form of a CrossmAb, which is a heterodimeric construct with two different Fabs binding to targets 1 and 2 fused to an Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains are swapped, such that CH1 is fused in line with VL and CL is fused in line with VH.

In some embodiments, the multispecific binding protein is in the form of Fit-Ig, which is a homodimeric construct in which a Fab that binds to antigen 2 is fused to the N-terminus of a HC Fab that binds to antigen 1. The construct contains a wild-type Fc.

Additional multispecific binding protein formats can be developed by combining the various CD33 binding fragment formats described herein.

In certain embodiments of the present disclosure, the third component for the multispecific binding proteins is an antibody constant region. In certain embodiments, each of the two immunoglobulin heavy chains of the antibody constant region comprises a constant region with an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a human IgG1 constant region. In certain embodiments, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439; and the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411 and K439.

In certain embodiments of the present invention, the NKG2D antigen binding site comprises:

(1) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 81 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 82 [ADI-29379];

(2) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 83 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 84 [ADI-29463];

(3) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 85, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

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99% or 100%) identical to A SEQ ID NO: 86 [ADI-27744];

(4) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 87 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 88 [ADI-27749];

(5) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 191 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to A SEQ ID NO: 88 [A49MI]; or (6) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 89 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 90 [ADI-29378].

In certain embodiments of the present invention, the NKG2D antigen binding site comprises:

(1) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 124 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 125 [ADI-27705];

(2) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 129 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 130 [ADI-27724];

(3) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 131 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 132 [ADI-27740];

(4) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 133 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 134 [ADI-27741];

(5) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 135 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to A of SEQ ID NO: 136 [ADI-27743];

(6) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 137 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to A of SEQ ID NO: 138 [ADI-28153];

(7) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 139 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 140 [ADI-28226 (C26)];

(8) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 141 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 142.

(9) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 143, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

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99% or 100%) identical to SEQ ID NO: 144.

(10) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 145, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 146.

(11) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 147, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 148.

(12) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 149, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 150.

(13) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 151, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 152.

(14) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 153, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 154.

(15) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 155, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 156.

(16) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 157, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 158.

(17) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 159, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 160.

(18) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 161, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 162.

(19) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 163, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 164.

(20) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 165, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 166.

(21) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 167, and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 168.

(22) a heavy chain variable domain containing an amino acid sequence

- 137 049630 at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 175, and a light chain variable domain comprising an amino acid sequence at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 176.

(23) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 583 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 584; or (24) a heavy chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 585 and a light chain variable domain comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 580.

Table 2 lists the peptide sequences of the variable heavy chain and variable light chain domains that can bind NKG2D in combination. Unless otherwise noted, the CDR sequences listed in Table 2 are as defined by Kabat. The NKG2D binding domains may differ in their binding affinity to NKG2D; however, all activate human NKG2D and NK cells.

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Table 2 Clones Amino acid sequence of the variable region of the heavy chain Amino acid sequence of the light chain variable region ADI- 29379 (E79) QVQLVQSGAEVKKPGASVKVSCKASG YTFTSYYMHWVRQAPGQGLEWMGIIN PSGGSTSYAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCARGAPNYGD TTHDYYYMDVWGKGTTVTVSS [SEQIDNO:81] CDR1: YTFTSYYMH [SEQ GO NO:93] (not by Kabat) or SYYMH [SEQ GO NO:566] CDR2: IINPSGGSTSYAQKFQG [SEQ ID NO:94] CDR3: ARGAPNYGDTTHDYYYMDV [SEQ ID NO:95] not no Kabat) or GAPNYGDTTHDYYYMDV [SEQ ID NO:567] EIVMTQSPATLSVSPGERATLSC RASQSVSSNLAWYQQKPGQAP RLLIYGASTRATGIPARFSGSGS GTEFTLTISSLQSEDFAVYYCQQ YDDWPFTFGGGTKVEIK [SEQ ID NO:82] CDR1: RASQSVSSNLA [SEQ ID NO:96] CDR2: GASTRAT [SEQ ID NO:97] CDR3: QQYDDWPFT [SEQ ID NO:98] ADI- 29463 (F63) QVQLVQSGAEVKKPGASVKVSCKASG YTFTGYYMHWVRQAPGQGLEWMGW INPNSGGTNYAQKFQGRVTMTRDTSIS TAYMELSRLRSDDTAVYYCARDTGEY YDTDDHGMDVWGQGTTVTVSS [SEQ ID NO:83] CDR1: YTFTGYYMH [SEQ ID NO:99] (not by Kabat numbering) or GYYMH [SEQ ID NO:568] CDR2: WINPNSGGTNYAQKFQG [SEQ ID NO: 100] CDR3: ARDTGEYYDTDDHGMDV [SEQ ID NO: 101] (not no Kabat) or DTGEYYDTDDHGMDV [SEQ ID NO:569] EIVLTQSPGTLSLSPGERATLSC RASQSVSSNLAWYQQKPGQAP RLLIYGASTRATGIPARFSGSGS GTEFTLTISSLQSEDFAVYYCQQ DDYWPPTFGGGTKVEIK [SEQ ID NO:84] CDR1: RASQSVSSNLA [SEQ ID NO: 102] CDR2: GASTRAT [SEQ ID NO: 103] CDR3: QQDDYWPPT [SEQ ID NO: 104] ADI- 27744 (A44) EVQLLESGGGLVQPGGSLRLSCAASGF TFSSYAMSWVRQAPGKGLEWVSAISG SGGSTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKDGGYYDS GAGDYWGQGTLVTVSS DIQMTQSPSSVSASVGDRVTITC RASQGIDSWLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSYPRTFGGGTKVEIK

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[SEQ ID NO:85] CDR1: FTFSSYAMS [SEQ ID NO: 105] (not no Kabat) or SYAMS [SEQ ID NO:570] CDR2: AISGSGGSTYYADSVKG [SEQ ID NO: 106] CDR3: AKDGGYYDSGAGDY [SEQ ID NO: 107] (not no Kabat) or DGGYYDSGAGDY [SEQ ID NO:571] [SEQ ID NO:86] CDR1: RASQGIDSWLA [SEQ ID NO: 108] CDR2: AASSLQS [SEQ ID NO: 109] CDR3: QQGVSYPRT [SEQ ID NO: 110] ADI- 27749 (A49) EVQLVESGGGLVKPGGSLRLSCAASGF TFSSYSMNWVRQAPGKGLEWVSSISSS SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPMGAAA GWFDPWGQGTLVTVSS [SEQ ID NO:87] CDR1: FTFSSYSMN [SEQ ID NO:111] or SYSMN [seq id no: 192] CDR2: SISSSSSYIYYADSVKG [SEQ ID NO: 112] CDR3: (not no Kabat) ARGAPMGAAAGWFDP [SEQ ID NO: 113] or GAPMGAAAGWFDP [SEQ ID NO: 193] DIQMTQSPSSVSASVGDRVTITC RASQGISSWLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK [SEQ ID NO:88] CDR1: RASQGISSWLA [SEQ ID NO: 114] CDR2: AASSLQS [SEQ ID NO: 115] CDR3: QQGVSFPRT [SEQ ID NO: 116] A49MI EVQLVESGGGLVKPGGSLRLSCAASGF TFSSYSMNWVRQAPGKGLEWVSSISSS SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPIGAAAG WFDPWGQGTLVTVSS [SEQ ID NO:191] CDR1: (not no Kabat) FTFSSYSMN [SEQ ID NO: 111] or SYSMN [seq id no: 192] CDR2: SISSSSSYIYYADSVKG [SEQ ID NO: 112] CDR3: (not no Kabat) DIQMTQSPSSVSASVGDRVTITC RASQGISSWLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK [SEQ ID NO:88] CDR1: RASQGISSWLA [SEQ ID NO: 114] CDR2: AASSLQS [SEQ ID NO: 115] CDR3: QQGVSFPRT

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ARGAPIGAAAGWFDP [SEQ ID NO: 194] or GAPIGAAAGWFDP [SEQ ID NO: 195] [SEQ ID NO: 116] A49MQ EVQLVESGGGLVKPGGSLRLSCAASGF TFSSYSMNWVRQAPGKGLEWVSSISSS SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPQGAAA GWFDPWGQGTLVTVSS (SEQ ID NO:586) CDR1 (not no Kabat) (SEQ ID NO: 111) FTFSSYSMN or CDR1 (SEQ ID NO: 192) - SYSMN CDR2 (SEQ ID NO: 112) - SISSSSSYIYYADSVKG CDR3 (not no Kabat) (SEQ ID NO:587) ARGAPQGAAAGWFDP or CDR3 (SEQ ID NO:588) - GAPQGAAAGWFDP DIQMTQSPSSVSASVGDRVTITC RASQGISSWLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK (SEQ ID NO:88) CDR1 (SEQ ID NO: 114) RASQGISSWLA CDR2 (SEQ ID NO: 115) AASSLQS CDR3 (SEQ ID NO: 116) QQGVSFPRT A49ML EVQLVESGGGLVKPGGSLRLSCAASGF TFSSYSMNWVRQAPGKGLEWVSSISSS SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPLGAAA GWFDPWGQGTLVTVSS (SEQ ID NO:589) CDR1 (not no Kabat) (SEQ ID NO: 111) FTFSSYSMN or CDR1 (SEQ ID NO: 192)- SYSMN CDR2 (SEQ ID NO: 112) - SISSSSSYIYYADSVKG CDR3 (not no Kabat) (SEQ ID NO:590) ARGAPLGAAAGWFDP or CDR3 (SEQ ID NO:591) - GAPLGAAAGWFDP DIQMTQSPSSVSASVGDRVTITC RASQGISSWLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK (SEQ ID NO:88) CDR1 (SEQ ID NO: 114) RASQGISSWLA CDR2 (SEQ ID NO: 115) AASSLQS CDR3 (SEQ ID NO: 116) QQGVSFPRT A49MF EVQLVESGGGLVKPGGSLRLSCAASGF TFSSYSMNWVRQAPGKGLEWVSSISSS DIQMTQSPSSVSASVGDRVTITTC RASQGISSWLAWYQQKPGKAP

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SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPFGAAA GWFDPWGQGTLVTVSS (SEQ ID NO:592) CDR1 (not no Kabat) (SEQ ID NO:111) - FTFSSYSMN or CDR1 (SEQ ID NO: 192) - SYSMN CDR2 (SEQ ID NO: 112) - SISSSSSYIYYADSVKG CDR3 (not no Kabat) (SEQ ID NO:593) ARGAPFGAAAGWFDP or CDR3 (SEQ ID NO:594) - GAPFGAAAGWFDP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK [SEQ ID NO:88] CDR1: RASQGISSWLA [SEQ ID NO: 114] CDR2: AASSLQS [SEQ ID NO: 115] CDR3: QQGVSFPRT [SEQ ID NO: 116] A49MV EVQLVESGGGLVKPGGSLRLSCAASGF TFSSYSMNWVRQAPGKGLEWVSSISSS SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPVGAAA GWFDPWGQGTLVTVS S (SEQ ID NO:595) CDR1 (not no Kabat) (SEQ ID NO: 111) FTFSSYSMN or CDR1 (SEQ ID NO: 192)- SYSMN CDR2 (SEQ ID NO: 112) - SISSSSSYIYYADSVKG CDR3 (not no Kabat) (SEQ ID NO:596) ARGAPVGAAAGWFDP or CDR3 (SEQ ID NO:597) - GAPVGAAAGWFDP DIQMTQSPSSVSASVGDRVTITC RASQGISSWLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK [SEQ ID NO:88] CDR1: RASQGISSWLA [SEQ ID NO: 114] CDR2: AASSLQS [SEQ ID NO: 115] CDR3: QQGVSFPRT [SEQ ID NO: 116] A49consensus oral EVQLVESGGGLVKPGGSLRLSCAASGF TFS S YS MN W VRQ APGKGLEW VS S IS S S SSYIYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGAPXGAAA GWFDPWGQGTLVTVSS where X represents M, L, I, V, Q or F (SEQIDNO:91) DIQMTQSPSSVSASVGDRVTITC RAS QGIS S WLAWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQ GVSFPRTFGGGTKVEIK [SEQ ID NO:88] CDR1: RASQGISSWLA

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CDR1 (not no Kabat) (SEQ ID NO:111) FTFSSYSMN or CDR1 (SEQ ID NO:192) - SYSMN CDR2 (SEQ ID NO:112) - SISSSSSYIYYADSVKG CDR3 (not no Kabat) (SEQ ID NO:92) ARGAPXGAAAGWFDP or CDR3 (SEQ ID NO:123) - GAPXGAAAGWFDP, where X is M, L, I, V, Q or F [SEQ ID NO: 114] CDR2: AASSLQS [SEQ ID NO: 115] CDR3: QQGVSFPRT [SEQ ID NO: 116] ADI- 29378 (E78) QVQLVQSGAEVKKPGASVKVSCKASG YTFTSYYMHWVRQAPGQGLEWMGIIN PSGGSTSYAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCAREGAGFAY GMDYYYMDVWGKGTTVTVSS [SEQ ID NO:89] CDR1: YTFTSYYMH [SEQ ID NO: 117] (not no Kabat) or SYYMH [SEQ ID NO:572] CDR2: IINPSGGSTSYAQKFQG [SEQ ID NO: 118] CDR3: AREGAGFAYGMDYYYMDV [SEQ ID NO: 119] (not no Kabat) or EGAGFAYGMDYYYMDV [SEQ ID NO:573] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSGSGS GTDFTLTISSLEPEDFAVYYCQQ SDNWPFTFGGGTKVEIK [SEQ ID NO:90] CDR1: RASQSVSSYLA [SEQ ID NO: 120] CDR2: DASNRAT [SEQ ID NO: 121] CDR3: QQSDNWPFT [SEQ ID NO: 122] ADI- 27705 [SEQ ID NO: 124] CDR1: GSFSGYYWS [SEQ ID NO: 126] (not no Kabat) or GYYWS [SEQ ID NO:574] CDR2: EIDHSGSTNYNPSLKS DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YNSYPITFGGGTKVEIK [SEQ ID NO: 125]

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[SEQ ID NO: 127] CDR3: ARARGPWSFDP [SEQ ID NO: 128] (not no Kabat) or ARGPWSFDP [SEQ ID NO:575] ADI- 27724 [SEQ ID NO: 129] EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLAWYQQKPGQA PRLLIYGASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQ YGSSPITFGGGTKVEIK [SEQ ID NO: 130] ADI27740 (A40) QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:131] DIQMTQSPSTLSASVGDRVTITC RASQSIGSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YHSFYTFGGGTKVEIK [SEQ ID NO: 132] ADI- 27741 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:133] DIQMTQSPSTLSASVGDRVTITC RASQSIGSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ SNSYYTFGGGTKVEIK [SEQ ID NO: 134] ADI- 27743 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:135] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YNSYPTFGGGTKVEIK [SEQ ID NO: 136] ADI- 28153 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWGFDP WGQGTLVTVSS [SEQ ID NO: 137] ELQMTQSPSSLSASVGDRVTITC RTSQSISSYLNWYQQKPGQPPK LLIYWASTRESGVPDRFSGSGSG TDFTLTISSLQPEDSATYYCQQS YDIPYTFGQGTKLEIK [SEQ ID NO: 138] ADI- QVQLQQWGAGLLKPSETLSLTCAVYG DIQMTQSPSTLSASVGDRVTITC

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28226 (C26) GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO: 139] RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YGSFPITFGGGTKVEIK [SEQ ID NO: 140] ADI- 28154 [SEQ ID NO: 141] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTDFTLTISSLQPDDFATYYCQQ SKEVPWTFGQGTKVEIK [SEQ ID NO: 142] ADI- 29399 [SEQ ID NO: 143] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YNSFPTFGGGTKVEIK [SEQ ID NO: 144] ADI- 29401 [SEQ ID NO: 145] DIQMTQSPSTLSASVGDRVTITC RASQSIGSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YDIYPTFGGGTKVEIK [SEQ ID NO: 146] ADI- 29403 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO: 147] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YDSYPTFGGGTKVEIK [SEQ ID NO: 148] ADI- 29405 [SEQ ID NO: 149] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YGSFPTFGGGTKVEIK [SEQ ID NO: 150]

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ADI- 29407 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:151] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YQSFPTFGGGTKVEIK [SEQ ID NO: 152] ADI- 29419 [SEQ ID NO: 153] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YSSFSTFGGGTKVEIK [SEQ ID NO: 154] ADI- 29421 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:155] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YESYSTFGGGTKVEIK [SEQ ID NO: 156] ADI- 29424 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:157] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YDSFITFGGGTKVEIK [SEQ ID NO: 158] ADI- 29425 [SEQ ID NO: 159] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YQSYPTFGGGTKVEIK [SEQ ID NO: 160] ADI- 29426 QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS DIQMTQSPSTLSASVGDRVTITC RASQSIGSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YHSFPTFGGGTKVEIK

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[SEQ ID NO: 161] [SEQ ID NO: 162] ADI- 29429 [SEQ ID NO: 163] DIQMTQSPSTLSASVGDRVTITC RASQSIGSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YELYSYTFGGGTKVEIK [SEQ ID NO: 164] ADI29447 (F47) [SEQ ID NO: 165] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTISSLQPDDFATYYCQQ YDTFITFGGGTKVEIK [SEQ ID NO: 166] ADI- 27727 QVQLVQSGAEVKKPGSSVKVSCKASG GTFSSYAISWVRQAPGQGLEWMGGIIP IFGTANYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCARGDSSIRHAY Y Y YGMD VWGQGTT VT VS S [SEQ ID NO:167] CDR1: GTFSSYAIS [SEQ ID NO: 169] (not no Kabat) or SYAIS [SEQ ID NO:576] CDR2: GIIPIFGTANYAQKFQG [SEQ ID NO: 170] CDR3: ARGDSSIRHAYYYYGMDV [SEQ ID NO: 171] (not no Kabat) or GDSSIRHAYYYYGMDV [SEQ ID NO:577] DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLAWYQQ KPGQPPKLLIYWASTRESGVPD RFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYSTPITFGGGTKVE IK [SEQ ID NO: 168] CDR1: KSSQSVLYSSNNKNYLA [SEQ ID NO: 172] CDR2: WASTRES [SEQ ID NO: 173] CDR3: QQYYSTPIT [SEQ ID NO: 174] ADI- 29443 (F43) QLQLQESGPGLVKPSETLSLTCTVSGG SISSSSYYWGWIRQPPGKGLEWIGSIYY S GS T Y YNPS LKSR VTIS VDT S KNQFS LK LSSVTAADTAVYYCARGSDRFHPYFD YWGQGTLVTVSS [SEQ ID NO: 175] CDR1: GSISSSSYYWG [SEQ ID NO: 177] (not no Kabat) or EIVLTQSPATLSLSPGERATLSC RASQSVSRYLAWYQQKPGQAP RLLIYD AS NRATGIP ARFS GS GS GTDFTLTISSLEPEDFAVYYCQQ FDTWPPTFGGGTKVEIK [SEQ ID NO: 176] CDR1: RASQSVSRYLA [SEQ ID NO: 180]

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SSSYYWG [SEQ ID NO:578] CDR2: SIYYSGSTYYNPSLKS [SEQ ID NO: 178] CDR3: ARGSDRFHPYFDY [SEQ ID NO: 179] (not no Kabat) or GSDRFHPYFDY [SEQ ID NO:579] CDR2: DASNRAT [SEQ ID NO:581] CDR3: QQFDTWPPT [SEQ ID NO:582] ADI29404 (F04) QVQLQQWGAGLLKPSETLSLTCAVYG GSFSGYYWSWIRQPPGKGLEWIGEIDH SGSTNYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCARARGPWSFDPW GQGTLVTVSS [SEQ ID NO:583] DIQMTQSPSTLSASVGDRVTITC RASQSISSWLAWYQQKPGKAP KLLIYKASSLESGVPSRFSGSGS GTEFTLTIS S LQPDDFAT YYCEQ YDSYPTFGGGTKVEIK [SEQ ID NO:584] ADI- 28200 QVQLVQSGAEVKKPGSSVKVSCKASG GTFSSYAISWVRQAPGQGLEWMGGIIP IFGTANYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYYCARRGRKASGS FYYYYGMDVWGQGTTVTVSS [SEQ ID NO:585] DIVMTQSPDSLAVSLGERATINC ESSQSLLNSGNQKNYLTWYQQ KPGQPPKPLIYWASTRESGVPD RFSGSGSGTDFTLTISSLQAEDV AVYYCQNDYSYPYTFGQGTKL EIK [SEQ ID NO:580]

The antibody molecule may have a heavy chain constant region selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4 (e.g., human). In another embodiment, the antibody molecule has a light chain constant region selected from, for example, the kappa or lambda light chain constant regions (e.g., human). The constant region may be altered, for example, mutated, to alter the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function and/or complement function). In one embodiment, the antibody has an effector function and can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has reduced or no ability to bind to an Fc receptor. For example, an isotype or subtype, fragment, or other mutant does not support binding to an Fc receptor, such as having a mutagenized or deleted Fc receptor binding region.

Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, interaction with CD16 is primarily focused on the amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406(6793):267-273). Mutations to increase or decrease binding affinity for CD16 can be selected based on known domains, for example, using phage display libraries or yeast surface-displayed cDNA libraries, or can be designed based on the known three-dimensional interaction structure.

In certain embodiments, the constant domain of the antibody comprises a CH2 domain and a CH3 domain of an IgG, such as a human IgG1 antibody. In some embodiments, mutations are introduced into the constant domain of the antibody to allow heterodimerization with another constant domain of the antibody. For example, if the constant domain of the antibody is derived from the constant domain of a human IgG1, the constant domain of the antibody may comprise an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to amino acids 234-332 of a human IgG1 antibody and differs at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439. All amino acid positions in the Fc domain or hinge region described herein are numbered according to EU numbering.

Assembly of the heavy chains of a heterodimeric antibody can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which can result in the assembly of homodimers of each antibody heavy chain as well as the assembly of heterodimers. Facilitation of preferential assembly of heterodimers can be achieved by incorporating different mutations into the CH3 domain of each antibody heavy chain constant region, as shown in U.S. Patent No. 13/494,870, ...

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16/028850, US 11/533709, US 12/875015, US 13/289934, US 14/773418, US 12/811207, US 13/866756, US 14/647480 and US 14/830336. For example, mutations can be made in the CH3 domain based on human IgG1 and include separate pairs of amino acid substitutions in the first polypeptide and the second polypeptide, which allows the two chains to selectively heterodimerize with each other. All amino acid substitution positions illustrated below are numbered according to the EU index according to Kabat.

In one scenario, an amino acid substitution in a first polypeptide replaces an original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in a second polypeptide replaces an original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T), or valine (V) such that the larger amino acid substitution (bump) is placed on the surface of the smaller amino acid substitutions (cavity). For example, one polypeptide may include a T366W substitution, and another may include three substitutions, including T366S, L368A, and Y407V.

The heavy chain variable domain of an antibody of the present invention may optionally be linked to an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 99%, or 100%) identical to an antibody constant region, such as an IgG constant region, including the hinge domain, CH2, and CH3 domains, with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a constant region of a human antibody, such as a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a constant region of an antibody of another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be included in a constant region compared to the constant region of human IgG1, such as Q347, Y349, L351, S354, E356, b357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and/or K439. Illustrative replacements include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, B356K, E357Q, E357L, E357W, K36OE, K360W, Q362E, S364K,

S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D,

K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392B, T394F, T394W, D399R, D399K,

D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D,

T411E, K439D and K439B.

In certain embodiments, mutations that can be included in the CH1 constant region of human IgG1 can be at amino acid residues V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In certain embodiments, mutations that can be included in the Ck constant region of human IgG1 can be at amino acid residues B123, F116, S176, V163, S174, and/or T164.

Alternatively, amino acid substitutions can be selected from the following sets of substitutions shown in Table 3.

Table 3 The first polypeptide The second polypeptide Set 1 S364E/F405A Y349K/T394F Set 2 S364H/D401K Y349T/T411E

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Table 3 The first polypeptide The second polypeptide Set 3 S364H/T394F Y349T/F405A Set 4 S364E/T394F Y349K/F405A Set 5 S364E/T411E Y349K/D401K Set 6 S364D/T394F Y349K/F405A Set 7 S364H/F405A Y349T/T394F Set 8 S364K/E357Q L368D/K370S Set 9 L368D/K370S S364K Set 10 L368E/K370S S364K Set 11 K360E/Q362E D401K Set 12 L368D/K370S S364K/E357L Set 13 K370S S364K/E357Q Set 14 F405L K409R Set 15 K409R F405L

Alternatively, amino acid substitutions can be selected from the following sets of substitutions shown in Table 4:

Table 4 The first polypeptide The second polypeptide Set 1 K409W D399V/F405T Set 2 Y349S E357W Set 3 K360E Q347R Set 4 K360E/K409W Q347R/D399V/F405T Set 5 Q347E/K360E/K409W Q347R/D399V/F405T Set 6 Y349S/K409W E357W/D399V/F405T

Alternatively, amino acid substitutions can be selected from the following sets of substitutions shown in Table 5:

Table 5 The first polypeptide The second polypeptide Set 1 T366K/L351K L351D/L368E Set 2 T366K/L351K L351D/Y349E Set 3 T366K/L351K L351D/Y349D Set 4 T366K/L351K L351D/Y349E/L368E Set 5 T366K/L351K L351D/Y349D/L368E Set 6 E356K/D399K K392D/K409D

Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6.

Table 6 The first polypeptide The second polypeptide L351Y, D399R, D399K, S400K, S400R, Y407A, Y407I, Y407V T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392M, K392V, K392F K392D, K392E, K409F, K409W, T411D and T411E

Alternatively, at least one amino acid substitution may be selected from the following set of substitutions shown in Table 7, wherein the position(s) shown in the First Polypeptide column are replaced with any known negatively charged amino acid, and the position(s) shown in the Second Polypeptide column are replaced with any known positively charged amino acid.

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Table 7 The first polypeptide Second polypeptide 5 KZ 92, KZ 70, K409 or K439 D399, E356 or E357

Alternatively, at least one amino acid substitution may be selected from the following set of substitutions shown in Table 8, wherein the position(s) listed in the First Polypeptide column are replaced with any known positively charged amino acid, and the position(s) listed in the Second Polypeptide column are replaced with any known negatively charged amino acid.

Table 8 The first polypeptide The second polypeptide D399, E356 or E357 K409, K439, KZ 70 or KZ 92

Alternatively, amino acid substitutions can be selected from the following set of substitutions shown in Table 9.

Table 9 The first polypeptide The second polypeptide T350V, L351Y, F405A or Y407V T35OV, T366L, K392L or T394W

Alternatively or in addition, the structural stability of heterodimeric heavy chains within multispecific binding proteins can be enhanced by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain, which forms an artificial disulfide bridge at the interface of the two polypeptides.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at position T366, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of T366, L368, and Y407.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of T366, L368 and Y407, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at position T366.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411; and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405, and T411; and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of L351, D399, S400 and Y407, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of T366, N390, K392, K409, and T411, and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of L351, D399, S400, and

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Y407.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Q347, Y349, K360 and K409, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Q347, E357, D399 and F405.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Q347, E357, D399 and F405, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Y349, K360, Q347 and K409.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of K370, K392, K409 and K439, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of D356, E357 and D399.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of D356, E357, and D399, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of K370, K392, K409, and K439.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of L351, E356, T366 and D399, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 at one or more positions selected from the group consisting of L351, E356, T366 and D399.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by a S354C substitution, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by a Y349C substitution.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by a Y349C substitution, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by a S354C substitution.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions K36OE and K409W, and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions Q347R, D399V, and F405T.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions O347R, D399V, and F405T, and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions K36OE and K409W.

In certain embodiments of the present disclosure, the amino acid sequence

- 152 049630 the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by T366W substitutions, and the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by T366S, T368A and Y407V substitutions.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions T366S, T368A, and Y407V, and wherein the amino acid sequence of another polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitution T366W.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions T350V, L351Y, F405A, and Y407V, and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions T350V, T366L, K392L, and T394W.

In certain embodiments of the present disclosure, the amino acid sequence of one polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions T350V, T366L, K392L, and T394W, and wherein the amino acid sequence of the other polypeptide chain of the constant region of the antibody differs from the amino acid sequence of the constant region of IgG1 by the substitutions T350V, L351Y, F405A, and Y407V.

The following are examples of CD33-binding F3'-TriNKETs comprising: a CD33-binding single-chain variable fragment (scFv) linked to an Fc domain through an Ala-Ser hinge; and an NKG2D-binding Fab fragment (A49, A49MI, A49MQ, A49ML, A49MF, or A49MV) comprising a heavy chain portion comprising a heavy chain variable domain (SEQ ID NO: 87 or SEQ ID NO: 191) and a CH1 domain, and a light chain portion comprising a light chain variable domain (SEQ ID NO: 88) and a light chain constant domain, wherein the heavy chain variable domain is linked to the CH1 domain and the CH1 domain is linked to the Fc domain. CDR sequences are underlined.

In each example, the Fc domain associated with the CD33-binding scFv comprises the substitutions Q347R, D399V, and F405T to form a heterodimer with the Fc domain associated with the Fab comprising the substitutions K36OE and K409W. These substitutions in the Fc domains are shown in bold and underlined in the sequences described below. Alternatively, in an exemplary embodiment, the Fc domain associated with the NKG2D-binding Fab fragment comprises the mutations Q347R, D399V, and F405T, and the Fc domain associated with the CD33-binding scFv comprises the corresponding mutations K36OE and K409W to form a heterodimer.

In addition, the Fc domain associated with the CD33-binding scFv contains a S354C substitution, and the Fc domain associated with the Fab contains a Y349C substitution, thereby stabilizing the interaction between the two Fc domains via an S-S bridge. These substitutions in the Fc domains are shown in bold, italics, and underlined in the sequences below.

Alternatively, in an exemplary embodiment, the Fc domain associated with the NKG2D-binding Fab fragment comprises a S354C substitution in the CH3 domain that forms a disulfide bond with a Y349C substitution on the Fc associated with the CD33-binding scFv.

A CD33-binding scFv of the present invention may comprise a heavy chain variable domain linked to a light chain variable domain by a (G4S)4 linker. The scFv is linked to the Fc domain via a hinge comprising Ala-Ser (bold and underlined). SEQ ID NOs: 188, 198, and 206-223 are exemplary sequences of such CD33-binding scFv polypeptides. The V _L and V H contained in the scFv (e.g., SEQ ID NOs: 188, 198, or 206-223) comprise a SS bridge of 100VL-44VH (resulting from G100C and G44C substitutions, respectively) (cysteine residues are shown in bold italics and underlined in the sequences below). (G4S) ₄ is the bolded sequence GGGGSGGGGSGGGGSGGGGS [SEQ ID NO: 186] in SEQ ID NOs: 188, 198, and 206-243.

Illustrative sequences of CD33-binding scFvs linked to the Fc domain via an Ala-Ser containing hinge are shown below.

- 153 049630 scFv (LC-HC)-FcAbi (replaces Q347R, D399V, F405T and S354C)

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFGCGTKVEIKGGGGSGGGGS GGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKCLEW VANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYD SSGYFVYYGMDVWGQGTTVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG [SEQ ID NO:224] scFv (HC-LC)-FcAbi (replacements Q347R, D399V, F405T and S354C)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKCLEWVANIKQ DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFV YYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRV TITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPD DFATYYCQQYESFPTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG [SEQ ID NO:225] scFv (LC-HC)-Fc Ab2 (replacements Q347R, D399V, F405Tu S354C)

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLES GVPSRFS GS GS GTEFTLTISSLQPDDFAT YYCQQLESYPLTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSEVOLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVROAPGKCLE WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAG ELDVWGQGTMVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALP APIEKTIS KAKGQPREPRVYTLPPCRDELTKNQVS LTCLVKGFYPSDIA V EWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQGNVFSCSVMHEALHNHY TQKSLSLSPG [SEQ ID NO:226] scFv (HC-LC)-Fc Ab2 (replacements Q347R, D399V, F405Tu S354C)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEWVANIKQ DGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSSGGGGSGGGGSGGGGSGGGGSDIOMTQSPSTLSASVGDRVTITCRASOSIS SWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQ LESYPLTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALP APIEKTIS KAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG [SEQ ID NO:227] scFv (LC-HC)-Fc H76 (replacements Q347R, D399V, F405Tu S354C)

DIOMTQSPSTLSASVGDRVTITCRASOSISSWLAWYOQKPGKAPKLLIYKASSLES GVPSRFS GS GS GTEFTLTISSLQPDDFAT YYCOOYDDLPTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFFSKYTMSWVRQAPGKCLE WVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYY DSSGYFVYYGMDVWGOGTTVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKE YKCKVSNKALP APIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG [SEQ ID NO: 197] scFv (HC-LC)-Fc H76 (replacements Q347R, D399V, F405Tu S354C)

EVQLLESGGGLVQPGGSLRLSCAASGTFFSKYTMSWVRQAPGKCLEWVSAIVGS GESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVY YGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVT ITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPD DFATYYCQQYDDLPTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

- 154 049630

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQGNVFSCSV MHEALHNHYTQKSLSLSPG [SEQ ID NO:228] scFc (LC-HC)-/c H76 (replacements K360E, K409Wu Y349C)

DIOMTQSPSTLSASVGDRVTTITCRASOSISSWLAWYQQKPGKAPKLLIYKASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFFSKYTMSWVRQAPGKCLE WVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYY DSSGYFVYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [SEQ ID NO:243] scFv (LC-HC)-Fc Ab4 (replacements Q347R, D399V, F405T and S354C)

DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLG SNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGCGTKVEIKGGG GSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVR QAPGQCLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYY CAREAADGFVGERYFDLWGRGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG [SEQ ID NO:229] scFv (HC-LC)-Fc AM (replaces Q347R, D399V, F405T and S354C)

QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQCLEWMGMI NPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERY FDLWGRGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCR SSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQDVALPITFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG [SEQ ID NO:230] scFv (LC-HC)-/c 107 (replaces Q347R, D399V, F405T and S354C)

DIOMTQSPSTLSASVGDRVTITCRASOSISSWLAWYOQKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKC

- 155 049630

LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNA GELDVWGQGTMVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG [SEQ ID NO: 187] scFv (HC-LC)-/ c 107 (replacements Q347R, D399V, F405Ti S354C)

EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKCLEWVATIKQ DGSEKSYVDSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSIS SWLAWYQOKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOQ SOSYPPITFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG [SEQ ID NO:231] scFc (LC-HC)-Fc 107 (replacements K360E, K409Wu Y349C)

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLOPDDFATYYCOOSQSYPPITFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKC LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNA GELDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [SEQ ID NO:242] scFv (LC-HC)-Fc Ab6 (replacements Q347R, D399V, F405Tu S354C)

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKCL EWVATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAG ELDVWGQGTMVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG [SEQ ID NO:232] scFv (HC-LC)-/ c Ab6 (Q347R replacements, D399V, F405T and S354C)

EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKCLEWVATIKR

- 156 049630

DGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSIS SWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQ SQSYPPITFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG [SEQ ID NO:233] scFv (LC-HC)-Fc Ab7 (replacements Q347R, D399V, F405T and S354C)

DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQ CLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAG YDDEDMDVWGKGTTVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG [SEQ ID NO:234] scFv (HC-LC)-/ c Ab7 (replacements Q347R, D399V, F405T and S354C)

QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQCLEWMGIIN PSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDV WGKGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRAS QGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQAHSYPLTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG [SEQ ID NO:235] scFv (LC-HC)-Fc Ab8 (replacements Q347R, D399V, F405T and S354C)

DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKCLE WVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYD S S GYFV YYGMD VWGQGTTVT VS S ASDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG [SEQ ID NO:236] scFv (HC-LC)-Fc Ab8 (replacements Q347R, D399V, F405T and S354C)

EVQLVESGGGLVKPGGSLRLSCAASGFFSSYAMSWVRQAPGKCLEWVSSISSSS

- 157 049630

EGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYY GMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTI TCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDD FATYYCQQYDDLPTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG [SEQ ID NO:237] scFv (LC-HC)-Fc Ab9 (replacements Q347R, D399V, F405T and S354C)

DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLK SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFFSSYWMSWVRQAPGKCL EWVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGP GIAYQGHFDYWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG [SEQ ID NO:238] scFv (HC-LC)-Fc Ab9 (replacements Q347R, D399V, F405T and S354C)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKCLEWVANINT DGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFD YWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRA SQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQVYDTPLTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG [SEQ ID NO:239] scFv (LC-HC)-Fc AblO (replacements Q347R, D399V, F405T and S354C)

EIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRA TGIPARFSGSGSGTDFTISSLEPEDFAVYYCQQYDNLPTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKCLE WIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGM DVWGQGTTVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG [SEQ ID NO:240] scFv (HC-LC)-Fc AblO (Q347R replacements, D399V, F405T and S354C)

QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKCLEWIGSIGYS GTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQG TTVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASHSVYS YLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQY DNLPTFGCGTKVEIKASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTIS KAKGQPREPRVYTLPPCRDELTKNQVS LTCLVKGF YPSDIA VE WESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG [SEQ ID NO:241]

The TriNKET of the present disclosure is A49-F3'-TriNKET-107 comprising a first polypeptide chain, referred to as scFv-Fc 107, which comprises a CD33-binding scFv linked to an Fc domain via an Ala-Ser linker; a second polypeptide chain, referred to as VH-CH1-Fc A49, which comprises an NKG2D-targeted heavy chain; and a third polypeptide chain, referred to as VL-CL A49, which comprises a NKG2D-targeted light chain. The amino acid sequence of scFv-Fc 107 comprises:

- 158 049630 scFv (LC-HC)-Fc 107 (replacements Q347R, D399V, F405T and S354C)

