EA046221B1 - CD33-BINDING POLYPEPTIDES AND THEIR APPLICATIONS - Google Patents

Description

Cross reference to related applications

This application claims the benefit of U.S. Provisional Application No. 62/843,408, filed May 4, 2019, and U.S. Provisional Application No. 62/844,359, filed May 7, 2019, each of which is incorporated herein by reference for all purposes.

Field of technology to which the invention relates

The present invention relates to CD33-binding polypeptides and methods of using CD33-binding polypeptides to modulate the biological activity of CD33. Such methods include, but are not limited to, methods for treating cancer.

State of the art

CD33, also known as p67, SIGLEC3 or SIGLEC-3, binds sialic acids and regulates inflammatory and immune responses of hematopoietic cells. CD33 may also influence the initiation, proliferation and progression of various hematological cancers. The CD33 antigen is expressed, for example, in more than 85% of acute myeloid leukemia (AML) cases. Thus, CD33 is a tumor-associated antigen suitable for targeted therapy of hematological cancers such as leukemia. Therefore, there is a therapeutic need for more potent CD33 antagonists.

The essence of the invention

Disclosed herein are CD33 binding polypeptides and methods of using CD33 binding polypeptides to treat, for example, leukemia. In some embodiments, the CD33 binding polypeptide contains at least one VHH domain. Some embodiments are presented below.

Embodiment 1. A polypeptide containing at least one VHH domain that binds to CD33 and which contains a CDR1 containing the amino acid sequence SEQ ID NO: 47, 3, 7, 11, 15, 19, 23, 27, 31, 35, 50, 53, 56, 59, 62, 65, 68 or 71; CDR2 containing the amino acid sequence SEQ ID NO: 48, 4, 8, 12, 16, 20, 24, 28, 32, 36, 51, 54, 57, 60, 63, 66, 69 or 72; and CDR3 containing the amino acid sequence SEQ ID NO: 49, 5, 9, 13, 17, 21, 25, 29, 33, 37, 52, 55, 58, 61, 64, 67, 70, or 73.

Embodiment 2. The polypeptide of Embodiment 1, wherein at least one VHH domain contains a CDR1 containing the amino acid sequence of SEQ ID NO: 47 or 3; CDR2 containing the amino acid sequence of SEQ ID NO: 48 or 4; and CDR3 containing the amino acid sequence of SEQ ID NO: 49 or 5.

Embodiment 3. The polypeptide of Embodiment 1 or Embodiment 2, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 7; CDR2 containing the amino acid sequence SEQ ID NO: 8; and CDR3 containing the amino acid sequence of SEQ ID NO: 9.

Embodiment 4. The polypeptide of any one of embodiments 1-3, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 11 or 50; CDR2 containing the amino acid sequence of SEQ ID NO: 12 or 51; and CDR3 containing the amino acid sequence of SEQ ID NO: 13 or 52.

Embodiment 5. The polypeptide of any one of embodiments 1-4, wherein at least one VHH domain contains a CDR1 containing the amino acid sequence of SEQ ID NO: 15, 53 or 56; CDR2 containing the amino acid sequence of SEQ ID NO: 16, 54 or 57; and CDR3 containing the amino acid sequence of SEQ ID NO: 17, 55 or 58.

Embodiment 6. The polypeptide of any one of embodiments 1-5, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 19 or 59; CDR2 containing the amino acid sequence SEQ ID NO: 20 or 60; and CDR3 containing the amino acid sequence of SEQ ID NO: 21 or 61.

Embodiment 7. The polypeptide of any one of embodiments 1-6, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 23 or 62; CDR2 containing the amino acid sequence of SEQ ID NO: 24 or 63; and CDR3 containing the amino acid sequence of SEQ ID NO: 25 or 64.

Embodiment 8. The polypeptide of any one of embodiments 1-7, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 27 or 65; CDR2 containing the amino acid sequence of SEQ ID NO: 28 or 66; and CDR3 containing the amino acid sequence of SEQ ID NO: 29 or 67.