DIQMTQSPSTLSASVGDRVTITCRASOSISSWLAWYQQKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKC LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNA GELDVWGOGTMVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG

[SEQ ID NO:187]

The amino acid sequence of the scFv portion of scFv-Fc 107 contains

107 scF v

DIOMTOSPSTLSASVGDRVTITCRASQSISSWLAWYOOKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOOSOSYPPITFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVOLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVROAPGKC LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLOMNSLRAEDTAVYYCARPLNA GELDVWGOGTMVTVSS

[SEQ ID NO: 188]

VH-CH1-Fc A49 comprises VH A49 [SEQ ID NO: 87] linked to the CH1 domain and the Fc domain (including the hinge region, CH2 and CH3 domains). The amino acid sequence of VH-CH1-Fc A49 comprises

VH-CH 1-Fc A49 (replacements K360E, K409W and Y349C)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVSSISSSS SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[SEQ ID NO: 189]

VH-CHl-Fc A49 (replacements for Q347R, D399V, F405T and S354C)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSS SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS L [SEQ ID NO:244]

VL-CL A49 comprises VL A49 [SEQ ID NO: 88] linked to the light chain constant domain (CL). The amino acid sequence of VL-CL A49 comprises:

VL-CLA49

DIOMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLO SGVPSRFSGSGSGTDFTISSLOPEDFATYYCOOGVSFPRTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLS KAD YEKHKVYACE VTHQGLS SPVTKSFNRGEC

[SEQ ID NO:190]

Another TriNKET of the present disclosure is A49MI-F3'-TriNKET-107, comprising a first polypeptide chain referred to as scFv-Fc 107, as described above [SEQ ID NO: 187]; a second polypeptide chain referred to as VH-CH1-Fc A49MI, which comprises an NKG2D-targeting heavy chain with an Fc domain; and a third polypeptide chain referred to as VL-CL A49, as described above [SEQ ID NO: 190]. A49MI-F3'-TriNKET-107 is identical to A49-F3'-TriNKET-107, except for an I to M substitution in CDR3 of the NKG2D-targeting VH. This substitution is shown in bold and italics (and underlined because it is part of the CDRs) in the sequence below. VH-CH1-Fc A49MI comprises VH A49MI [SEQ ID NO: 191] linked to the CH1 domain and the Fc domain (including the hinge region, CH2 and CH3 domains). The amino acid sequence of VH-CH1-Fc A49MI comprises

- 159 049630

VH-CHl-Fc A49MI (replaces K360E, K409W« Y349C)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVSSISSSS SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[SEQ ID NO:196]

VH-CHl-Fc A49MI (replaces Q347R. D399V, F405T and S354C)

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWVSSISSSS SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWG QGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL [SEQ ID NO:245]

Another TriNKET of the present disclosure is A49-F3'-TriNKET-107(si) comprising a first polypeptide chain, referred to as scFv-Fc(si)107, which comprises a CD33-binding scFv linked to a silent Fc domain via an Ala-Ser linker; a second polypeptide chain, referred to as VH-CH1-Fc(si)A49, which comprises an NKG2D-targeting heavy chain with a silent Fc domain; and a third polypeptide chain, referred to as VL-CL A49, as described above [SEQ ID NO: 190]. A49-F3'-TriNKET-107(si) is identical to A49-F3'-TriNKET-107 except for the L234A, L235A, and P329A substitutions in both Fc domains. These substitutions are shown in bold and italics in the two sequences below. The amino acid sequence of scFv-Fc(si) 107 contains scFv-Fc 107

DIQMTQSPSTLSASVGDRVTITCRASOSISSWLAWYQQKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOOSOSYPPITFGCGTKVEIKGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKC LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNA GELDVWGQGTMVTVSSASDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALGAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG

[SEQ ID NO:204]

The amino acid sequence of the scFv portion of scFv-Fc(si) 107 is identical to the amino acid sequence of the scFv portion of scFv-Fc 107 as described above [SEQ ID NO: 188].

The amino acid sequence of VH-CH1-Fc(si) A49 contains

VH-CHl-Fc(si) A49

EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSS SYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP REPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [SEQ ID NO:205]

Another TriNKET of the present disclosure is A49-F3'-TriNKET-H76 comprising a first polypeptide chain referred to as scFv-Fc H76, which comprises a CD33-binding scFv linked to an Fc domain via an Ala-Ser linker; a second polypeptide chain referred to as VHCH1-Fc A49, as described above [SEQ ID NO: 189]; and a third polypeptide chain referred to as VL-CL A49, as described above [SEQ ID NO: 190]. The amino acid sequence of scFv-Fc H76 comprises

- 160 049630 scFv-Fc H76

DIQMTQSPSTLSASVGDRVTTITCRASOSISSWLAWYQQKPGKAPKLLIYKASSLES

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCOQYDDLPTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFFSKYTMSWVRQAPGKCLE WVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYY DS S GYFVYYGMD VWGOGTTVT VS ASDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG

[SEQ ID NO:197]

The amino acid sequence of the scFv portion of scFv-Fc H76 contains

H76 scFv

DIQMTQSPSTLSASVGDRVTTITCRASOSISSWLAWYOQKPGKAPKLLIYKASSLES

GVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGCGTKVEIKGGGGSGGGG SGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGTFFSKYTMSWVRQAPGKCLE WVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYY DS S GYFV YYGMD VWGOGTTVT VS S

[SEQ ID NO: 198]

In other embodiments of the present disclosure, a CD33-binding scFv of a multispecific binding protein (e.g., TriNKET) comprises CDR1, CDR2, and CDR3 of a heavy chain variable domain and CDR1, CDR2, and CDR3 of a light chain variable domain of any of the antibodies shown in Table 1. In certain embodiments, the amino acid sequence of the heavy chain variable domain is identical to the VH sequence of the antibody in Table 1, except for a Cys substitution at position 44, and the amino acid sequence of the light chain variable domain is identical to the VL sequence of the same antibody, except for a Cys substitution at position 100. In certain embodiments, the heavy chain variable domain is connected to the light chain variable domain by a linker (G4S) ₄ . The heavy chain variable domain may be N-terminal to the light chain variable domain or C-terminal to the light chain variable domain. ScFv is linked to the Fc domain via an Ala-Ser containing hinge.

The multispecific binding proteins described above can be produced using recombinant DNA technology well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain can be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain can be cloned into a second expression vector; a third nucleic acid sequence encoding a first immunoglobulin light chain can be cloned into a third expression vector; a fourth nucleic acid sequence encoding a second immunoglobulin light chain can be cloned into a fourth expression vector; the first, second, third and fourth expression vectors can be stably transfected together into host cells to produce multimeric proteins.

To achieve the highest yield of multispecific binding proteins, various ratios of the first, second, third and fourth expression vectors can be tested to determine the optimal ratio for transfection into host cells. Following transfection, individual clones can be isolated to create a cell bank using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy or Clonepix.

The clones can be cultured under conditions suitable for bioreactor scale-up and maintenance of expression of the multispecific protein. The multispecific binding proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

In certain embodiments, the antibody binds CD33 with a K _D of 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or lower, as measured using standard binding assays, such as surface plasmon resonance or biolayer interferometry. In certain embodiments, the antibody binds EB13 from a subject's body fluid, tissue, and/or cell.

Competitive assays are known in the art for determining whether an antibody binds to or competes for binding to the same epitope as a disclosed antibody, e.g., Ab1 antibody, Ab2 antibody, Ab3 antibody, Ab4 antibody, or Ab5 antibody. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., BIAcore assay), biolayer interferometry, and flow cytometry.

- 161 049630 metry.

Typically, a competitive assay involves the use of an antigen (e.g., human CD33 protein or fragment thereof) bound to a solid surface or expressed on the surface of a cell, a CD33-binding antibody to be tested, and a reference antibody. The reference antibody is labeled and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1x, 5x, 10x, 20x, or 100x). Antibodies identified in a competition assay (e.g., competing antibodies) include antibodies that bind to the same epitope or similar (e.g., overlapping) epitopes as the reference antibody, and antibodies that bind to an adjacent epitope that is close enough to the epitope bound by the reference antibody to cause steric hindrance.

A competition assay can be performed in both directions to ensure that the presence of the label does not interfere with or otherwise inhibit binding. For example, in the first direction, the reference antibody is labeled and the test antibody is unlabeled, and in the second direction, the test antibody is labeled and the reference antibody is unlabeled.

A test antibody competes with a reference antibody for specific binding to an antigen if an excess of one antibody (e.g., 1x, 5x, 10x, 20x, or 100x) inhibits binding of the other antibody by, e.g., at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay.

Two antibodies may be defined as binding to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies may be defined as binding to overlapping epitopes if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

The antibodies described herein may be further optimized (e.g., affinity matured) to improve biochemical characteristics, including affinity and/or specificity, improve biophysical properties, including aggregation, stability, precipitation, and/or non-specific interactions, and/or reduce immunogenicity. Affinity maturation procedures are known to those skilled in the art. For example, diversity may be introduced into an immunoglobulin heavy chain and/or an immunoglobulin light chain by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis, and/or site-directed mutagenesis.

In certain embodiments, the isolated human antibodies contain one or more somatic mutations. In these cases, the antibodies may be modified to a human germline sequence to optimize the antibody (e.g., through a process called germline gene modification).

Typically, an optimized antibody has at least the same or substantially the same affinity for an antigen as the non-optimized (or parent) antibody from which it was derived. Preferably, an optimized antibody has a higher affinity for an antigen when compared to the parent antibody.

If the antibody is intended for use as a therapeutic agent, it can be conjugated to an effector agent, such as a small molecule toxin or a radionuclide, using standard in vitro conjugation chemistry. If the effector agent is a polypeptide, the antibody can be chemically conjugated to the effector or attached to the effector as a fusion protein. The construction of fusion proteins is within the skill of the art.

The antibody may be conjugated to an effector moiety, such as a small molecule toxin or a radionuclide, using standard in vitro conjugation chemistry. If the effector moiety is a polypeptide, the antibody may be chemically conjugated to the effector or attached to the effector as a fusion protein. The construction of fusion proteins is within the skill of the art.

In certain embodiments, a protein (e.g., a multispecific binding protein) of the present disclosure is substantially not internalized by a CD33-expressing cell. A low level of internalization can improve the pharmacokinetics of the protein, thereby reducing the dose required for interaction of CD33-expressing target cells with effector cells (e.g., NK cells). Internalization can be measured by any method known in the art, such as the methods described in Examples 5 and 10 of the present disclosure. For example, in certain embodiments, internalization of the protein (e.g., a multispecific binding protein) by EOL-1 cells is less than 25%, 30%, 35%, 40%, 45%, or 50% after a two-hour incubation, as assessed by the methods described herein. In certain embodiments, internalization of a protein (e.g., a multispecific binding protein) by EOL-1 cells is less than 25%, 30%, 35%, 40%, 45%, or 50% after 24 hour incubation, as assessed

- 162 049630 by the methods described herein. In certain embodiments, internalization of a protein (e.g., a multispecific binding protein) by Molm-13 cells is less than 25%, 30%, 35%, 40%, 45%, or 50% after a two-hour incubation, as assessed by the methods described herein.

KHYG-1 cells express surface NKG2D but do not express CD16. In certain embodiments, TriNKET-mediated killing of Molm-13 and THP-1 cells is dependent on NKG2D-mediated activation of KHYG-1 effector cells. In certain embodiments, TriNKETs of the present invention mediate killing of Molm-13 by KHYG-1 (Figure 15A), EOL-1 (Figure 16), and THP-1 (Figure 17A) human AML target cell lines.

In certain embodiments, TriNKETs of the present disclosure mediate cytotoxicity of resting human NK cells against human Molm-13 or THP-1 AML cells. FIGS. 15B and 38A show that TriNKETs of the present invention mediate killing of human Molm-13 AML cells by resting human NK cells. The ratio of resting human effector NK cells (E) to target cells (T) (E:T) was 10:1 in FIG. 15B and 5:1 in FIG. 38A. The E:T ratio may reflect differences in maximum % lysis.

In certain embodiments, TriNKETs of the present disclosure mediate the killing of human EOL-1 AML cells by resting human NK cells. Fig. 38B shows that TriNKETs mediate the killing of human EOL-1 cells by resting human NK cells with an E:T of 5:1. In certain embodiments, TriNKETs of the present disclosure mediate the killing of THP-1 target cells that express high affinity FcγRI by resting human NK cells. Figs. 17B, 38C, and 38D show that TriNKETs mediate the killing of human THP-1 AML cells by resting human NK cells using an E:T of 5:1.

In certain embodiments, TriNKETs of the present disclosure mediate killing of Molm-13 target cells by CD8+ T cells. Figs. 40A-40B show that TriNKETs mediate killing of Molm-13 cells by human CD8+ T cells at an E:T of 50:1.

A protein containing an antigen-binding site that competes with the CD33-binding sites described herein

In one aspect, the present invention provides a protein that comprises an antigen-binding site that competes with the CD33-binding sites described herein for binding to CD33. In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 1 and an antibody light chain having the amino acid sequence of SEQ ID NO: 2.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 3 and an antibody light chain having the amino acid sequence of SEQ ID NO: 4.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 5 and an antibody light chain having the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 7 and an antibody light chain having the amino acid sequence of SEQ ID NO: 8.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 9 and an antibody light chain having the amino acid sequence of SEQ ID NO: 10.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 11 and an antibody light chain having the amino acid sequence of SEQ ID NO: 12.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having an amino acid sequence

- 163 049630 the body of SEQ ID NO: 13, and a light chain of an antibody having the amino acid sequence of SEQ ID NO: 14.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 15 and an antibody light chain having the amino acid sequence of SEQ ID NO: 16.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 17 and an antibody light chain having the amino acid sequence of SEQ ID NO: 18.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody heavy chain having the amino acid sequence of SEQ ID NO: 19 and an antibody light chain having the amino acid sequence of SEQ ID NO: 20.

In certain embodiments, the antigen-binding site of the protein that competes with CD33 binding sites comprises a heavy chain variable domain having an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identical to the amino acid sequence of SEQ ID NO: 1. or 100%) identical to SEQ ID NO: 2. In certain embodiments, the antigen-binding site of the protein that competes with CD33 binding sites comprises a heavy chain variable domain having an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 4.

In certain embodiments, the antigen-binding site of the protein that competes with CD33 binding sites comprises a heavy chain variable domain that has an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 6.

In certain embodiments, the antigen-binding site of the protein that competes with the CD33 binding sites comprises a heavy chain variable domain that has an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain of an antibody that includes an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 8.

In certain embodiments, the antigen-binding site of the protein that competes with the CD33 binding sites comprises a heavy chain variable domain having an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 10.

In certain embodiments, the antigen-binding site of the protein that competes with CD33 binding sites comprises a heavy chain variable domain that has an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 12.

In certain embodiments, the antigen-binding site of the protein that competes with CD33 binding sites comprises a heavy chain variable domain having an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%,

- 164 049630

93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13, and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14.

In certain embodiments, the antigen-binding site of the protein that competes with the CD33 binding sites comprises a heavy chain variable domain that has an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain of an antibody that includes an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 16.

In certain embodiments, the antigen-binding site of the protein that competes with CD33 binding sites comprises a heavy chain variable domain having an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain of an antibody that comprises an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 18.

In certain embodiments, the antigen-binding site of the protein that competes with the CD33 binding sites comprises a heavy chain variable domain that has an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19, and a light chain variable domain of an antibody that includes an amino acid sequence that is at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 20.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antigen-binding site that binds a tumor-associated antigen.

In certain embodiments, the present invention provides a protein that comprises an antigen-binding site that competes for binding to human and cynomolgus monkey CD33 with an antibody that comprises an antibody constant region or a portion thereof capable of binding CD16.

CAR T Cells, CD33/CD3-Directed Bispecific T Cell Activators, Immunocytokines, Antibody-Drug Conjugates, and Immunotoxins Another aspect of the present disclosure provides a molecule or complex comprising an antigen-binding site that binds CD33 as described herein. Exemplary molecules or complexes include, but are not limited to, chimeric antigen receptors (CARs), T cell activators (e.g., CD33/CD3-directed bispecific T cell activators), immunocytokines, antibody-drug conjugates, and immunotoxins.

Any antigen binding site that binds CD33 as described herein may be used, including, but not limited to, an antigen binding site that binds CD33 as described in Section I. Antigen Binding Site. In certain embodiments, the amino acid sequence(s) of the antigen binding site that binds CD33 are provided in Table 1. In certain embodiments, the antigen binding site that binds CD33 is an scFv. In certain embodiments, the scFv comprises an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an amino acid sequence selected from SEQ ID NO: 188, SEQ ID NO: 198, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ

ID NO:210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID

NO

NO

NO

NO

NO

NO

NO

216, SEQ ID NO

222, SEQ ID NO

451, SEQ ID NO

457, SEQ ID NO

463, SEQ ID NO

469, SEQ ID NO

475, SEQ ID NO

217, SEQ ID NO

223, SEQ ID NO

452, SEQ ID NO

458, SEQ ID NO

464, SEQ ID NO

470, SEQ ID NO

476, SEQ ID NO

218, SEQ ID NO

447, SEQ ID NO

453, SEQ ID NO

459, SEQ ID NO

465, SEQ ID NO

471, SEQ ID NO

477, SEQ ID NO

219, SEQ ID NO

448, SEQ ID NO

454, SEQ ID NO

460, SEQ ID NO

466, SEQ ID NO

472, SEQ ID NO

478, SEQ ID NO

220, SEQ ID NO

449, SEQ ID NO

455, SEQ ID NO

461, SEQ ID NO

467, SEQ ID NO

473, SEQ ID NO

479, SEQ ID NO

221, SEQ ID

450, SEQ ID

456, SEQ ID

462, SEQ ID

468, SEQ ID

474, SEQ ID

480, SEQ ID

NO: 481, SEQ ID NO: 482, SEQ ID NO: 483, and SEQ ID NO: 484. In certain embodiments, the scFv comprises an amino acid sequence selected from SEQ ID NO:

SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO

SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO

SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO

208, SEQ ID NO: 209, SEQ ID NO

214, SEQ ID NO: 215, SEQ ID NO

220, SEQ ID NO: 221, SEQ ID NO

188, SEQ ID NO: 198, 210, SEQ ID NO: 211, 216, SEQ ID NO: 217, 222, SEQ ID NO: 223,

- 165 049630

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

SEQ ID NO

447, SEQ ID NO

453, SEQ ID NO

459, SEQ ID NO

465, SEQ ID NO

471, SEQ ID NO

477, SEQ ID NO

448, SEQ ID NO

454, SEQ ID NO

460, SEQ ID NO

466, SEQ ID NO

472, SEQ ID NO

478, SEQ ID NO

449, SEQ ID NO

455, SEQ ID NO

461, SEQ ID NO

467, SEQ ID NO

473, SEQ ID NO

479, SEQ ID NO

450, SEQ ID NO

456, SEQ ID NO

462, SEQ ID NO

468, SEQ ID NO

474, SEQ ID NO

480, SEQ ID NO

451, SEQ ID NO: 452,

457, SEQ ID NO: 458,

463, SEQ ID NO: 464,

469, SEQ ID NO: 470,

475, SEQ ID NO: 476,

481, SEQ ID NO: 482,

SEQ ID NO: 483 and SEQ ID NO: 484.