Embodiment 9. The polypeptide of any one of embodiments 1-8, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 31 or 68; CDR2 containing the amino acid sequence of SEQ ID NO: 32 or 69; and CDR3 containing the amino acid sequence of SEQ ID NO: 33 or 70.

Embodiment 10. The polypeptide of any one of embodiments 1-9, wherein at least one VHH domain comprises a CDR1 containing the amino acid sequence of SEQ ID NO: 35 or 71; CDR2 containing the amino acid sequence of SEQ ID NO: 36 or 72;

- 1 046221 and CDR3 containing the amino acid sequence SEQ ID NO: 37 or 73.

Embodiment 11. The polypeptide of any one of embodiments 1-10, wherein at least one VHH domain comprises CDR1, CDR2 and CDR3 containing, respectively, the amino acid sequences of SEQ ID NOs: 47, 48 and 49; SEQ ID NO: 3, 4 and 5; SEQ ID NO: 7, 8 and 9;

SEQ ID NO: 11, 12 and 13; SEQ ID NO: 15, 16 and 17; SEQ ID NO: 19, 20 and 21; SEQ ID NO: 23, 24 and 25;

SEQ ID NO: 27, 28 and 29; SEQ ID NO: 31, 32 and 33; SEQ ID NO: 35, 36 and 37; SEQ ID NO: 50, 51 and 52;

SEQ ID NO: 53, 54 and 55; SEQ ID NO: 56, 57 and 58; SEQ ID NO: 59, 60 and 61; SEQ ID NO: 62, 63 and 64;

SEQ ID NO: 65, 66 and 67; SEQ ID NO: 68, 69 and 70; or SEQ ID NO: 71, 72 and 73.

Embodiment 12. The polypeptide of any one of embodiments 1-11, wherein at least one VHH domain is humanized.

Embodiment 13. The polypeptide of any one of embodiments 1-12, wherein at least one VHH domain contains an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 99 identical. % amino acid sequence SEQ ID NO: 38, 114, 39, 40, 41, 42, 43, 44, 45, 46, 115, 116, 117, 118, 119, 120, 121 or 122.

Embodiment 14. The polypeptide of any one of embodiments 1-12, wherein at least one VHH domain contains the amino acid sequence SEQ ID NO: 38, 114, 39, 40, 41, 42, 43, 44, 45, 46, 115 , 116, 117, 118, 119, 120, 121 or 122.

Embodiment 15. The polypeptide of any one of embodiments 1-12, wherein at least one VHH domain contains an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or at least 99 identical. % amino acid sequence SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 123, 124, 125, 126, 127, 128, 129, 130 or 131.

Embodiment 16. The polypeptide of any one of embodiments 1-12, wherein at least one VHH domain contains the amino acid sequence SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 123, 124 , 125, 126, 127, 128, 129, 130 or 131.

Embodiment 17. The polypeptide of any one of embodiments 1-16, comprising two VHH domains.

Embodiment 18. The polypeptide of any one of embodiments 1-16, comprising three VHH domains.

Embodiment 19. The polypeptide of any one of embodiments 1-18, comprising at least one binding domain that binds an antigen other than CD33.

Embodiment 20. The polypeptide of Embodiment 19 comprising at least one binding domain that binds CD3, T-cell receptor (TCR)a, TCRe, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D.

Embodiment 21. The polypeptide of embodiment 17 or 18, wherein each VHH domain binds to CD33.

Embodiment 22. The polypeptide of Embodiment 21, wherein each VHH domain contains the same amino acid sequences CDR1, CDR2 and CDR3.

Embodiment 23. The polypeptide of Embodiment 21, wherein each VHH domain contains the same VHH sequence.

Embodiment 24. The polypeptide of any one of embodiments 1-16, comprising a single VHH domain.

Embodiment 25. The polypeptide of any one of embodiments 1-24, comprising an Fc region.

Embodiment 26. The polypeptide of Embodiment 25, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 74-109.

Embodiment 27. The polypeptide of Embodiment 25 or Embodiment 26 that forms a dimer under physiological conditions.

Embodiment 28. The polypeptide of any one of embodiments 1-27, wherein CD33 is human CD33.

Embodiment 29. The polypeptide of Embodiment 28, wherein human CD33 comprises the sequence SEQ ID NO: 1.

Embodiment 30. An immunoconjugate comprising the polypeptide of any one of embodiments 1-29 and a cytotoxic agent.

Embodiment 31. The immunoconjugate of Embodiment 30, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamycin, pyrrolobenzodiazepine, and enediyne antibiotic.

Embodiment 32. A pharmaceutical composition comprising the polypeptide of any one of Embodiments 1-29 or the immunoconjugate of Embodiment 30 or Embodiment 31 and a pharmaceutically acceptable carrier.

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Embodiment 33. An isolated nucleic acid encoding the polypeptide of any one of embodiments 1-29.

Embodiment 34. A vector containing the nucleic acid of Embodiment 33.

Embodiment 35. A host cell containing the nucleic acid of Embodiment 33 or the vector of Embodiment 34.

Embodiment 36. A host cell that expresses the polypeptide of any one of embodiments 1-29.

Embodiment 37. The method of producing a polypeptide as in any one of Embodiments 1-29, comprising incubating a host cell as in Embodiment 35 or Embodiment 36 under conditions suitable for expression of the polypeptide.

Embodiment 38. The method of Embodiment 37, further comprising isolating the polypeptide.

Embodiment 39. A method of treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of the polypeptide of any one of Embodiments 1-29, the immunoconjugate of Embodiment 30 or Embodiment 31, or the pharmaceutical composition of Embodiment 32.

Embodiment 40. The method of Embodiment 39, wherein the cancer is selected from lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade/follicular non-Hodgkin's lymphoma (NHL); NHL from small lymphocytes (ML); moderate malignancy/follicular NHL; diffuse NHL of moderate malignancy; immunoblastic NHL of high grade; high-grade lymphoblastic NHL; NHL from small non-divided cells with a high degree of malignancy; volumetric NHL; mantle cell lymphomas; AIDS-related lymphoma; Waldenström's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; and chronic myeloblastic leukemia.

Embodiment 41. The method of embodiment 39 or 40, wherein the cancer is acute myeloid leukemia (AML).

Embodiment 42. The method of any one of embodiments 39-41, further comprising administering an additional therapeutic agent.

Embodiment 43. The method of Embodiment 42, wherein the additional therapeutic agent is an anticancer agent.

Embodiment 44. The method of Embodiment 43, wherein the anticancer agent is selected from a chemotherapeutic agent, a biological anticancer agent, radiation therapy, CAR-T therapy, and an oncolytic virus.

Embodiment 45. The method of any one of embodiments 39-44, wherein the cancer is a CD33-expressing cancer.

Brief description of drawings

In fig. 1 shows biolayer interferometry data for hz1E4v2 compared to other CD33-binding sdAbs disclosed herein.