In certain embodiments, an antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 181, 46, and 182, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively. In certain embodiments, the antigen-binding site comprises a heavy chain variable domain having an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the antigen binding site comprises an scFv comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 188.

In other embodiments of any of the above aspects in this subsection, the antigen-binding site that binds CD33 comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 183, 34, and 184, respectively; and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences represented by the amino acid sequences of SEQ ID NOs: 36, 185, and 38, respectively. In certain embodiments, the antigen-binding site comprises a heavy chain variable domain having an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the antigen-binding site comprises an scFv comprising an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 198.

Chimeric antigen receptors (CARs)

In certain embodiments, the present invention provides a CD33-targeted CAR comprising an antigen-binding site that binds CD33 as described herein (see, e.g., Table 1). The CD33-targeted CAR may comprise a Fab fragment or an scFv.

The term chimeric antigen receptor or alternatively CAR refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule (also referred to herein as a primary signaling domain).

Accordingly, in certain embodiments, the CAR comprises an extracellular antigen-binding site that binds CD33 as described herein, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain. In certain embodiments, the CAR further comprises one or more functional signaling domains derived from at least one costimulatory molecule (also referred to as a costimulatory signaling domain).

In one embodiment, the CAR comprises a chimeric fusion protein comprising a CD33 binding domain (e.g., a CD33 binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain, as set forth in Table 1 as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain. In one embodiment, the CAR comprises a chimeric fusion protein comprising a CD33 binding domain (e.g., a CD33 binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain, as set forth in Table 1 as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a costimulatory signaling domain and a primary signaling domain. In one aspect, a CAR comprises a chimeric fusion protein comprising a CD33 binding domain (e.g., a CD33 binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain, as defined in Table 1 as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two costimulatory signaling domains and a primary signaling domain.

- 166 049630 domain. In one embodiment, the CAR comprises a chimeric fusion protein comprising a CD33 binding domain, a heavy chain variable domain, and a light chain variable domain, as listed in Table 1 as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two costimulatory signaling domains and a primary signaling domain.

With respect to the transmembrane domain, in various embodiments, the CAR is engineered to comprise a transmembrane domain that is fused to an extracellular domain of the CAR. In one embodiment, the transmembrane domain is a domain that is naturally associated with one of the domains in the CAR. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid association of such domains with transmembrane domains of the same or different surface membrane proteins in order to minimize interactions with other members of the receptor complex. In another embodiment, the transmembrane domain is capable of homodimerizing with another CAR on the surface of CAR T cells. In another embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted in a manner that minimizes interactions with the binding domains of a native binding partner present in the same CAR T cell.

The transmembrane domain may be derived from any naturally occurring membrane-bound or transmembrane protein. In one embodiment, the transmembrane region is capable of transmitting a signal to the intracellular domain(s) whenever the CAR binds to a target. In certain embodiments, the transmembrane domain comprises the transmembrane region(s) of one or more proteins selected from the group consisting of TCR α-chain, TCR β-chain, TCR ζ-chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain comprises the transmembrane region(s) of one or more proteins selected from the group consisting of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Re, IL2Ry, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D and NKG2C.

The extracellular CD33 binding domain (e.g., the CD33 binding domain of a scFv) may be linked to the transmembrane domain via a hinge region. Various hinges may be used, including, but not limited to, a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a Gly-Ser linker, a ( _G4S ) ₄ linker, a KIR2DS2 hinge, and a CD8a hinge.

The intracellular signaling domain of the CAR of the invention is responsible for activating at least one of the specialized functions of the immune cell (e.g., cytolytic activity or helper activity, including cytokine secretion, of a T cell) into which the CAR has been placed. Thus, as used herein, the term intracellular signaling domain refers to the portion of the protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain may typically be used, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces an effector function signal. Thus, the term intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

The intracellular signaling domain of a CAR comprises a primary signaling domain (i.e., a functional signaling domain derived from a stimulatory molecule) and one or more costimulatory signaling domains (i.e., functional signaling domains derived from at least one costimulatory molecule).

As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell, such as a T cell, NK cell, or B cell, that provides cytoplasmic signaling sequence(s) that regulates immune cell activation in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one embodiment, the signal is a primary signal that is initiated, for example, by binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, and that results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc.

Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAMs containing primary intracellular signaling domains that act in a stimulatory manner include:

- 167 049 630 are assigned for specific use in the present invention, include sequences derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10 and DAP12. In one embodiment, the primary signaling domain in any one or more CARs of the invention comprises a cytoplasmic signal sequence derived from CD3 zeta.

In some embodiments, the primary signaling domain is a functional signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, and/or CD3 zeta. In an embodiment, the intracellular signaling domain comprises a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and/or DAP12. In a specific embodiment, the primary signaling domain is a functional signaling domain of the zeta chain associated with the T cell receptor complex.

As used herein, the term "costimulatory molecule" refers to a recognizable binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligands, that is required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), CD2, CD7, CD258 (LIGHT), NKG2C, B7-H3, and a ligand that specifically binds to CD83, etc. Additional examples of such costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SeLPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp and a ligand that specifically binds to CD83. In some embodiments, the costimulatory signaling domain of a CAR is a functional signaling domain of a costimulatory molecule described herein, such as OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a/CD18), ICOS, and 4-1BB (CD137), or any combination thereof.

In the context of this document, the term signaling domain refers to a functional portion of a protein that acts by transmitting information within a cell, regulating cellular activity through specific signaling pathways, generating second messengers, or acting as effectors in response to such messengers.

The cytoplasmic signal sequences in the cytoplasmic signal portion of the CAR of the invention may be linked to each other in a random or predetermined order. Optionally, a short oligo- or polypeptide linker, for example, 2 to 10 amino acids in length, may form the linkage.

In another aspect of the present invention, there is provided a nucleic acid encoding a CAR targeting CD33 as described herein. The nucleic acid is used to express the CAR in an effector cell (e.g., a T cell) by introducing the nucleic acid into the cell.

Exemplary nucleic acid sequences encoding extracellular portions of a CAR are described herein, for example, as SEQ ID NOs: 246-265. Modifications can be made to the sequence to create an equivalent or improved version of the nucleic acid sequence, for example, by changing one or more codons in accordance with a codon degeneracy table. A DNA codon degeneracy table is provided in Table 10.

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Table 10. Amino acid codons Amino acids One letter code Three letter code Codons Alanine A Ala GC GC GC GC A C G U Cysteine WITH Cys UG UG C U Aspartic acid D Asp GA GA C U Glutamic acid E Glu GA GA A G Phenylalanine F Phe UU UU C U Glycine G Gly GG GG GG GG A C G U G istidine AND His CA CA C U Isoleucine I Iso AU AU AU A C U Lysine TO Lys AA AA A G Leucine L Leu UU UU CU CU CU CU A G A C G U Methionine M Met AU G Asparagine N Asn AA AA C U Proline R Pro CC CC CC CC A C G U Glutamine Q Gin CA CA A G Arginine R Arg AG AG CG CG CG CG A G A C G U Serine S Ser AG AG UC UC UC UC C U A C G U Threonine T Thr AC AC AC AC A C G U Valin V Vai GU GU GU GU A C G U Tryptophan W Trp UG G Tyrosine Y Tyr UA UA C U

In certain embodiments, the nucleic acid is a DNA molecule (e.g., a cDNA molecule). In certain embodiments, the nucleic acid further comprises an expression control sequence (e.g., a promoter and/or enhancer) operably linked to the CAR coding sequence. In certain embodiments, the invention provides a vector comprising the nucleic acid. The vector may be a viral vector (e.g., an AAV vector, a lentiviral vector, or an adenoviral vector) or a non-viral vector (e.g., a plasmid).

In certain embodiments, the nucleic acid is an RNA molecule (e.g., an mRNA molecule). A method of creating mRNA for use in transfection may include in vitro transcription of a template with specially designed primers, followed by the addition of

- 169 049 630 em polyA to produce an RNA construct comprising 3' and 5' untranslated sequences, a 5' cap and/or an internal ribosome entry site (IRES), a nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. The RNA molecule may be further modified to enhance translation efficiency and/or stability, such as disclosed in U.S. Patents 8,278,036; 8,883,506; and 8,716,465. The RNA molecules thus produced can efficiently transfect a variety of cell types.

In one embodiment, the nucleic acid encodes an amino acid sequence comprising a signal peptide at the amino terminus of the CAR. Such a signal peptide may promote localization of the CAR to the cell surface when expressed in an effector cell and is cleaved from the CAR during cellular processing. In one embodiment, the nucleic acid encodes an amino acid sequence comprising a signal peptide at the N-terminus of an extracellular CD33 binding domain (e.g., a CD33 binding scFv domain).

RNA or DNA can be introduced into target cells using any of a number of different methods, such as commercially available methods that include, but are not limited to, electroporation, transfection, cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biological particle delivery systems such as gene guns (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

In another aspect of the present invention, there is provided an immune effector cell expressing a CAR targeting CD33. There is also provided an immune effector cell comprising a nucleic acid encoding a CAR targeting CD33. Immune effector cells include, but are not limited to, T cells and NK cells. In certain embodiments, the T cell is selected from a CD8+ T cell, a CD4+ T cell, and an NKT cell. The T cell or NK cell may be a primary cell or a cell line.

Immune effector cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors by methods known in the art. Immune effector cells can also be differentiated in vitro from pluripotent or multipotent cells (e.g., hematopoietic stem cells). In some embodiments, the present invention provides a pluripotent or multipotent cell (e.g., hematopoietic stem cell) expressing a CAR targeting CD33 or comprising a nucleic acid as described herein.

In certain embodiments, immune effector cells are isolated and/or purified. For example, regulatory T cells can be removed from a T cell population using a CD25 binding ligand. Effector cells expressing a checkpoint protein (e.g., PD-1, LAG-3, or TIM-3) can be removed by similar methods. In certain embodiments, effector cells are isolated in a positive selection step. For example, a T cell population can be isolated by incubation with anti-CD3/anti-CD28 conjugated beads. Other cell surface markers such as IFN-7, TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin can also be used for positive selection.

Immune effector cells can be activated and expanded generally using methods known in the art, such as those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication Nos. 2006/0121005 and 2016/0340406. For example, in certain embodiments, T cells can be expanded and/or activated by contact with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. The cells can be expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are expanded for a period of 4 to 9 days. For long-term cell culture, multiple stimulation cycles may be desirable (e.g., culturing for a period of 60 days or more). In certain embodiments, the cell culture comprises serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFe, TNF-α, or a combination thereof. Other cell growth additives known to those skilled in the art, such as surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol, may also be included in the cell culture. In certain embodiments, the immune effector cell of the present invention is a cell obtained by in vitro expansion.

Additional embodiments of a CAR targeting CD33 (e.g., a regulated CAR), a nucleic acid encoding the CAR, and effector cells expressing the CAR or co

- 170 049 630 containing nucleic acid are disclosed in U.S. Patent Nos. 7,446,190 and 9,181,527, U.S. Patent Application Publication Nos. 2016/0340406 and 2017/0049819, and International Patent Application Publication No. WO2018/140725.

CD33/CD3-targeted bispecific T cell activators

In certain embodiments, the present invention provides a CD33/CD3 targeted bispecific T cell activator comprising an antigen binding site that binds CD33 as described herein. In certain embodiments, the CD33/CD3 targeted bispecific T cell activator comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identical to SEQ ID NO: 187. In certain embodiments, the CD33/CD3 targeted bispecific T cell activator comprises at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) amino acid sequence identical to SEQ ID NO: 197.

In certain embodiments, the CD33/CD3-targeted bispecific T cell activator further comprises an antigen-binding site that binds CD3. Exemplary antigen-binding sites that bind CD3 are disclosed in International Patent Application Publication Nos. WO 2014/051433 and WO 2017/097723.

In another aspect, the present invention provides a nucleic acid encoding at least one CD33/CD3-targeted bispecific T cell activator polypeptide, wherein the polypeptide comprises an antigen-binding site that binds CD33. In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide that, when expressed, is at the N-terminus of one or more CD33/CD3-targeted bispecific T cell activator polypeptides. Also provided is a vector (e.g., a viral vector) comprising the nucleic acid, a producer cell comprising the nucleic acid or vector, and a producer cell expressing the CD33/CD3-targeted bispecific T cell activator.

Immunocytokines

In certain embodiments, the present invention provides an immunocytokine comprising an antigen-binding site that binds CD33 as described herein and a cytokine. Any cytokine (e.g., proinflammatory cytokines) known in the art can be used, including, but not limited to, IL-2, IL-4, IL-10, IL-12, IL-15, TNF, IFNa, IFNy, and GM-CSF. More exemplary cytokines are disclosed in U.S. Patent No. 9,567,399. In certain embodiments, the antigen-binding site is linked to the cytokine by chemical conjugation (e.g., covalent or non-covalent chemical conjugation). In certain embodiments, the antigen-binding site is linked to the cytokine by a polypeptide fusion. The immunocytokine can further comprise an Fc domain linked to the antigen-binding site that binds CD33. In certain embodiments, the immunocytokine comprises an amino acid sequence that is at least 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 187. In certain embodiments, the immunocytokine comprises an amino acid sequence that is at least 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 197. In certain embodiments, the cytokine is linked to the Fc domain directly or through a linker.

In another aspect, the present invention provides a nucleic acid encoding at least one immunocytokine polypeptide, wherein the polypeptide comprises an antigen-binding site that binds CD33. In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide that, when expressed, is at the N-terminus of one or more immunocytokine polypeptides. Also provided is a vector (e.g., a viral vector) comprising the nucleic acid, a producer cell comprising the nucleic acid or vector, and a producer cell expressing the immunocytokine.

Antibody-drug conjugates

In certain embodiments, the present invention provides an antibody-drug conjugate comprising an antigen-binding site that binds CD33 as described herein and a cytotoxic drug moiety. Exemplary cytotoxic drug moieties are disclosed in International Patent Application Publication Nos. WO 2014/160160 and WO 2015/143382. In certain embodiments, the cytotoxic drug moiety is selected from auristatin, N-acetyl-γ-calicheamicin, a maytansinoid, pyrrolobenzodiazepine, and SN-38. The antigen-binding site may be linked to the cytotoxic drug moiety by chemical conjugation (e.g., covalent or non-covalent chemical conjugation). In certain embodiments, the antibody-drug conjugate further comprises an Fc domain linked to the antigen-binding site that binds CD33. In certain embodiments, the antibody-drug conjugate

- 171 049630 the agent comprises an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 187. In certain embodiments, the antibody-drug conjugate comprises an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 197. In certain embodiments, the cytotoxic drug moiety is linked to the Fc domain directly or via a linker.

Immunotoxins

In certain embodiments, the present invention provides an immunotoxin comprising an antigen-binding site that binds CD33 as described herein and a cytotoxic peptide fragment. Any cytotoxic peptide fragment known in the art may be used, including, but not limited to, ricin, diphtheria toxin, and Pseudomonas exotoxin A. More exemplary cytotoxic peptides are disclosed in International Patent Application Publication Nos. WO 2012/154530 and WO 2014/164680. In certain embodiments, the cytotoxic peptide fragment is linked to the protein by chemical conjugation (e.g., covalent or non-covalent chemical conjugation). In certain embodiments, the cytotoxic peptide fragment is linked to the protein by polypeptide fusion. The immunotoxin may further comprise an Fc domain linked to the antigen-binding site that binds CD33. In certain embodiments, the immunotoxin comprises an amino acid sequence that is at least 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 187. In certain embodiments, the immunotoxin comprises an amino acid sequence that is at least 90% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 197. In certain embodiments, the cytotoxic peptide fragment is linked to the Fc domain directly or through a linker.

In another aspect of the present invention, there is provided a nucleic acid encoding at least one immunotoxin polypeptide, wherein the polypeptide comprises an antigen-binding site that binds CD33. In certain embodiments, the nucleic acid further comprises a nucleotide sequence encoding a signal peptide that, when expressed, is at the N-terminus of one or more immunotoxin polypeptides. There is also provided a vector (e.g., a viral vector) comprising the nucleic acid, a producer cell comprising the nucleic acid or vector, and a producer cell expressing the immunotoxin.

Therapeutic compositions and their use

The present invention provides methods for treating cancer using the multispecific binding protein described herein and/or the pharmaceutical composition described herein. The methods can be used to treat various cancers that express CD33 by administering to a patient in need thereof a therapeutically effective amount of the multispecific binding protein described herein.

The therapeutic method can be characterized according to the type of cancer to be treated. For example, in certain embodiments of the invention, the cancers are AML, myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloid leukemia, and ALL.

For example, in certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer. In yet other embodiments, the cancer is a vascularized tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratoses, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, anal rectal cancer, anal canal cancer, rectal cancer, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bile duct cancer, bone cancer, bone marrow cancer, bronchial cancer, bronchial gland carcinoma, neuroendocrine tumors, cholangiocarcinoma, chondrosarcoma, papilloma/choroidal carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, gastrointestinal cancer, duodenal cancer, endocrine cancer, endodermal tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer, epithelial cell cancer, Ewing's sarcoma, eye and orbital cancer, gynecologic cancer, focal nodular hyperplasia, gallbladder cancer, gastric antral cancer, gastric vault cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangiomas, adenoma

- 172 049630 liver, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, intrahepatic bile duct cancer, invasive squamous cell carcinoma, small bowel cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo maligna melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic carcinoma, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cancer tract, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, nonepithelial skin cancer, non-Hodgkin's lymphoma, small cell carcinoma, oligodendroglial carcinoma, oral cavity cancer, osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumors, plasmacytoma, pseudosarcoma, lung blastoma, rectal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous cancer, paranasal sinus cancer, skin cancer, small cell carcinoma, small bowel cancer, smooth muscle cancer, soft tissue cancer, somatostatin-secreting tumor, spine cancer, squamous cell carcinoma, striated muscle cancer, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, cancer ureter, urethra cancer, bladder cancer, urinary bladder cancer

In other specific embodiments, the cancer is a non-Hodgkin's lymphoma, such as a B-cell lymphoma or a T-cell lymphoma. In specific embodiments, the non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In other specific embodiments, the non-Hodgkin lymphoma is a T-cell lymphoma, such as T-cell progenitor lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer cell lymphoma/T-cell lymphoma, enteropathic type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

A cancer to be treated can be characterized by the presence of a particular antigen expressed on the surface of the cancer cell. In certain embodiments, the cancer cell can express one or more of the following, in addition to CD33: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3, HER4/ERBB4, MUC1, TROP2, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, Mage-As, B7.1, B7.2, CTLA4, and PD1.

In embodiments of the present invention, the cancer to be treated is selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), myeloproliferative neoplasms (MPN), lymphoma, non-Hodgkin's lymphomas, and classical Hodgkin's lymphoma.