In fig. 2A-2M show the binding of certain single domain antibodies (sdAbs) to CD33 expressed on MOLM-13 or HEK 293 cells. MOLM-13 indicates MOL-13 cells transfected with a plasmid encoding CD33 as described in Example 2. CD33m indicates the truncated CD33 protein (SEQ ID NO: 113). CD33M stands for full-length CD33 protein (SEQ ID NO: 112). HEK 293 or parental HEK 293 refers to untransfected HEK 293 cells. FIG. Figure 2A shows the binding of 1E4-IgG1 to CD33. In fig. 2B shows the binding of hz1E4v2-IgG1 to CD33. In fig. Figure 2C shows the binding of 1H9-IgG1 to CD33. In fig. Figure 2D shows the binding of hz1H9v2-IgG1 to CD33. In fig. Figure 2E shows the binding of hz1G3v3-IgG1 to CD33. In fig. 2F shows the binding of 1C7-IgG1 to CD33. In fig. Figure 2G shows the binding of hz1C7v1-IgG1 to CD33. In fig. 2H shows the binding of hz1C7v11-IgG1 to CD33. In fig. 2I shows the binding of hzA07v4-IgG1 to CD33. In fig. Figure 2J shows the binding of hzB07v7-IgG1 to CD33. In fig. 2K shows the binding of hzG11v2-IgG1 to CD33. In fig. 2L shows the binding of F02-IgG1 to CD33. In fig. 2M shows the binding of hzF02v18-IgG1 to CD33.

Detailed description

Embodiments provided herein relate to CD33-binding polypeptides and their use in various methods of treating cancer.

Definitions and Various Embodiments

The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described.

All references cited herein, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference as if each individual reference were specifically and separately identified as being incorporated by reference in its entirety.

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The methods and procedures disclosed or referred to herein are generally well known and conventional methods commonly used by those skilled in the art, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual , 3rd. edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al., eds. (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor, eds. (1995)), Harlow and Lane, eds. (1988), ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998), Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998), Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8), J. Wiley and Sons; Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993); and their updated versions.

Unless otherwise specified, scientific and technical terms used in the context of the present invention have the meanings commonly used by those skilled in the art. In addition, unless the context otherwise requires or is expressly stated, terms in the singular are intended to include the plural and terms in the plural are intended to include the singular. In the event of any conflict in definitions between different sources or references, the definition given herein shall control.

Typically, the numbering of residues in the immunoglobulin heavy chain corresponds to the EU index according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The Kabat EU index refers to the EU numbering of human IgG1 antibody residues.

It should be appreciated that embodiments of the invention disclosed herein include embodiments consisting of and/or essentially consisting of. As used herein, the singular forms include plural references unless otherwise indicated. The use of the term or in this specification does not imply that the alternatives are mutually exclusive.

In this application, the use of or means and/or, unless otherwise explicitly stated or obvious to a person skilled in the art. In the context of a multiple-dependency claim, the use of or refers to more than one preceding independent or dependent claim.

The phrase reference sample, reference cell, or reference tissue means a sample with at least one known characteristic that can be used as a comparison to a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. The reference sample can be used to determine the level of protein and/or mRNA observed, for example, in healthy tissue, as opposed to the level of protein and/or mRNA in a sample with unknown characteristics. In some embodiments, the reference sample is obtained from the same subject, but from a different part than the one being tested. In some embodiments, the reference sample is taken from an area of tissue surrounding or adjacent to the cancer. In some embodiments, the reference sample is obtained not from a subject, but from a subject known to have or not to have the disorder of interest (eg, a specific cancer or CD33-related disorder). In some embodiments, the reference sample is obtained from the same subject, but before the onset of cancer. In some embodiments, the reference sample is derived from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample will indicate a level at which one skilled in the art would be able to determine, given the present disclosure, that the molecule is absent and/or at a low level. When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample will indicate a level at which one of ordinary skill in the art would be able to determine, given the present disclosure, that the molecule is present at some level.

Terms benefit, clinical benefit, susceptibility and therapeutic responsiveness

- 4 046221 effects, as used herein in the context of the beneficial effect or response to administration of a therapeutic agent, can be measured by assessing various endpoints, for example, inhibition to some extent of disease progression, including slowing and stopping; reducing the number of episodes and/or symptoms of the disease; reducing the size of the lesion; inhibiting (i.e. reducing, slowing or completely stopping) the infiltration of pathogenic cells into adjacent peripheral organs and/or tissues; inhibiting (i.e. reducing, slowing down or completely stopping) the spread of the disease; alleviating to some extent one or more symptoms associated with the disorder; increasing the duration of relapse-free course after treatment, for example, progression-free survival; increased overall survival; higher response speed; and/or reduction in mortality at a certain point in time after treatment. A subject or cancer that does not respond or does not respond is a subject or cancer that does not meet the above susceptibility requirements.

The terms nucleic acid molecule, nucleic acid and polynucleotide can be used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. Nucleic acid sequence refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

The terms polypeptide and protein are used interchangeably to refer to a polymer composed of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. This definition covers both full-length proteins and their fragments. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Moreover, for purposes of the present disclosure, a polypeptide refers to a protein that includes modifications such as deletions, additions and substitutions (generally conservative in nature) in the native sequence, provided that the protein retains the desired activity. These modifications may be intentional, such as the result of site-directed mutagenesis, or may be accidental, such as the result of mutations in the protein-producing hosts or errors in the PCR amplification process.

CD33 as used herein refers to any native mature CD33 that results from processing of a CD33 precursor in a cell. The term includes CD33 from any vertebrate source, including mammals such as primates (eg, humans and cynomolgus or rhesus monkeys) and rodents (eg, mice and rats), unless otherwise noted. The term also includes naturally occurring variants of CD33, such as splice variants or allelic variants. A non-limiting exemplary amino acid sequence of human CD33 is shown, for example, in UniProt under accession number P20138. The mature form of CD33 may lack a signal peptide (eg, the mature form of CD33 may lack amino acids 1-17 with UniProt accession number P20138). See SEQ ID NO: 1.

The term specifically binds to an antigen or epitope is a term that is well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit specific binding or preferential binding if it reacts or binds more often, faster, stays bound longer, and/or binds with higher affinity to a particular cell or substance than to alternative cells or substances. A single-domain antibody (sdAb) or VHH-containing polypeptide specifically binds or binds preferentially to a target if it binds with higher affinity, avidity, is more readily bound, and/or remains bound longer than other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a CD33 epitope is a sdAb or VHH-containing polypeptide that binds to that epitope with higher affinity, avidity, and is more readily bound and/or longer bound than with other CD33 epitopes or re-CD33 epitopes. It will also be understood by reading this definition that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, specific binding or preferential binding does not necessarily require (although may include) exclusive binding. Typically, but not necessarily, a linking reference means a preferred binding. Specificity refers to the ability of a binding protein to selectively bind antigen.

The terms inhibition or inhibit refer to the reduction or elimination of a phenotypic trait, or to the reduction or elimination of the frequency, extent, or likelihood of that trait. To reduce or inhibit means to reduce, reduce or suppress activity, function and/or quantity compared to a reference. In some embodiments, reduction or inhibition means the ability to cause an overall reduction of 10% or more. In some embodiments, reducing or inhibiting means

- 5 046221 ability to cause a general reduction of 50% or more. In some embodiments, reduction or inhibition means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amount is suppressed or reduced over a period of time compared to a control over the same period of time. As used herein, the term inhibit CD33 activity refers to a decrease in CD33 activity, such as binding to sialic acid. In some embodiments, inhibit refers to a decrease in CD33 activity compared to CD33 activity in the absence of the modulator. In some embodiments, a CD33 binding polypeptide disclosed herein inhibits the binding of CD33 to sialic acid.