In some embodiments of the present invention, the cancer to be treated is AML. In some embodiments of the present invention, AML is selected from undifferentiated acute myeloid leukemia, acute myeloid leukemia with minimal maturation, acute myeloid leukemia with maturation, acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, acute myelomonocytic leukemia with eosinophilia, acute monocytic leukemia, acute erythroid leukemia, acute megakaryoblastic leukemia (AMCL), acute basophilic leukemia, acute panmyelosis with fibrosis, and blastic plasmacytoid dendritic cell neoplasia (BPDC). In some embodiments, AML is characterized by expression of CLL-1 on AML leukemia stem cells (LSCs). In some embodiments, the LSCs in a subject with AML further express a membrane marker selected from CD34, CD38, CD123, TIM3, CD25, CD32, and CD96. In some embodiments, AML is characterized by minimal residual disease (MRD). In some embodiments, AML MRD is characterized by the presence or absence of a mutation selected from FLT3-ITD (Fms-like tyrosine kinase 3 internal tandem duplications (ITD)), NPM1 (nucleophosmin 1), DNMT3A (DNMT3A DNA methyltransferase gene), and IDH (isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2)).

In certain embodiments of the present invention, MDS is selected from MDS with multilineage dysplasia (MDS-MLD), MDS with unilineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS), MDS with excess blasts (MDS-EB), MDS with deletion of del(5q), and MDS unclassified (MDS-U).

- 173 049630

It is contemplated that the multispecific binding proteins and/or pharmaceutical compositions of the present invention can be used to treat a variety of cancers, not limited to cancers in which the cancer cells express CD33. For example, in certain embodiments, the multispecific binding proteins and/or pharmaceutical compositions described herein can be used to treat a cancer that is associated with immune cells that express CD33. CD33 is expressed in many myeloid cell lines, and tumor-infiltrating myeloid cells (e.g., tumor-associated macrophages) can promote cancer progression and metastasis. Accordingly, the methods described herein can be used to treat a variety of cancers in which CD33 is expressed, whether on cancer cells or on immune cells.

In certain embodiments, the multispecific binding proteins and/or pharmaceutical compositions of the present invention can be used to treat a cancer that expresses an Fc receptor with a higher binding affinity for Fc (e.g., IgG1 Fc) than CD16. In certain embodiments, the Fc receptor is FcyRI. In certain embodiments, the Fc receptor is expressed on cancer cells and/or other cells in the tumor microenvironment.

In certain embodiments, the patient has effector cells (e.g., NK cells) that express a CD16 variant with a V158F substitution. In certain embodiments, the patient has a single nucleotide polymorphism (SNP) in the CD16 gene that causes a V158F substitution. In certain embodiments, the patient has such a SNP in only one allele. In certain embodiments, the patient has such a SNP or SNPs in both alleles.

Combination therapy

In another aspect of the present invention, a combination therapy is provided. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents for the treatment of cancer.

Illustrative therapeutic agents that may be used as part of combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane, sisofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor and variants of the above agents that may exhibit differential binding to the cognate receptor and increased or decreased serum half-life.

An additional class of agents that may be used as part of combination therapy in the treatment of cancer are immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include agents that inhibit one or more of: (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the U.S. Food and Drug Administration for the treatment of melanoma.

Other agents that may be used as part of combination therapy in the treatment of cancer include monoclonal antibody agents that target non-checkpoint targets (eg, Herceptin) and non-cytotoxic agents (eg, tyrosine kinase inhibitors).

Still other categories of anticancer agents include, for example: (i) an inhibitor selected from an ALK inhibitor, an ATR inhibitor, an A2A antagonist, a base excision repair inhibitor, a Bcr-Abl tyrosine kinase inhibitor, a Bruton tyrosine kinase inhibitor, a CDC7 inhibitor, a CHK1 inhibitor, a cyclin-dependent kinase inhibitor, a DNA-PK inhibitor, an inhibitor of both DNA-PK and mTOR, a DNMT1 inhibitor, a DNMT1 plus 2-chloro-deoxyadenosine inhibitor, an HDAC inhibitor, a Hedgehog signaling pathway inhibitor, an IDO inhibitor, a JAK inhibitor, an mTOR inhibitor, a MEK inhibitor, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and and DHODH, a proteasome inhibitor, a topoisomerase II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor and a WEE1 inhibitor; (ii) an OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25 or ICOS agonist and (iii) a cytokine selected from IL-12, IL-15, GM-CSF and G-CSF.

- 174 049630

The proteins of the invention can also be used as an adjunct to surgical removal of the primary lesion.

The amount of the multispecific binding protein and the additional therapeutic agent and the relative time of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering a combined therapy to a patient in need of such administration, the therapeutic agents in combination or the pharmaceutical composition or compositions containing the therapeutic agents can be administered in any order, such as, for example, sequentially, together, together, simultaneously, and the like. In addition, for example, the multispecific binding protein can be administered at the time when the additional therapeutic agent(s) exert their prophylactic or therapeutic effect, or vice versa.

Pharmaceutical compositions

The present invention also relates to pharmaceutical compositions that comprise a therapeutically effective amount of the protein described herein. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a summary of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990).

In one aspect of the present disclosure, there is provided a protein composition that comprises a CD33 binding site as described herein and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 5; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 7; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 11; and a light chain variable domain having an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 13; and

- 175 049630 a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 15; and a light chain variable domain having an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the formulation comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 17; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the composition comprises a protein that comprises an antigen-binding site with a heavy chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 19; and a light chain variable domain having an amino acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 20.

The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can be included in the composition for proper formulation. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a summary of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990).

For example, the present invention may exist in an aqueous pharmaceutical formulation comprising a therapeutically effective amount of a protein in a buffered solution forming the formulation. Aqueous carriers may include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In certain embodiments, an aqueous formulation is prepared comprising a protein described herein in a pH buffered solution. The pH of the formulations is typically from 3 to 11, more preferably from 5 to 9 or from 6 to 8, and most preferably from 7 to 8, such as from 7 to 7.5. Ranges including intermediate pH values of the above pH values are also part of this disclosure. For example, ranges of values are intended to be included in which combinations of any of the above values serve as upper and/or lower limits. Examples of buffers that will adjust the pH in this range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid buffers. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3 mg/mL citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL sodium citrate (e.g., 0.305 mg/mL), about 1.5 mg/mL disodium phosphate dihydrate (e.g., 1.53 mg/mL), about 0.9 mg/mL sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg/mL sodium chloride (e.g., 6.165 mg/mL). In certain embodiments, the buffer system comprises 1-1.5 mg/mL citric acid, 0.25-0.5 mg/mL sodium citrate, 1.25-1.75 mg/mL disodium phosphate dihydrate, 0.7-1.1 mg/mL sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg/mL sodium chloride. The pH of the liquid formulation can be adjusted by adding a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

In some embodiments, the formulation includes an aqueous carrier that is pharmaceutically acceptable (safe and non-toxic for human administration) and suitable for preparing a liquid formulation. Exemplary carriers include sterile water for injection (SWI), bacteriostatic water for injection (BWFI), a pH buffer solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

A polyol that acts as a tonicity agent and can stabilize the antibody may also be included in the formulation. The polyol is added to the formulation in an amount that can vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous

- 176 049630 the composition can be isotonic. The amount of added polyol can also be varied depending on the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (e.g., mannitol) can be added compared to a disaccharide (such as trehalose). In certain embodiments, the polyol that can be used in the composition as a tonic agent is mannitol. In certain embodiments, the concentration of mannitol can be from about 5 to about 20 mg/mL. In certain embodiments, the concentration of mannitol can be from about 7.5 to 15 mg/mL. In certain embodiments, the concentration of mannitol can be about 10-14 mg/mL. In certain embodiments, the concentration of mannitol can be about 12 mg/mL. In certain embodiments, polyol sorbitol can be included in the composition.

A detergent or surfactant may also be added to the formulation. Examples of detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of detergent added is such that it reduces aggregation of the antibody in the formulation and/or minimizes particle formation in the formulation and/or reduces adsorption. In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may comprise the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edi., 1996). In certain embodiments, the formulation may contain from about 0.1 to about 10 mg/mL polysorbate 80 or from about 0.5 to about 5 mg/mL. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

In certain embodiments, the liquid formulation of the invention may be prepared as a 10 mg/mL solution in combination with sugar at stabilizing levels. In certain embodiments, the liquid formulation may be prepared in an aqueous vehicle. In certain embodiments, the stabilizer may be added in an amount no greater than that which would result in an undesirable or unsuitable viscosity for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In some embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative, which are added to the formulations described herein to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multiple-use (multiple dose) formulation.

In certain embodiments of the present disclosure, a formulation with an enhanced shelf life is provided comprising a protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

Deamidation is a common product variant of peptides and proteins that can occur during fermentation, cell harvest/clarification, purification, drug substance/drug product storage, and during sample analysis. Deamidation is the loss of NH3 from the protein to form a succinimide intermediate, which can undergo hydrolysis. The succinimide intermediate results in a 17 unit mass loss of the parent peptide. Subsequent hydrolysis results in an 18 unit mass gain. Isolation of the succinimide intermediate is difficult due to instability in aqueous conditions. Thus, deamidation is typically detected by a 1 unit mass gain. Deamidation of asparagine results in the formation of either aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local conformation of the polypeptide, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the rate of deamidation. The succession of Gly and Ser to Asn in protein sequences results in higher sensitivity to deamidation. In certain embodiments, the liquid formulation of the present disclosure may be stored under pH and humidity conditions to prevent deamination of the protein product.

In certain embodiments, the formulation is a lyophilized formulation. In certain embodiments, the formulation is lyophilized and is contained in about 12-60 vials. In certain embodiments, the formulation is lyophilized and may contain 45 mg of the lyophilized formulation per vial. In certain embodiments, about 40 mg to about 100 mg of the lyophilized formulation is contained in a single vial. In certain embodiments, the lyophilized formulation from 12, 27, or 45 vials is combined to provide a therapeutic dose of the protein in a medicament for intravenous administration. The formulation may be a liquid formulation. In certain embodiments, the liquid formulation is stored in an amount of about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the liquid formulation is stored in an amount of about 600 mg/vial. In certain embodiments, the liquid formulation is stored in an amount of about 250 mg/vial.

In certain embodiments, the lyophilized formulation comprises proteins described

- 177 049630 herein, and a lyoprotectant. The lyoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative. The amount of sucrose or maltose used to stabilize the lyophilized drug may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose may be from 1:2 to 1:5.

In certain embodiments, the pH of the formulation prior to lyophilization may be adjusted by adding a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide. Prior to lyophilization, the pH of the solution comprising the protein of the present invention may be adjusted from 6 to 8. In certain embodiments, the pH range for the lyophilized drug may be from 7 to 8.

In certain embodiments, a bulking agent may be added. A bulking agent is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized mixture (e.g., facilitates the formation of a substantially homogeneous lyophilized mass that maintains an open-cell structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.

In certain embodiments, the lyophilized protein product comprises an aqueous carrier. The aqueous carrier of interest is pharmaceutically acceptable (e.g., safe and non-toxic for human administration) and useful in preparing a liquid formulation after lyophilization. Exemplary diluents include sterile water for injection (SWI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution. In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with either sterile water for injection, USP (SWI) or 0.9% sodium chloride injection, USP. During reconstitution, the lyophilized powder is dissolved in the solution. In certain embodiments, the lyophilized protein product of the present disclosure comprises about 4.5 mL of water for injection and is diluted with 0.9% saline (sodium chloride solution).

The protein compositions may be sterilized by conventional sterilization methods or may be sterilized by filtration. The resulting aqueous solutions may be packaged for use as is or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier before administration. The resulting solid form compositions may be packaged in several single dose units, each containing a fixed amount of the above agent or agents. The solid form composition may also be packaged in a container for an arbitrary amount.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, a composition and a route of administration that are not toxic to the patient.

The specific dose may be the same for each patient, such as 50-5000 mg of protein. Alternatively, the patient dose may be adjusted based on the approximate body weight or body surface area of the patient. Other factors in determining the appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and health of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely performed by those skilled in the art, particularly in light of the dosage information and assays described herein. The dosage may also be determined using known assays for determining usable doses in conjunction with appropriate dose-response data. The dosage for an individual patient may be adjusted as the disease progresses. Blood levels of the target construct or complex in the patient may be measured to see if the dosage needs to be adjusted to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which target constructs and/or complexes and their dosages will be most likely to be effective for a given individual (Schmitz et al., Clinica. Chimica. Acta. 308: 43-53, 2001; Steimer et al., Clinica. Chimica. Acta. 308: 33-41, 2001).

Typically, dosages based on body weight are from about 0.01 to about 100 mg per kg of body weight, such as from about 0.01 to about 100 mg per kg of body weight, from about 0.01 to about 50 mg/kg, from

- 178 049630 about 0.01 mcg to about 10 mg/kg body weight, from about 0.01 mcg to about 1 mg/kg body weight, from about 0.01 mcg to about 100 mcg/kg body weight, from about 0.01 mcg to about 50 mcg/kg body weight, from about 0.01 mcg to about 10 mcg/kg body weight, from about 0.01 mcg to about 1 mcg/kg body weight, from about 0.01 mcg to about 0.1 mcg/kg body weight, from about 0.1 mcg to about 100 mg/kg body weight, from about 0.1 mcg to about 50 mg/kg body weight, from about 0.1 mcg to about 10 mg/kg body weight, from about 0.1 mcg up to about 1 mg/kg body weight, from about 0.1 mcg to about 100 mcg/kg body weight, from about 0.1 mcg to about 10 mcg/kg body weight, from about 0.1 mcg to about 1 mcg/kg body weight, from about 1 mcg to about 100 mg/kg body weight, from about 1 mcg to about 50 mg/kg body weight, from about 1 mcg to about 10 mg/kg body weight, from about 1 mcg to about 1 mg/kg body weight, from about 1 mcg to about 100 mcg/kg body weight, from about 1 mcg to about 50 mcg/kg body weight, from about 1 mcg to about 10 mcg/kg body weight, from about 10 mcg to about 100 mg/kg body weight, from about 10 mcg to about 50 mg/kg body weight, from about 10 mcg to about 10 mg/kg body weight, from about 10 mcg to about 1 mg/kg body weight, from about 10 mcg to about 100 mcg/kg body weight, from about 10 mcg to about 50 mcg/kg body weight, from about 50 mcg to about 100 mg/kg body weight, from about 50 mcg to about 50 mg/kg body weight, from about 50 mcg to about 10 mg/kg body weight, from about 50 mcg to about 100 mcg/kg body weight, from about 100 mcg to about 100 mg/kg body weight, from about 100 mcg to about 50 mg/kg body weight, from about 100 mcg to about 10 mg/kg body weight, from about 100 mcg to about 1 mg/kg body weight, from about 1 mg to about 100 mg/kg body weight, from about 1 mg to about 50 mg/kg body weight, from about 1 mg to about 10 mg/kg body weight, from about 10 mg to about 100 mg/kg body weight, from about 10 mg to about 50 mg/kg body weight, from about 50 mg to about 100 mg/kg body weight. Doses can be administered one or more times daily, weekly, monthly or annually, or even once every 2 to 20 years. Those skilled in the art can readily estimate the frequency of repetition for dosing based on the measured residence time and concentrations of the target construct or complex in body fluids or tissues. The administration according to the present invention can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct injection into the lesion. The administration can be carried out one or more times per day, one or more times per week, one or more times per month and one or more times per year.

The above description presents several aspects and embodiments of the invention. The patent application specifically contemplates all combinations and permutations of aspects and embodiments.

Throughout this specification, where compositions are described as having, including, or containing particular components, or where processes and methods are described as having, including, or containing particular steps, it is intended that there are additional compositions of the present invention that consist essentially of or consist of the recited components, and that there are processes and methods of the present invention that consist essentially of or consist of the recited processing steps.

In an application where it is stated that an element or component is included and/or selected from a list of listed elements or components, it should be understood that the element or component may be any of the listed elements or components, or the element or component may be selected from a group consisting of two or more listed elements or components.

In addition, it should be understood that the elements and/or features of a composition or method described herein may be combined in a variety of ways without departing from the spirit and scope of the present invention, whether expressed or implied herein. For example, if reference is made to a particular compound, that compound may be used in various embodiments of the compositions of the present invention and/or in the methods of the present invention, unless the context otherwise requires. In other words, embodiments have been described and illustrated in this application in a manner that allows a clear and precise content of the application to be written and depicted, but it is intended and will be understood that embodiments may be combined or separated in various ways without departing from the spirit of the present invention. For example, it is understood that all features described and illustrated herein may be applied to all aspects of the invention described and illustrated herein.

It should be understood that the expression at least one of includes individually each of the listed objects after the expression and various combinations of two or more of the listed objects, unless otherwise clear from the context and use. It should be understood that the expression and/or in relation to three or more listed objects has the same meaning, unless otherwise clear from the context.

The use of the terms include, includes, including, have, has, having, contain, contains or containing, including their grammatical equivalents, should generally be understood as open-ended and non-limiting, e.g. not excluding additional unlisted elements or steps, unless otherwise specifically stated or clear from the context.

When the term about is used before a quantitative value, the present invention also

- 179 049630 includes the specific quantity value itself, unless otherwise specifically stated. In the context of this document, the term about refers to a deviation of ±10% from the nominal value, unless otherwise stated or implied.

It should be understood that the order of the steps or the order in which certain actions are performed is immaterial as long as the present invention remains usable. Moreover, two or more steps or actions may be performed simultaneously.

The use of any and all examples or exemplary language herein, such as, for example, or including, is intended merely to better illustrate the present invention and does not limit the scope of the invention unless otherwise stated. No language in the description should be construed as indicating any unreported element as essential to the practice of the present invention.

Examples

The following examples are merely illustrative and are not intended to limit the scope or spirit of the invention in any way.

Example 1. Kinetics and binding affinity to different CD33 variants

The kinetics and affinity of a series of anti-CD33 Fab fragments targeting different CD33 variants (human CD33 ECD, cynomolgus CD33 ECD, human CD33 V domain, human CD33 C domain, and selected CD33 SNPs) were assessed by surface plasmon resonance using a Biacore 8K instrument (GE Healthcare). The anti-Fab antibody was immobilized on a CM5 chip using standard coupling chemistry. CD33 FAB was captured on the anti-Fab chip at a density of ~100 EU. Solutions containing different concentrations of soluble monomeric CD33 or its domains were injected over the captured FAB and control surfaces at 30 μl/min at 37°C. Surfaces were regenerated between cycles by rapid injection of 10 mM glycine, pH 1.8. To obtain kinetic rate constants, doubly corrected data were fitted to a 1:1 interaction model using Biacore 8K Evaluation software (GE Healthcare). The equilibrium binding constant K _D was determined from the ratio of binding rate constants kd/k _a .

Kinetics and Affinity Analysis Based on the Octet Platform ForteBio affinity measurements were performed on an Octet HTX as described in Estep et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs 5(2), 270–278 (2013). Briefly, ForteBio affinity measurements were performed by loading IgG online onto ANS sensors. Sensors were equilibrated offline in assay buffer for 30 min and then monitored online for 60 s to establish baseline. IgG-loaded sensors were exposed to 100 nM antigen for 3 min and then transferred to assay buffer for 3 min to measure the off-rate. All kinetics were analyzed using a 1:1 binding model. Results are presented in Table 11.

Table 11. Kinetic parameters of human CD33 binding to IgG antibodies measured by BLI

Name ADI IgG K _D (M) k _on (l/M/c) k _off (l/c) ADI-10154 3.70E-09 2.46E+05 8.97E-04 ADI-10155 5.30E-09 2.29E+05 1.21E-03 ADI-10157 4.20E-09 7.99E+04 3.36E-04 ADI-10158 2.60E-09 5.86E+05 1.55E-03 ADI-10159 5.80E-10 3.48E+05 2.00E-04 ADI-10160 5.10E-09 6.38E+05 3.25E-03 ADI-10161 3.70E-09 5.87E+05 2.17E-03 ADI-10163 6.80E-09 6.65E+05 4.53E-03 ADI-10164 4.30E-09 3.44E+05 1.48E-03 ADI-10165 4.00E-09 7.79E+05 3.11E-03

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ADI-10167 3.90E-09 1.00E+06 3.93E-03 ADI-10168 1.00E-08 4.76E+05 4.78E-03 ADI-10173 9.70E-09 3.24E+05 3.14E-03 ADI-10177 3.60E-09 6.01E+05 2.14E-03 ADI-11776 2.94E-10 6.79E+05 2.00E-04 ADI-11801 4.57E-10 4.38E+05 2.00E-04 ADI-11802 4.53E-10 4.41E+05 2.00E-04 ADI-11807 3.07E-10 6.51E+05 2.00E-04 ADI-11809 3.13E-10 6.38E+05 2.00E-04 ADI-11812 1.91E-09 1.66E+05 3.15E-04 ADI-11815 1.44E-09 1.59E+05 2.29E-04 ADI-11819 2.43E-10 8.24E+05 2.00E-04 ADI-11825 5.40E-10 6.96E+05 3.76E-04 ADI-11826 3.93E-10 6.19E+05 2.43E-04 ADI-11828 8.10E-10 6.58E+05 5.33E-04 ADI-11830 1.20E-09 7.70E+05 9.23E-04 ADI-11835 2.81E-10 8.44E+05 2.37E-04 ADI-11839 2.45E-09 7.04E+05 1.72E-03 Lintuzumab 2.21E-09 4.31E+05 9.52E-04

Example 2. Anti-CD33 antibodies bind to human CD33 with high affinity and cross-react with cynomolgus monkey CD33

Despite the relatively high homology between human and cynomolgus monkey CD33 (87% in ECD), most commercially available CD33 antibodies, such as lintuzumab, mylotarg, etc., do not cross-react with cynomolgus monkey CD33. Figure 2 shows an alignment of full-length human and cynomolgus monkey CD33, highlighting the differences in the primary sequence in the ECD domain.

The binding affinity of 29 Fab fragments to human and cynomolgus monkey CD33 ECD was determined by Biacore analysis. Eight of the 29 antibodies showed cross-reactivity with cynomolgus monkey CD33. The binding kinetic parameters are shown in Table 12. The data were compared with lintuzumab. Some antibodies showed affinity >100-fold higher than lintuzumab.

Binding of CD33 mAb Fab fragments to the extracellular domain (ECD) of human CD33 was measured by Biacore at 37°C. The Biacore profile of ADI-10159 is shown in Fig. 3A; the Biacore profile of ADI-10177 is shown in Fig. 3B; the Biacore profile of ADI-11776 is shown in Fig. 3C; the Biacore profile of ADI-11801 in Fig. 3D; the Biacore profile of ADI-11807 is shown in Fig. 3E; the Biacore profile of ADI11809 is shown in Fig. 3F;

The Biacore profile of ADI-11815 is shown in Fig. 3G; the Biacore profile of ADI-11819 is shown in Fig. 3H; the Biacore profile of ADI-11830 is shown in Fig. 3I; the Biacore profile of ADI-11835 is shown in Fig. 3J; and the Biacore profile of the lintuzumab Fab fragment is shown in Fig. 3K.

Binding of CD33 mAb Fab fragments to cynomolgus monkey CD33 ECD was measured by Biacore at 37°C. The Biacore profile of ADI-10159 is shown in Fig. 4A; the Biacore profile of ADI10177 is shown in Fig. 4B; the Biacore profile of ADI-11776 is shown in Fig. 4C; the Biacore profile of ADI11807 is shown in Fig. 4D; the Biacore profile of ADI-11809 is shown in Fig. 4E; the Biacore profile of ADI-11819 is shown in Fig. 4F; the Biacore profile of ADI-11830 is shown in Fig. 4G; and the Biacore profile of ADI-11835 is shown in Fig. 4H.

Binding of CD33 mAb Fab fragments to the V and C domains of human CD33 was measured by Biacore at 37°C. Figures 5A–5J show binding to the V domain; panels K–T show binding to the C domain. Both Figures 5A and 5K show Biacore profiles of ADI-10159; both Figures 5B and 5L show Biacore profiles of ADI-10177; both Figures 5C and 5M show Biacore profiles of ADI-11776; both Figures 5D and 5N show Biacore profiles of ADI-11801; both Figures 5E and 5O show Biacore profiles of ADI-11807; both Figures 5F and 5P both show profiles of Biacore ADI-11809; 5G and 5Q both show profiles of Biacore ADI-11815; 5H and 5R both show profiles of Biacore ADI-11819; 5I and 5S both show profiles of Biacore ADI11830; and 5J and 5T both show profiles of Biacore ADI-11835.

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Table 12. Kinetic parameters of binding of human CD33 ECD and cynomolgus monkey CD33 ECD to Fab measured by SPR at 37°C. No binding is defined as no signal at a maximum concentration of 100 nM.

Antibody Human CD33 CD33 of the cynomolgus macaque ka (1/Ms) ka (1/s) K _D (nM) ka(1/Ms) ka (1/s) K _D (nM) ADI-10159 4.29e5 1.95e-3 4.53 5.28e5 4.36e-2 74.9 ADI-10177 5.58e5 6.36e-3 11.4 3.53e5 7.54e-3 21.4 ADI-11776 1.6e6 1.24e-3 0.78 2.62e6 4.72e-3 1.81 ADI-11807 1.14e6 3.32e-4 0.29 9.98e5 1.51e-4 0,15 ADI-11809 1.02e6 1.23e-6 0,0012 1.06e6 1.26e-4 0,118 ADI-11819 2.84e6 5.65e-5 0,02 3.82e6 1.90e-2 4.96 ADI-11830 1.99e6 5.47e-3 2.75 1.34e6 3.64e-2 27.8 ADI-11835 2.6e6 1.43e-3 0.53 2.76e6 1.89e-2 6.86 ADI-11815 3.39e5 7.91e-5 0.23 Lack of binding ADI-11801 7.56e5 4.17e-4 0.55 Lack of binding ADI-10152 2.46e5 9.14e-3 37.2 Lack of binding ADI-10154 1.92e5 8.33e-3 43.4 Lack of binding ADI-10155 1.70e5 6.85e-3 40.2 Lack of binding ADI-10157 2.82e6 7.60e-3 2.69 Lack of binding ADI-10158 4.75e5 1.49e-2 3.14 Lack of binding ADI-10160 6.37e5 2.69e-2 42.2 Lack of binding ADI-10161 5.74e5 1.18e-2 20.6 Lack of binding ADI-10163 5.15e5 2.75e-2 53.5 Lack of binding ADI-10164 3.19e5 1.01e-2 31,5 Lack of binding ADI-10165 5.91e5 1.88e-2 31.8 Lack of binding ADI-10167 1.25e6 3.07e-2 24.4 Lack of binding ADI-10168 3.75e5 1.75e-2 46.6 Lack of binding ADI-10173 2.12e5 1.25e-2 58.8 Lack of binding ADI-11802 4.9e5 1.54e-3 3.14 Lack of binding ADI-11812 3.96e5 4.86e-4 1.23 Lack of binding ADI-11825 2.13e6 3.8e-3 1.78 Lack of binding ADI-11826 1.76e6 4.63e-3 2.63 Lack of binding ADI-11828 1.32e6 3.51e-3 2.67 1.03e6 5.48e-2 53.5 ADI-11839 8.74e5 1.62e-2 18.6 Lack of binding Lintuzumab 7.35e5 1.22e-2 16.7 Lack of binding

Mapping the binding interface to individual CD33 domains

The binding interface between the Fab fragment of each anti-CD33 antibody and CD33 was mapped. Figures 5A–5T show binding of the Fab fragments of the various anti-CD33 antibodies to individual domains of human CD33 (the V domain and the C domain). No binding to the C domain was observed for any of the antibodies tested. ADI-11815 did not bind to either the V or C domain, suggesting that it requires a unique conformational epitope.

Table 13 shows a comparison of binding kinetics to the entire human CD33 ECD and the V domain. ADI-10159, ADI-11176, ADI-11807, ADI-11830, ADI-11835, ADI-11801, ADI-10155, ADI-11802, ADI11825, ADI-11826, ADI-11828, and ADI-11839 show similar kinetics, suggesting that the epitope of these antibodies is entirely located in the V domain. Decreased binding to the V domain was observed for ADI-10177, ADI-11809, ADI-11819, ADI-10157, ADI-10158, and ADI-10164, suggesting that these antibodies bind to a conformational epitope located partially within the V domain.

The kinetics of binding to the human CD33 C domain were also measured using ADI-10152, ADI10154, ADI-10155, ADI-10157, ADI-10158, ADI-10160, ADI-10161, ADI-10163, ADI-10164, ADI-10165, ADI-10167, ADI-10168, ADI-10173, ADI-11802, ADI-11812, ADI-11825, ADI-11826, ADI-11828 and ADI-182 049630

11839. None of these antibodies bound the C domain of human CD33.

Table 13. Biacore analysis of FAB binding to recombinant full-length ECD and human CD33 V domain was performed at 37°C. The asterisk indicates antibodies that bind to a conformational epitope partially located in the V domain._________________

Antibody Human CD33 V-domain Human CD33 1¾ (1/Mc) kd (1/s) K _D (nM) Ml/Mc) kd (1/s) K _D (nM) ADI-10159 9.05e5 5.0e-3 5.56 4.29e5 1.95e-3 4.53 ADI- 10177* 1.56e6 8.57e-2 54.8 5.58e5 6.36e-3 11.4 ADI-11776 4.52e6 1.43e-2 0.33 1.6b 1.24e-3 0.78 ADI-11807 3.07e6 8.47e-4 0.28 1.14e6 3.32e-4 0.29 ADI- 11809* 2.6e6 7.69e-4 0,3 1.02e6 1.23e-6 0,0012 ADI-11819 6.75e6 9.16e-4 0.14 2.84e6 5.65e-5 0,02 ADI-11830 6.67e6 2.99e-2 4,5 1.99e6 5.47e-3 2.75 ADI-11835 7.6e6 8.68e-3 1,14 2.6e6 1.43e-3 0.53 ADI-11815 Lack of binding 3.39e5 7.91e-5 0.23 ADI-11801 1.8e6 1.25e-3 0.69 7.56e5 4.17e-4 0.55 ADI-10152 4.88e5 4.09e-2 100 2.46e5 9.14e-3 37.2 ADI-10154 1.52e6 3.35e-1 220 1.92e5 8.33e-3 43.4 ADI-10155 5.48e5 4.67e-2 85.2 1.70e5 6.85e-3 40.2 ADI- 10157* 1.12e5 1.51e-2 134 2.82e6 7.60e-3 2.69 ADI- 10158* 1.04e6 1.33e-1 127 4.75e5 1.49e-2 3.14 ADI-10160 1.59e6 3.27e-1 206 6.37e5 2.69e-2 42.2 ADI-10161 1.29e6 1.24e-1 95.8 5.74e5 1.18e-2 20.6 ADI-10163 Lack of binding 5.15e5 2.75e-2 53.5 ADI-10164 1.29e6 3.7e-1 286 3.19e5 1.01e-2 31,5 ADI-10165 Lack of binding 5.91e5 1.88e-2 31.8 ADI-10167 Lack of binding 1.25e6 3.07e-2 24.4 ADI-10168 Lack of binding 3.75e5 1.75e-2 46.6 ADI-10173 Lack of binding 2.12e5 1.25e-2 58.8 ADI-11802 1.34b 4.35e-3 3.2 4.9e5 1.54e-3 3.14 ADI-11812 Lack of binding 3.96e5 4.86e-4 1.23 ADI-11825 4.93e6 1.95e-2 3.95 2.13e6 3.8e-3 1.78 ADI-11826 5.12e6 2.25e-3 4.38 1.76e6 4.63e-3 2.63 ADI-11828 3.37b 2.13e-2 6.3 1.32e6 3.51e-3 2.67 ADI-11839 9.19e6 4.1e-1 44.6 8.74e5 1.62e-2 18.6

Antibodies recognize CD33 regardless of its glycosylation status.

The ability of anti-CD33 antibodies to recognize glycosylated CD33 was analyzed. Table 14 shows that the antibodies recognize the V domain regardless of its glycosylation status. Human CD33 is heavily glycosylated at 2 glycosylation sites located within the V domain. Differences in the glycosylation level of CD33 in different cells have been reported in the literature. Glycosylation may potentially impair antibody binding to the target. In some samples, the V domain was deglycosylated with PNGase prior to testing. Deglycosylation status was confirmed by SDS-PAGE shift and MS. All antibodies tested, presented in Table 14, bound to deglycosylated V CD33 similarly to the fully glycosylated version, except ADI10163, ADI-10165, ADI-10167, and ADI-10173.

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Table 14. Biacore analysis of FAB binding to fully glycosylated versus deglycosylated V domain performed at 37°C

Antibody Deglycosylated V-domain V-domain ka(l/Mc) kd (1/c) K _D (hM) U (1/Mc) kd (1/c) K _D (hM) ADI- 10159 9.78e5 1.2e-2 12.3 9.05e5 5.0e-3 5.56 ADI- 10177 1.88e6 7.01e-2 38.2 1.56e6 8.57e-2 54.8 ADI- 11776 4.85e6 1.36e-3 0.28 4.52e6 1.43e-2 0.33 ADI- 11807 3.9e6 7.45e-4 0.19 3.07e6 8.47e-4 0.28 ADI- 11809 3.4e6 7.11e-4 0.21 2.6e6 7.69e-4 0,3 ADI- 11819 9.15e6 1.03e-3 0.11 6.75e6 9.16e-4 0.14 ADI- 11830 6.8e6 2.69e-2 3.95 6.67e6 2.99e-2 4,5 ADI- 7.94e6 7.79e-3 0.98 7.6e6 8.68e-3 1,14

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11835 ADI- 11801 2.1e6 1.15e-3 0.55 1.8e6 1.25e-3 0.69 ADI- 10152 1.05e5 7.7e-2 76.8 4.88e5 4.09e-2 100 ADI- 10154 3.13e5 6.55e-2 209 1.52e6 3.35e-1 220 ADI- 10155 1.25e6 1.32e-1 105 5.48e5 4.67e-2 85.2 ADI- 10157 4.11e5 2.61e-2 63.8 1.12e5 1.51e-2 134 ADI- 10158 2.63e6 2.31e-1 88.1 1.04e6 1.33e-1 127 ADI- 10160 1.7e6 2.25e-1 133 1.59e6 3.27e-1 206 ADI- 10161 2.29e6 1.96e-1 85.6 1.29e6 1.24e-1 95.8 ADI- 10163 1.68e6 2.56e-1 152 Lack of binding ADI- 10164 8.11e5 1.16e-1 143 1.29e6 3.7e-1 286 ADI- 10165 1.3e6 2.03e-1 156 Lack of binding ADI- 10167 1.66e6 1.28e-1 77.8 Lack of binding ADI- 10168 Lack of binding Lack of binding ADI- 10173 6.74e5 1.31e-1 200 Lack of binding ADI- 11802 1.57e6 4.17e-3 2.65 1.34e6 4.35e-3 3.2 ADI- 11812 Lack of binding Lack of binding ADI- 5.78e6 1.8e-2 3.11 4.93e6 1.95e-2 3.95 11825 ADI- 11826 5.37e6 1.98e-2 3.69 5.12e6 2.25e-3 4.38 ADI- 11828 3.588e6 1.89e-2 4.87 3.37b 2.13e-2 6.3 ADI- 11839 1.65e6 1.16e-1 70.3 9.19e6 4.1e-1 44.6

Anti-CD33 antibodies bind to the R69G CD33 SNP.

The ability of CD33 antibodies to recognize the R69G mutation in CD33 was analyzed. Although several SNPs have been described for CD33, R69G is particularly significant, occurring in 39-42% of the population. Table 15 shows antibody binding to human CD33 containing the R69G mutation.

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Table 15. Biacore analysis of FAB binding to R69G CD33 Antibody R69G Human CD33 Human CD33 ka (1/Ms) kd (1/s) K _D (nM) ka(1/Ms) kd (1/s) K _D (nM) ADI-10159 4.26e5 2.43e-3 5.68 4.29e5 1.95e-3 4.53 ADI-10177 2.3e5 2.01e-3 87.4 5.58e5 6.36e-3 11.4 ADI-11776 1.98e6 4.26e-4 0.22 1.6e6 1.24e-3 0.78 ADI-11807 5.97e5 2.59e-4 0.43 1.14e6 3.32e-4 0.29 ADI-11809 8.15e5 1.79e-4 0.22 1.02e6 1.23e-6 0,0012 ADI-11819 2.78e6 2.44e-4 0.09 2.84e6 5.65e-5 0,02 ADI-11830 2.27e6 6.63e-3 2.94 1.99e6 5.47e-3 2.75 ADI-11835 3.07e6 2.05e-3 0.67 2.6e6 1.43e-3 0.53 ADI-11815 3.00e5 1.3e-3 4.34 3.39e5 7.91e-5 0.23 ADI-11801 Lack of binding 7.56e5 4.17e-4 0.55 ADI-10152 2.88e5 2.18e-2 75.8 2.46e5 9.14e-3 37.2 ADI-10154 1.53e5 2.83e-2 186 1.92e5 8.33e-3 43.4 ADI-10155 2.33e5 1.05e-2 45,0 1.70e5 6.85e-3 40.2 ADI-10157 1.56e6 2.87e-2 17.8 2.82e6 7.60e-3 2.69 ADI-10158 5.30e5 3.31e-2 62.3 4.75e5 1.49e-2 3.14 ADI-10160 8.36e5 5.33e-2 63.7 6.37e5 2.69e-2 42.2 ADI-10161 6.29e5 3.07e-2 48.9 5.74e5 1.18e-2 20.6 ADI-10163 5.59e5 5.9e-2 106 5.15e5 2.75e-2 53.5 ADI-10164 3.08e5 3.5e-2 113 3.19e5 1.01e-2 31,5 ADI-10165 6.9e5 5.02e-2 72.7 5.91e5 1.88e-2 31.8 ADI-10167 1.22eb 8.74e-2 71.6 1.25eb 3.07e-2 24.4 ADI-10168 4.85e5 3.07e-2 63.5 3.75e5 1.75e-2 46.6 ADI-10173 Lack of binding 2.12e5 1.25e-2 58.8 ADI-11802 Lack of binding 4.9e5 1.54e-3 3.14 ADI-11812 3.38e5 2.28e-3 6.74 3.96e5 4.86e-4 1.23 ADI-11825 1.86e6 6.95e-3 3.74 2.13e6 3.8e-3 1.78 ADI-11826 1.89e6 7.23e-3 3.83 1.76e6 4.63e-3 2.63 ADI-11828 1.16e6 7.00e-3 6.03 1.32e6 3.51e-3 2.67 ADI-11839 Lack of binding 8.74e5 1.62e-2 18.6 Antibodies to CD33 bind to the S128N SNP of CD33.