As used herein, the term epitope refers to a site on a target molecule (eg, an antigen, such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (eg, an sdAb or a VHH-containing polypeptide) binds. Epitopes often include chemically active surface groups of molecules, such as amino acids, polypeptides, or sugar side chains, and have certain three-dimensional structure characteristics as well as specific charge characteristics. Epitopes can be formed from either contiguous and/or adjacent residues (eg, amino acids, nucleotides, sugars, lipid moiety) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moiety) are typically retained when exposed to denaturing solvents, whereas epitopes formed from tertiary stacking are typically lost when treated with denaturing solvents. An epitope may include, without limitation, at least 3, at least 5, or 810 residues (eg, amino acids or nucleotides). In some embodiments, the epitope is less than 20 residues (eg, amino acids or nucleotides) in length, less than 15 residues, or less than 12 residues. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be identified by a certain minimum distance to a CDR residue on an antigen-binding molecule. In some embodiments, an epitope may be identified by the above distance and further limited to those residues that participate in a bond (eg, a hydrogen bond) between the antigen binding molecule residue and the antigen residue. An epitope can also be identified using different types of scans, for example an alanine or arginine scan can indicate one or more residues with which an antigen binding molecule can interact. Unless explicitly stated otherwise, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the presence of such a set means the presence of a minimal series (or set of species) of epitopes. Thus, in some embodiments, the set of residues identified as an epitope refers to the minimum epitope relevant to the antigen, rather than an exhaustive list of epitope residues on the antigen.

A non-linear epitope or conformational epitope includes non-contiguous polypeptides, amino acids and/or sugars in an antigenic protein to which an epitope-specific antigen binding molecule binds. In some embodiments, at least one of the residues will not be adjacent to other specified residues of the epitope; however, one or more residues may also be adjacent to other residues.

A linear epitope contains contiguous polypeptides, amino acids and/or sugars in an antigenic protein to which an epitope-specific antigen-binding molecule binds. It should be noted that in some embodiments, each of the residues in the linear epitope is not required to be directly associated with (or involved in the formation of a linkage with) an antigen binding molecule. In some embodiments, the linear epitopes may be from immunizations with a peptide actually consisting of the linear epitope sequence, or from structural regions of the protein that are relatively separate from the rest of the protein (such that the antigen binding molecule can interact at least substantially only with this region of the sequence ).

The term antibody is used in the broadest sense and covers a variety of polypeptides that contain antibody-like antigen-binding domains, including, but not limited to, traditional antibodies (typically containing at least one heavy chain and at least one light chain), single-domain antibodies (sdAbs, containing at least one VHH domain and Fc region), VHH-containing polypeptides (polypeptides containing at least one VHH domain) and fragments of any of the above, provided that they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include, but are not limited to, heavy chain constant domains (including CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain, CL, while the hinge mediates dimerization) and Fc regions (including hinge, CH2, and CH3, where the hinge mediates dimerization).

The term antibody also includes, without limitation, chimeric antibodies, humanized antibodies, and antibodies from various species such as camel (including llama), shark, mouse, human, Javan

- 6 046221 macaque, etc.

As used herein, the term antigen binding domain refers to the portion of an antibody sufficient to bind an antigen. In some embodiments, the antigen binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Thus, in some embodiments, the antigen binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3, and any portions of FR1 and/or FR4 necessary to maintain antigen binding, and a light chain variable region comprising CDR1 -FR2CDR2-FR3-CDR3 and any parts of FR1 and/or FR4 necessary to maintain antigen binding. In some embodiments, the antigen binding domain of the sdAb or VHH-containing polypeptide contains three VHH CDR domains. Thus, in some embodiments, the antigen binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain containing CDR1-FR2-CDR2-FR3-CDR3, and any portions of FR1 and/or FR4 necessary to maintain binding to the antigen.

The term VHH, or VHH domain, or VHH antigen binding domain as used herein refers to the antigen binding portion of a single domain antibody, such as a camel antibody or a shark antibody. In some embodiments, the VHH contains three CDRs and four framework regions, designated FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In some embodiments, the VHH may be truncated at the N-terminus or C-terminus such that it contains only the FR1 and/or FR4 portion, or may lack one or both of these framework regions, provided that the VHH substantially retains antigen binding and specificity to it.