The ability of anti-CD33 antibodies to recognize the S128N mutation in CD33 was analyzed. Table 16 shows the binding of antibodies to human CD33 containing the S128N mutation. The binding affinity of ADI-10152, ADI-10154, ADI-10157, ADI-10158, ADI-10163, ADI-10164, ADI-10165, ADI-10167, ADI10168, and ADI-10173 to human CD33 was impaired by the S128N SNP.

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Table 16. Biacore analysis of FAB binding to S128N CD33

Antibody S128N Human CD33 Human CD33 ka(1/Ms) ka(1/s) K _D (nM) ka(1/Ms) ka(1/s) K _D (nM) ADI-10152 2.74e5 3.39e-2 124 2.46e5 9.14e-3 37.2 ADI-10154 1.93e5 5.39e-2 279 1.92e5 8.33e-3 43.4 ADI-10155 1.87e5 1.59e-2 85 1.70e5 6.85e-3 40.2 ADI-10157 5.65e4 9.24e-3 163 2.82e6 7.60e-3 2.69 ADI-10158 5.73e5 4.17e-2 72.8 4.75e5 1.49e-2 3.14 ADI-10160 7.86e5 6.73e-2 85.7 6.37e5 2.69e-2 42.2 ADI-10161 6.05e5 3.85e-2 63.6 5.74e5 1.18e-2 20.6 ADI-10163 5.07e5 7.48e-2 147 5.15e5 2.75e-2 53.5 ADI-10164 1.93e5 5.39e-2 279 3.19e5 1.01e-2 31,5 ADI-10165 6.41e5 5.82e-2 90.7 5.91e5 1.88e-2 31.8 ADI-10167 1.28e6 1.27e-1 98.6 1.25e6 3.07e-2 24.4 ADI-10168 4.04e5 4.69e-2 116 3.75e5 1.75e-2 46.6 ADI-10173 1.83e5 3.42e-2 187 2.12e5 1.25e-2 58.8 ADI-11802 6.62e5 5.90e-4 0,809 4.9e5 1.54e-3 3.14 ADI-11812 5.56e5 1.12e-3 2.01 3.96e5 4.86e-4 1.23 ADI-11825 2.10e6 6.49e-3 3.09 2.13e6 3.8e-3 1.78 ADI-11826 2.17e6 5.74e-3 2.65 1.76e6 4.63e-3 2.63 ADI-11828 1.37e6 7.42e-3 5.41 1.32e6 3.51e-3 2.67 ADI-11839 9.63e5 2.84e-2 29,5 8.74e5 1.62e-2 18.6

ADI-11815 recognizes a unique conformational epitope.

The binding epitope of the CD33 binding domain of ADI-11815 was analyzed. Figure 6 and Table 17 demonstrate that ADI-11815 has a unique conformational epitope. This antibody binds to the full-length human CD33 ECD but not to individual domains and is not cross-blocked by lintuzumab.

Table 17. Kinetic parameters of Fab ADI-11815 binding to different domains and

SNP R69G human CD33

Human CM3 V-domain S-to me ONP R6K CM3 human I ί s (1/Md ko IV4 to |Ш| k. to to (Pms) _more | |nm| k, to to tlVMd |1Л| |нМ| Asp to 11/4 to |nM| AD]11M5 3.3^5 7.91 ¢-5 0.23 Lack of connection bath Lack of connection bath 1.3Y-3 4.34 No

ADI-11801 binds to a unique epitope that includes R69.

The binding epitope of the CD33 binding domain, including ADI-11815, was analyzed. Figure 7 and Table 18 show the epitope on CD33 that is recognized by ADI-11801. Its binding to the human CD33 ECD is abolished by the R69G mutation.

Table 18. Kinetics of binding of Fab ADI-11801 to different domains of CD33 and R69G SNP

Human CD33 V-domain C-domain OHP R69G CD33 human HI μι “And |1/ms| to IM4 to ( .14 ) to IM4 ko (-Ki) to |i/mc| to IVc| to ClM J to |1/MC| to IVc| to 1-14 ) 4)1-11801 7.56t5 4.17^4 0.55 1.80c6 1.25c-3 0.69 Lack of connection vanya Lack of connection vanya No

Example 3. Evaluation of TriNKET binding to a cell expressing human NKG2D

The ability of TriNKETs, which include an NKG2D-binding domain and a CD33-binding domain, to bind to NKG2D was analyzed. EL4 cells transduced with human NKG2D cells and human KHYG-1 cells were used to test binding to cell-expressed human NKG2D. TriNKETs were diluted to maximal concentrations and then serially. Dilutions of mAb or TriNKETs were used to stain cells and binding of TriNKETs or mAbs was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and MFI of binding was normalized to secondary antibody controls to obtain fold-change values over background.

Figures 10A-10B show the binding of CD33-targeted TriNKETs to human NKG2D expressed on EL4 (top) or KHYG-1 (bottom) cells. The FOB binding signal was similar for both

- 187 049 630 for both EL4-hNKG2D and KHYG-1 cells, the binding rank order between clones was also conserved across the two cell lines. Fig. 10A shows the binding of CD33-targeted TriNKETs to human NKG2D expressed on EL4 cells. Fig. 10B shows the binding of CD33-targeted TriNKETs to human NKG2D expressed on KHYG-1 cells. The FOB binding signal was similar for both EL4-hNKG2D and KHYG-1 cells, the binding rank order between clones was also conserved across the two cell lines.

Example 4. Evaluation of TriNKET or mAb binding to a cell expressing human cancer antigens

The ability of TriNKETs, which include an NKG2D-binding domain and a CD33-binding domain, to bind to CD33 was analyzed. Molm-13, a human AML cell line, was used to assess binding of mAbs to cell-surface expressed CD33. The mAb was diluted to 2 μg/ml and dilutions of the mAb were used to stain cells. Bound antibody was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and binding to cell-expressed CD33 was compared with stained isotypes and unstained cell populations.

Human cancer cell lines expressing CD33 were used to assess binding to the tumor antigen TriNKET derived from various clones targeting NKG2D. The human AML cell line Molm-13 was used to assess TriNKET binding to cells expressing CD33. TriNKET was diluted and incubated with the respective cells. TriNKET binding was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry, and MFI of binding to cell-expressed CD33 was normalized to secondary antibody controls to obtain fold-over values.

The ability of TriNKET CD33 to bind CD33 expressed on Molm-13 cells was tested. Figure 8 shows the binding of CD33-targeted TriNKETs to Molm-13 cells. The MFI of binding of six anti-CD33 antibodies to CD33 expressed on Molm-13 cells is shown. All six antibodies bound to cell-expressed CD33. Five of the six antibodies exhibited a higher MFI binding signal compared to lintuzumab.

The ability of CD33 TriNKETs to induce resting NK cell-mediated killing of Molm-13 AML cells was tested. Four different CD33-targeting domains were used with five NKG2D-targeting domains to generate a total of 20 different TriNKETs. Regardless of the NKG2D-targeting domain used in a TriNKET, binding to CD33 was conserved for one CD33-targeting domain.

Example 5. Evaluation of TriNKET or mAb internalization

Internalization of TriNKET following binding to cell surface CD33 was analyzed. Molm-13, a human AML cell line, was used to assess the internalization of mAbs bound to cell surface expressed CD33. The mAbs were diluted to 2 μg/mL and the mAb dilutions were used to stain the cells. Following surface staining, CD33 samples were split, half of the sample was placed at 37°C overnight to facilitate internalization and the other half of the sample-bound antibody was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were fixed after secondary antibody staining and stored at 4°C overnight for analysis the following day. After 24 h at 37°C, samples were removed from the incubator and the bound antibody on the cell surface was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Samples were fixed and all samples were analyzed on the same day. Antibody internalization was calculated as follows: % internalization = (1-(MFI of sample at 24 h/initial MFI))*100%.

Figure 9 shows the internalization of anti-CD33 antibodies bound to the surface of Molm-13 cells after 24 h. All anti-CD33 antibodies showed similar internalization after 24 h. Lintuzumab showed slightly higher internalization compared to the other anti-CD33 antibodies.

Example 6. Activation of primary NK cells using TriNKET

The ability of TriNKETs, which include an NKG2D-binding domain and a CD33-binding domain, to activate primary NK cells was investigated. PBMCs were isolated from human peripheral blood buffy coats by density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a magnetic bead negative selection method and the purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were cultured in medium containing 100 ng/ml IL-2 for activation or starved overnight of the cytokine. IL-2-activated NK cells were used after 24-48 h; resting NK cells were always used the day after purification.

Human cancer cell lines expressing a cancer target of interest, with

- 188 049630 were harvested from the culture and the cells were adjusted to 2 x 10 ⁶ /mL. Monoclonal antibodies or TriNKETs targeting the cancer target of interest were diluted in the culture medium. Resting and/or activated NK cells were harvested from the culture, the cells were washed and resuspended at 2 x 10 ⁶ /mL in the culture medium. IL-2 and fluorophore-conjugated anti-CD107a were added to the NK cells to activate the culture. Brefeldin-A and monensin were diluted in the culture medium to block protein trafficking out of the cell for intracellular cytokine staining. To a 96-well plate, 50 μL of tumor targets, mAb/TriNKET, BFA/monensin and NK cells were added to achieve a total culture volume of 200 μL. The plate was cultured for 4 hours before samples were prepared for FACS analysis.

After 4 h of culture activation, cells were prepared for flow cytometric analysis using fluorophore-conjugated antibodies to CD3, CD56, and IFNy. CD107a and IFNy staining were analyzed in CD3-CD56+ populations to assess NK cell activation.

Figure 12 shows TriNKET-mediated activation of resting human NK cells in coculture with CD33-expressing THP-1 AML cells. Human NK cell activation was assessed using IFNγ production and CD107a degranulation as activation markers. All TriNKETs and mAbs activated human NK cells above isotype controls. Similar activity was observed with the four different CD33 antibodies. TriNKET activity was dependent on the NKG2D-targeting clone, with some clones providing better TriNKET-mediated activation than others. NKG2D clones had similar activity with each of the CD33-targeting domains.

Example 7. Primary assay of human NK cell cytotoxicity

The ability of TriNKETs, which include an NKG2D-binding domain and a CD33-binding domain, to induce NK cell cytotoxicity against CD33-expressing cells was investigated. PBMCs were isolated from human peripheral blood buffy coats by density gradient centrifugation. Isolated PBMCs were washed and prepared for NK cell isolation. NK cells were isolated using a magnetic bead negative selection method and the purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NK cells were cultured in medium containing 100 ng/ml IL-2 or starved overnight of the cytokine. IL-2-activated or resting NK cells were used the following day in cytotoxicity assays.

KHYG-1 cells were maintained in 10% HI-FBS-RPMI-1640 with 10 ng/ml IL-2. The day before use as effector cells in killing assays, KHYG-1 cells were harvested from culture and the cells were washed out of the IL-2 containing medium. After washing, KHYG-1 cells were resuspended in 10% HIFBS-RPMI-1640 and left overnight without cytokine.

DELFIA Cytotoxicity Assay

Human cancer cell lines expressing the target of interest were harvested from culture, washed with HBS, and resuspended in growth medium at a concentration of ¹⁰⁶ /mL for labeling with BATDA reagent (Perkin Elmer AD0116). The manufacturer's instructions were followed for labeling target cells. After labeling, cells were washed three times with HBS and resuspended in growth medium at a concentration of 0.5 x 1.0 x 105/mL. To prepare background wells, an aliquot of labeled cells was set aside and the cells were centrifuged from the medium. 100 μL of medium was carefully added to the wells in triplicate to avoid damaging the pelleted cells. 100 μL of BATDA-labeled cells were added to each well of a 96-well plate. Wells were saved to allow spontaneous release from target cells and wells were prepared for maximal target cell lysis by addition of 1% Triton-X. Monoclonal antibodies or TriNKET against the tumor target of interest were diluted in culture medium and 50 μl of the diluted mAb or TriNKET were added to each well. Resting and/or activated NK cells were collected from the culture, cells were washed and resuspended at 105-2.0 ^{x 106} /ml in culture medium depending on the desired E:T ratio. 50 μl of NK cells were added to each well of the plate to give a total culture volume of 200 μl. The plate was incubated at 37°C with 5% _CO2 for 2-3 h prior to assay development.

After culturing for 2-3 h, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200 g for 5 min. 20 μl of the culture supernatant was transferred to a clean microplate provided by the manufacturer and 200 μl of room temperature europium solution were added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 min. Data were read from the plate using a Victor 3 or SpectraMax i3X instrument. % specific lysis was calculated as follows: % specific lysis = ((experimental release - spontaneous release) / (Maximum release Spontaneous release)) * 100%.

Figures 13 and 4 show human NK cell killing of Molm-13 (Figure 13) and THP-1 (Figure 14) target cells mediated by CD33-targeted TriNKETs. Human NK cell killing of Molm-13 AML target cells mediated by CD33-targeted TriNKETs was analyzed.

- 189 049630 (Fig. 13). Resting NK effector cells were used with Molm-13 target cells. Activated human effector NK cells produced higher background killing compared to resting human effector NK cells. With effector NK cells, TriNKETs were able to enhance lysis against Molm-13 AML target cells. Similar activity of each TriNKET was observed with resting and activated human NK cells as well as with Molm-13 target cells.

The killing of THP-1 AML target cells by human NK cells mediated by CD33-targeted TriNKETs was analyzed (Fig. 14). Activated human effector NK cells were used with THP-1 cells (Fig. 14), resulting in higher background killing compared to resting human effector NK cells. With effector NK cells, TriNKETs were able to enhance lysis against THP-1 AML target cells. Similar activity of each TriNKET was observed with resting and activated human NK cells, as well as with THP-1 target cells.

Thus, with effector NK cells, TriNKETs were able to enhance lysis against the AML target cells Molm-13 (Fig. 13) and THP-1 (Fig. 14). Similar activity of each TriNKET was observed with resting and activated human NK cells, as well as with the target cells Molm-13 (Fig. 13) and THP-1 (Fig. 14).

Figures 15A, 16, and 17A show the killing of human AML target cells Molm-13 (Figure 15A), EOL-1 (Figure 16), and THP-1 (Figure 17A) by KHYG-1 effector cells, respectively. KHYG-1 cells were shown to express surface NKG2D but not CD16. Therefore, TriNKET-mediated killing here is dependent on NKG2D-mediated activation of KHYG-1 effector cells. TriNKETs were able to mediate the killing of all three human AML target cell lines by KHYG-1 effector cells. Similarly, TriNKET activity was demonstrated on all three target cell lines.

TriNKET-mediated cytotoxicity of resting human NK cells was also tested. Figures 15B and 17B show that TriNKETs also mediated cytotoxicity of resting human NK cells against Molm-13 (Figure 15B) and THP-1 (Figure 17B) human AML cells. Figure 15B shows that TriNKETs mediated killing of Molm-13 AML cells by resting human NK cells. In Figure 15B, the ratio of resting human effector NK cells (E) to target cells (T) (E:T) was 10:1. The E:T ratio may reflect differences in the maximum % lysis.

TriNKETs also mediated killing of THP-1 target cells that express high-affinity FcγRI by resting human NK cells. Figure 17B shows that TriNKETs mediated killing of THP-1 AML cells by resting human NK cells with an E:T of 5:1.

Example 8. Evaluation of TriNKET Binding to Cells Expressing Human NKG2D 107 mAb was identified as a monoclonal antibody with high affinity binding to CD33. The amino acid sequences of the heavy and light chains of 107-F405L, an Fc variant of 107, are shown below. 107-F405L mAb included a Leu to Phe substitution at position 405 (according to EU numbering) in the CH3 Fc domain. Lys may optionally be included at the C-terminus of the heavy chain.

Heavy Chain mAh 107-F405L

EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQ DGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[SEQ ID NO:199]

Light chain mAh 107-F4№L

DIQMTOSPSTLSASVGDRVTITCRASOSISSWLAWYOQKPGKAPKLLIYEASSLES GVPSRFSGSGSGTEFTLTISSLOPDDFATYYCOOSQSYPPrrFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[SEQ ID NO:200]

A49-F3'-TriNKET-107, a derivative of TriNKET mAb 107, is described in the P-Multispecific Binding Proteins section. The amino acid sequences of this TriNKET are shown in SEQ ID NOs: 187, 189, and 190.

The ability of A49-F3'-TriNKET-107 to bind to NKG2D was assessed using EL4 cells transduced with human NKG2D (EL4-hNKG2D). Briefly, mAbs 49-F3'TriNKET-107 and 107-F405L were serially diluted. EL4-hNKG2D cells were incubated with diluted TriNKET or mAb solutions. Binding of TriNKET or mAb to EL4 cells was detected using a secondary

- 190 049630 fluorophore-conjugated anti-human IgG antibody. Cells were analyzed by flow cytometry and fold-over values were calculated by normalizing the MFI of binding to the MFI of a control group in which cells were incubated with secondary antibody only.

As shown in Fig. 35, 107-F405L mAb showed no binding to EL4-hNKG2D cells. A49-F3'-TriNKET-I07 showed weak binding to EL4-hNKG2D cells, not reaching saturation even at high concentrations of 100 μg/mL.

Example 9. Evaluation of TriNKET binding to cells expressing human CD33

The ability of A49-F3'-TriNKET-107 to bind to CD33 was assessed using human cancer cell lines expressing CD33. Briefly, human AML cell lines Mv4-11 and Molm-13, which expressed CD33, were incubated with serially diluted solutions of A49-F3'-TriNKET-107 and 107-F405L mAb. Binding of A49-F3'-TriNKET-107 or 107-F405L mAb to AML cells was detected using a fluorophore-conjugated anti-human IgG secondary antibody. Cells were analyzed by flow cytometry and fold changes over background were calculated by normalizing the MFI of binding to the MFI of a control group in which cells were incubated with the secondary antibody only.

As shown in Fig. 36A and 36B, A49-F3'-TriNKET-107 exhibited a three- to fourfold decrease in binding affinity compared to c107-F405L mAb on both Molm-13 and Mv4-11 cells. A49-F3'-TriNKET-107 was also found to bind to cells at a higher maximum fold relative to background compared to 107-F405L mAb.