The terms single domain antibody and sdAb are used interchangeably as used herein to refer to an antibody comprising at least one monomeric domain, such as a VHH domain, without a light chain and an Fc region. In some embodiments, the sdAb is a dimer of two polypeptides, where each polypeptide contains at least one VHH domain and an Fc region. As used herein, the terms single domain antibody and sdAb encompass polypeptides that contain multiple VHH domains, such as a polypeptide having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2 and VHH3 may be the same or different.

The term VHH-containing polypeptide refers to a polypeptide that contains at least one VHH domain. In some embodiments, the VHH polypeptide contains two, three, or four, or more VHH domains, where each VHH domain may be the same, or the VHH domains may be different. In some embodiments, the VHH-containing polypeptide contains an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as a sdAb. Additionally, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH-containing polypeptides that are also sdAbs include VHH ₁ -Fc, VHH ₁ -VHH ₂ -Fc, and VHH ₁ -VHH ₂ -VHH ₃ -Fc, where VHH ₁ , VHH 2 and VHH ₃ may be the same or different . In some embodiments of such structures, one VHH may be linked to another VHH via a linker, or one VHH may be linked to an Fc via a linker. In some such embodiments, the linker contains 1-20 amino acids, preferably 1-20 amino acids, predominantly consisting of glycine and optionally serine. In some embodiments, when the VHH-containing polypeptide contains an Fc, it forms a dimer. Thus, the structure of VHH ₁ -VHH ₂ -Fc, in the case of dimer formation, is considered tetravalent (ie, the dimer has four VHH domains). Likewise, the VHH ₁ -VHH ₂ -VHH ₃ -Fc structure, when formed as a dimer, is considered hexavalent (ie the dimer has six VHH domains).

The term monoclonal antibody refers to an antibody (including an sdAb or VHH-containing polypeptide) from a substantially homogeneous population of antibodies, i.e. the individual antibodies that make up such a population are identical, except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific and directed against a single antigenic site. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on an antigen. The modifier “monoclonal” indicates the nature of the antibody as being derived from an essentially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be produced by hybridoma technology, first described by Kohler and Milstein, 1975, Nature, 256:495, or can be produced by recombinant DNA methods described in US Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries, obtained by methods described, for example, in McCafferty et al., 1990, Nature, 348:552-554.

The term CDR means a complementarity determining region as determined by one skilled in the art by at least one identification method. In some embodiments, CDRs may be defined in accordance with any of the Chothia numbering schemes,

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Kabat, combinations according to Kabat and Chotia, AbM determination and/or contact determination. VHH contains three CDRs, designated CDR1, CDR2 and CDR3.

As used herein, the term heavy chain constant region refers to a region containing at least three heavy chain constant domains, CH1, hinge, CH2 and CH3. The term heavy chain constant region certainly includes non-function-altering deletions and substitutions within domains unless otherwise noted. Non-limiting exemplary heavy chain constant regions include γ, δ, and a. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an a constant region is an IgA antibody. Also, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Some isotypes may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (γ ₁ constant region), IgG2 (γ ₂ constant region), IgG3 (γ ₃ constant region) and IgG4 (γ ₄ constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing the a1 constant region) and IgA2 antibodies (containing the _a2 constant region); and IgM antibodies include, but are not limited to, IgM1 and IgM2.

As used herein, the term Fc region refers to the portion of the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region contains a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region may be any of the antibody heavy chain constant region isotypes discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

An acceptor human framework as used herein is a framework containing the amino acid sequence of a heavy chain variable domain ( _VH ) framework derived from a human immunoglobulin framework or a human consensus framework as discussed herein. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence, or it may contain amino acid sequence substitutions. In some embodiments, the number of amino acid substitutions is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 in all human framework regions in a single antigen binding domain, such as VHH .