Example 10. TriNKET Internalization Assessment

Internalization of A49-F3'-TriNKET-107 and 107-F405L mAbs to CD33 was assessed using human AML cell lines EOL-1 and Molm-13 that express CD33 on the cell surface. Briefly, cells were incubated with 2 μg/ml A49-F3'-TriNKET-107 or 107-F405L mAb for 20 min at room temperature. Cell samples were then divided into three aliquots. The first and second aliquots were placed at 37°C for 2 and 24 h, respectively, to allow antibody internalization. Cells were then incubated with fluorophore-conjugated anti-human IgG secondary antibody and fixed for flow cytometric analysis. A third portion of the cell samples used to establish the baseline were incubated with a fluorophore-conjugated anti-human IgG secondary antibody without incubation at 37°C. Cells were fixed after secondary antibody staining and stored at 4°C for analysis the following day (when the first half of the samples were ready). The amount of A49-F3'-TriNKET-107 and 107-F405L mAb bound to the cell surface was analyzed by flow cytometry on the same day. Antibody internalization was calculated as follows: % internalization = (1 - (MFI of 24-hour sample / MFI of initial sample)) x 100%.

As shown in Fig. 37A and 37B, the internalization of A49-F3'-TriNKET-107 and 107-F405L mAb after CD33 exposure increased over time in EOL-1 and Molm-13 cells. In both cells, 107F405L mAb was internalized faster and to a greater extent than A49-F3'-TriNKET-107.

Example 11. Activation of primary NK cells using TriNKET

The ability of A49-F3'-TriNKET-107 to induce cytotoxicity of primary NK cells against human AML cells was assessed using the DELFIA cytotoxicity assay. Briefly, PBMCs were isolated from human peripheral blood buffy coats by density gradient centrifugation. Isolated PBMCs were washed and NK cells were isolated using a magnetic bead negative selection method. The purity of isolated NK cells was typically >90% CD3CD56+. Isolated NK cells were starved overnight of cytokine.

The next day, human AML cell lines Molm-13, THP-1 and EOL-1 were harvested from the culture. AML cells were washed with HBS and resuspended in growth medium at a concentration of 106 cells/mL for labeling with BATDA reagent (Perkin Elmer AD0116) according to the manufacturer's instructions. After labeling, AML cells were washed three times with HBS and resuspended in culture medium at a concentration of 0.5-1.0x105 cells/mL. 100 μL of BATDA-labeled cells were added to each well of a 96-well plate.

The TriNKETs or mAbs to be tested were diluted in culture medium and 50 μl of the diluted TriNKETs or mAbs were added to each well. Resting NK cells were collected from the culture, washed and resuspended at 105 -2.0 x 106 cells/ml in culture medium depending on the desired E:T ratio of 5:1. 50 μl of NK cells were added to each well of the plate to give a total culture volume of 200 μl. The plate was incubated at 37°C with 5% _CO2 for 2-3 h.

After culturing, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200xg for 5 min. 20 μl of the culture supernatant was transferred to a clean microplate provided by the manufacturer. The supernatant from labeled cells incubated alone was used to measure spontaneous TDA release. The supernatant of labeled cells incubated with 1% Triton-X was used to measure maximal lysis of target cells. The supernatant from labeled cells before incubation for 2-3 h was used for background measurements and quality control purposes.

- 191 049630

200 μl of room temperature europium solution was added to each well containing the culture supernatant. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 min. Fluorescence was measured using a Victor 3 or SpectraMax i3X instrument.

Fluorescence levels represent target cell lysis. % specific lysis values were calculated as follows: % specific lysis = ((experimental release - spontaneous release)/(maximum release - spontaneous release) x 100%).

A49-F3'-TriNKET-107 and several mAbs were tested in this assay. The mAbs include 107-F405L mAb, 107-DE mAb, lintuzumab-GA, and 280-31-01(mut)DE. 107-DE mAb is a variant of 107 mAb with S239D and I332E substitutions in Fc to enhance ADCC activity (shown in bold and underlined in the sequence below). The amino acid sequence of the 107-DE heavy chain is shown below, optionally with a Lys at the C terminus.

Heavy chain mAb 107-DE

EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQ

IDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGO GTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[SEQ ID NO:201]

The light chain of mAb 107-DE is identical to the light chain of mAb 107-F405L [SEQ ID NO: 200].

280-31-01 (mut)-DE is a variant of the 280-31-01 (mut) antibody clone described in WO2012045752 with S239D and I332E substitutions in the Fc to enhance ADCC activity (shown in bold and underlined in the sequence below). The amino acid sequences of the heavy and light chains of 280-31-01 (mut)-DE are shown below, optionally with a Lys at the C-terminus of the heavy chain.

Heavy chain 2S0-31-Ol(mut)-DE

OVOLVOSGAEVKKPGSSVKVSCKASGGTFSDYAISWVROAPGOGLEWMGRIIPI LGVADYAOKFOGRVTITADKSTRTAYMELSSLRSEDTAVYYCARNWADAFDIWGQGT MVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:202)

Light chain 2£0-31-Ol(mut)-DE

DIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFDSSITFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:203)

SNPs in the CD16 gene can result in V158 or F158 variants of the human CD16 protein. CD16 F158 is known to have reduced CD16 Fc binding affinity than CD16 V158, thereby reducing antibody-dependent cell-mediated cytotoxicity (ADCC). Consequently, NK cells expressing CD16-F158 are less sensitive to CD16 stimulation than NK cells expressing CD16-V158. Indeed, as shown in Fig. 38A, mAb 107-F405L resulted in only low levels of killing of Molm-13 cells by NK cells expressing only the low-affinity variant of CD16 (CD16 ^F/F ). In comparison, A49-F3'-TriNKET-107 mediated more potent NK cell killing with higher specific lysis of Molm-13 cells, likely due to its additional ability to interact with NK cells via NKG2D binding. Similarly, when incubated with NK cells that carried this SNP on one allele (CD16 ^V/F ), A49F3'-TriNKET-107 was more potent in killing EOL-1 cells than 107F405L mAb (Fig. 38B).

THP-1 cells express FcγRI, which can bind to IgG1 Fc with high affinity. Competition for Fc binding by target cells may further reduce NK cell killing. Therefore, as shown in Fig. 38C and 38D, when THP-1 cells were incubated with NK

- 192 049630 CD16 ^F/F cells in the presence of 107-F405L or lintuzumab-GA mAb, no specific lysis of THP-1 cells was observed. Even 107-DE, an ADCC-enhancing variant of 107, failed to induce NK cell cytotoxicity. Among the mAbs tested, only 280-31-01(mut)-DE, an ADCC-enhancing variant of the 280-31-01 (mut) antibody clone described in WO2012045752, showed cell killing activity at high concentrations. Notably, A49-F3'-TriNKET107 mediated more potent NK cell killing with higher specific lysis of THP-1 cells, likely due to its additional ability to interact with NK cells via NKG2D binding.

Example 12. Activation of primary CD8+ T cells using TriNKET

NKG2D is expressed on NK cells and many T cells, including CD8+ T cells. The ability of A49-F3'-TriNKET-107 to induce CD8+ T cell cytotoxicity against human AML cells was assessed using the DELFIA cytotoxicity assay.

Briefly, PBMCs were isolated from human peripheral blood buffy coats by density gradient centrifugation. Isolated PBMCs were stimulated with 1 μg/ml concanavalin A (ConA) at 37°C for 18 h. ConA was then removed and PBMCs were cultured with 25 U/ml IL-2 at 37°C for 4 days. CD8+ T cells were purified by magnetic bead negative selection and then cultured in medium containing 10 ng/ml IL-15 at 37°C for 7-13 days.

The primary human effector CD8+ T cells obtained above were characterized for cellular markers. The cells were stained with fluorophore-conjugated antibodies against CD3, CD8, NKG2D, and CD16 and analyzed by flow cytometry. As shown in Fig. 39, the isolated CD8+ T cells were highly pure, as more than 99% of them were positive for CD3, CD8, and NKG2D expression and negative for CD16 expression.

To assess the ability of A49-F3'-TriNKET-107 to induce cytotoxicity in primary CD8+ T cells, Molm-13 cells were harvested from culture, washed, and resuspended in culture medium at a concentration of 106 cells/ml. Cells were labeled with BATDA reagent (Perkin Elmer AD0116) according to the manufacturer's instructions. After labeling, cells were washed three times with HBS and resuspended in culture medium at a concentration of 0.5x105 cells/ml. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. 50 μl of serially diluted monoclonal antibodies or TriNKET were added to each well.

CD8+ effector T cells were harvested from the culture, washed and resuspended at 5x106 cells/ml in culture medium. 50 μl of CD8+ T cells were added to each well of the plate to achieve an E:T ratio of 50:1 and a total culture volume of 200 μl. The plate was incubated at 37°C with 5% _CO2 for 3.5 h. After incubation, the cells were pelleted by centrifugation at 500xg for 5 min. 20 μl of the culture supernatant was transferred to a clean microplate provided by the manufacturer. The supernatant from labeled cells incubated alone was used to measure spontaneous TDA release. The supernatant of labeled cells incubated with 1% Triton-X was used to measure maximal target cell lysis.

200 μl of room temperature europium solution was added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 min. Fluorescence was measured using a SpectraMax i3X instrument.

Fluorescence levels represent target cell lysis. % specific lysis values were calculated as follows: % specific lysis = ((experimental release - spontaneous release)/(maximum release - spontaneous release) x 100%).

As shown in Figs. 40A and 40B, A49-F3'-TriNKET-107 enhanced the cytotoxic activity of primary human CD8+ T cells in a dose-dependent manner. A49-F3'-TriNKET-H76, a protein described in Section II - Multispecific Binding Proteins (having polypeptides comprising the sequences of SEQ ID NOS: 197, 189, and 190), was also active under these conditions, but exhibited less activity than A49-F3'-TriNKET-107. Monoclonal antibody 107-F405L and non-targeted TriNKET did not exhibit this activity.

Example 13. TriNKET binding to monocytes

CD33 expression on blood cells was assessed by flow cytometry using the method described in Example 9. Briefly, human whole blood was incubated with A49-F3'-TriNKET-107 or a human IgG1 isotype control antibody conjugated to a fluorophore. Binding of A49-F3'TriNKET-107 or the isotype control antibody was detected by flow cytometry. To assess binding levels on specific cell types, fluorophore-conjugated antibodies that bind to surface markers of NK cells, CD8+ T cells, CD4 ⁺ T cells, B cells, and monocytes were added to the incubation, and the presence or absence of binding of these antibodies was used to gate the flow cytometric data analysis.

As shown in Fig. 41A-41E, the binding of A49-F3'-TriNKET-107 to NK cells was weak compared to the non-targeting human IgG1 isotype control antibody, whereas

- 193 049630 strong binding of TriNKET to CD33+ monocytes.

Example 14. Long-term NK cell cytotoxicity mediated by TriNKET

NK cells have the natural ability to sense and kill transformed or stressed cells but not healthy cells. We tested the ability of A49-F3'-TriNKET-107 to preserve the natural selective cytotoxicity of NK cells using Molm-13 AML cells and primary human monocytes as target cells. Molm-13 cells were obtained from the DSMZ cell bank. Primary human monocytes were isolated from human peripheral blood. Briefly, PBMCs were isolated from human peripheral blood buffy coats by density gradient centrifugation. Monocytes were isolated by negative selection. CD33 expression on primary human monocytes and Molm-13 AML cells was confirmed by flow cytometric analysis (Figs. 42A–42B).

Primary human NK cells were isolated from human peripheral blood. Briefly, PBMCs were isolated from human peripheral blood buffy coats by density gradient centrifugation. NK cells were isolated by negative selection. To distinguish target cells from NK cells in coculture, target cells were labeled with a fluorescent tag. Specifically, isolated monocytes were labeled with IncuCyte CytoLight Rapid live cell labeling reagent according to the manufacturer's recommendations. Molm-13 cells were infected with a lentivirus encoding nuclear GFP, and cells with stable expression were selected with puromycin.

Isolated NK cells and target cells were mixed at an E:T ratio of 10:1 in the presence of 20 nM A49-F3'-TriNKET-107. Non-specific activation of NK cells in co-cultures was performed in parallel as a positive control for AML cell killing. The mixture was added to an Ibidi µ-slide. Time-lapse images for the phase and green channels were collected every hour, 3 images per sample, using an IncuCyte S3 instrument. Images were analyzed using IncuCyte S3 software. Live target cells were detected by green fluorescence, and the number of green cells at each time point was normalized to the number of green cells at time 0 from the same sample.

As shown in Fig. 43A and 43B, Molm-13 AML cells were able to proliferate in the presence of NK cells alone, but the growth of target cells was significantly inhibited by A49-F3'-TriNKET-107. In contrast, A49-F3'-TriNKET-107 did not mediate human NK cell killing of normal monocytes in long-term co-culture. The activity of A49-F3'-TriNKET-107 was similar to that of PMA+ionomycin, which also retains the natural selectivity of NK cells. These results suggest that A49-F3'-TriNKET-107 selectively compromised cancer cells and potentially had a broad therapeutic window.

Inclusion by reference

The entire disclosure of each of the patent documents and scientific articles referenced in this document is incorporated by reference for all purposes.

Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the above embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims (24)

1. Protein containing: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises a heavy chain complementarity determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO: 111, a heavy chain complementarity determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO: 112, and a heavy chain complementarity determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO: 113; and a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 114, a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 115, and a light chain CDR3 sequence comprising the amino acid sequences of SEQ ID NO: 116; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 45, a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 46, and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 47; and a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 48, a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 50. - 194 049630 chain comprising the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 50; and (c) an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, wherein the Fab fragment of said first antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, and wherein the scFv of said second antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16. 2. Protein containing: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises a heavy chain complementarity determining region 1 (CDR1) sequence comprising the amino acid sequence of SEQ ID NO: 192, a heavy chain complementarity determining region 2 (CDR2) sequence comprising the amino acid sequence of SEQ ID NO: 112, and a heavy chain complementarity determining region 3 (CDR3) sequence comprising the amino acid sequence of SEQ ID NO: 193; and a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 114, a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 115, and a light chain CDR3 sequence comprising the amino acid sequences of SEQ ID NO: 116; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 181, a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 46, and a heavy chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 182; and a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 48, a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 49, and a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 50; and (c) an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, wherein the Fab fragment of said first antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, and wherein the scFv of said second antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16. 3. Protein containing: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 88; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10, wherein the heavy chain variable region of said scFv further comprises one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 9 located within the framework region of the heavy chain variable domain of said scFv, and wherein the light chain variable region of said scFv further comprises one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 10 located within the framework region of the light chain variable domain of said scFv; and (c) an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, wherein the Fab fragment of said first antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, and wherein the scFv of said second antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16. 4. The protein according to claim 1 or 2, wherein said Fab fragment of said first antigen-binding site that binds NKG2D comprises an amino acid sequence of a heavy chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 87, and an amino acid sequence of a light chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 88; 5. The protein of claim 1 or 2, wherein the second antigen-binding site that binds CD33 comprises an amino acid sequence of the heavy chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 9; and an amino acid sequence of the light chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 10. - 195 049630 6. The protein of any one of claims 1 to 5, wherein the C-terminus of said Fab fragment of said first antigen-binding site is linked to an Fc domain of an antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16. 7. The protein according to any one of claims 1 to 6, wherein the variable domain of the light chain of the scFv of the second antigen-binding site is located at the N-terminus of the variable domain of the heavy chain of the scFv. 8. The protein according to any one of claims 1 to 7, wherein the variable domain of the light chain of the scFv of the second antigen-binding site is linked to the variable domain of the heavy chain of the scFv via a linker and wherein the linker comprises the amino acid sequence of SEQ ID NO: 186. 9. The protein according to any one of claims 1 to 8, wherein a disulfide bridge is formed between the variable domain of the heavy chain of the scFv of said second antigen-binding site and the variable domain of the light chain of the scFv of said second antigen-binding site. 10. The protein of claim 9, wherein said disulfide bridge is formed between C44 of the variable domain of the heavy chain and C100 of the variable domain of the light chain, numbered according to Kabat numbering. 11. The protein of claim 9, wherein said disulfide bridge is formed between said one amino acid substitution in the variable domain of the heavy chain of the scFv of said second antigen-binding site and said one amino acid substitution in the variable domain of the light chain of the scFv of said second antigen-binding site. 12. The protein of any one of claims 3 and 6-11, wherein said one amino acid substitution in the variable domain of the heavy chain of the scFv of said second antigen-binding site is G44C, numbered according to Kabat numbering, and said one amino acid substitution in the variable domain of the light chain of the scFv of said second antigen-binding site is G100C, numbered according to Kabat numbering. 13. The protein of any one of claims 1 to 12, wherein the C-terminus of the variable domain of the heavy chain of the scFv of the second antigen-binding site is linked to the N-terminus of the Fc domain of the antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16. 14. The protein of any one of claims 1 to 13, wherein the C-terminus of the variable domain of the heavy chain of the scFv of the second antigen-binding site is linked to the N-terminus of the Fc domain of the antibody sufficient to bind CD16, or a portion thereof sufficient to bind CD16, via a linker containing Ala-Ser or Gly-Ala-Ser. 15. The protein according to any one of claims 1 to 14, wherein the scFv of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO: 188. 16. The protein of any one of claims 1 to 15, wherein the Fc domain of the antibody sufficient to bind CD16 comprises the hinge region and the CH2 domain of a human IgG1 antibody. 17. The protein of any one of claims 1 to 16, wherein the Fc domain of the antibody sufficient to bind CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody, according to EU numbering. 18. The protein of any one of claims 1 to 17, wherein the Fc domain of the antibody sufficient to bind CD16 comprises an amino acid sequence that is at least 90% identical to the Fc domain of human IgG1 and differs from the Fc domain of human IgG1 in one or more positions Q347, Y349, T350, L351, S354, D356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 or K439, according to EU numbering. 19. The protein of any one of claims 1 to 17, wherein the Fc domain of the antibody sufficient to bind CD16 is the Fc domain of human IgG1 containing substitutions to form a heterodimer. 20. The protein of any one of claims 1 to 17, wherein the Fc domain of the antibody sufficient to bind CD16 is the Fc domain of human IgG1 containing the substitutions Y349C, K36OE and K409W, according to EU numbering. 21. The protein of any one of claims 1 to 17, wherein the Fc domain of the antibody sufficient to bind CD16 is the Fc domain of human IgG1 comprising the substitutions S354C, Q347R, D399V and F405T, according to EU numbering. 22. The protein according to any one of claims 1 to 21, wherein the protein comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 189; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 187; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 190, wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, wherein the third polypeptide is linked to the first polypeptide via a disulfide bond. 23. A composition for treating a cancer expressing CD33, comprising a protein according to any one of claims 1 to 22. 24. A pharmaceutical composition for treating cancer expressing CD33, comprising the protein of any one of claims 1 to 22 and a pharmaceutically acceptable carrier.