Affinity refers to the strength of the aggregate non-covalent interactions between one binding site of a molecule (eg, an antibody such as an sdAb, or a VHH-containing polypeptide) and its binding partner (eg, an antigen). The affinity or apparent affinity of a molecule X for its partner Y can usually be represented by the dissociation constant (Kd) or _Kdapparent , respectively. Affinity can be measured by conventional methods known in the art (such as, for example, Kd ELISA, KinExA, flow cytometry and/or surface plasmon resonance), including those presented herein. Such methods include, but are not limited to, BIAcore®, Octet®, or flow cytometry.

The term Kd as used herein refers to the equilibrium dissociation constant of the antigen binding molecule/antigen interaction. The term Kd as used herein includes Kd and Kd _-apparent .

In some embodiments, the K _d of an antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and by fitting the average fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (Prism Software graphical panel). In some such embodiments, Kd is Kd _-apparent .

The term biological activity refers to any one or more biological properties of a molecule (naturally occurring, found in vivo, or introduced or produced by recombinant means).

An agonist or activating antibody is an antibody that increases and/or activates the biological activity of a target antigen. In some embodiments, the agonistic antibody binds to the antigen and increases its biological activity by at least about 20, 40, 60, 80, 85% or more.

An antagonist, blocking or neutralizing antibody is an antibody that inhibits, reduces and/or inactivates the biological activity of a target antigen. In some embodiments, the neutralizing antibody binds to the antigen and reduces its biological activity by at least about 20, 40, 60, 80, 85, 90, 95, 99% or more.

- 8 046221 sdAb or VHH-containing polypeptide with affinity matured refers to an sdAb or VHH-containing polypeptide with one or more changes in one or more CDRs compared to the original sdAb or VHH-containing polypeptide that does not have such changes, wherein such the changes result in improved affinity of the sdAb or VHH-containing polypeptide for the antigen.

As used herein, the term humanized VHH refers to a VHH in which one or more framework regions are substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues of non-human origin. In addition, the humanized VHH may contain residues not found in either the original VHH or the human framework sequences, but which are included to further refine and optimize the characteristics of the VHH-containing sdAb polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. It will be understood that the humanized sequence can be identified by the primary sequence and does not necessarily indicate the process by which the antibody itself was created.

An effector-positive Fc region has the effector function of a native sequence Fc region. Examples of effector functions include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; inhibition of cell surface receptors (eg, B cell receptor); and B cell activation, etc. Such effector functions typically require that the Fc region be combined with a binding domain (eg, an antibody variable domain), and these functions can be assessed using various assays.

The native sequence Fc region contains an amino acid sequence identical to the amino acid sequence of the naturally occurring Fc region. Native sequence human Fc region includes native sequence human IgG1 Fc region (non-A and A allotypes); Fc region of human IgG2 with native sequence; Fc region of human IgG3 with native sequence; and the native sequence Fc region of human IgG4, as well as naturally occurring variants thereof.

The Fc region variant contains an amino acid sequence that differs from the native sequence Fc region sequence due to at least one amino acid modification. In some embodiments, the variant Fc region comprises an amino acid sequence that differs from the native sequence Fc region by virtue of at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution relative to the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the Fc region of the native sequence or in the Fc region of the parent polypeptide. In some embodiments, an Fc region variant provided herein may have at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Fc region of the native sequence and/or the Fc region of the parent polypeptide.

Fc receptor or FcR describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcyR is a native human FcR. In some embodiments, the FcR is an FcR that binds to an IgG antibody (gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB (inhibitory receptor), which have similar amino acid sequences and differ mainly in their cytoplasmic domains. The activating receptor FcyRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol., 15:203234 (1997)). A review of FcR is presented, for example, in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). The term FcR as used herein includes other FcRs, including FcRs that may be identified in the future. For example, the term Fc receptor or FcR also includes the neonatal FcRn receptor, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976); and Kim et al., J. Immunol., 24 :249 (1994)) and regulation of immunoglobulin homeostasis. Methods for measuring FcRn binding are known (see, for example, Ghetie and Ward, Immunol., Today,

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18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem., 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

The term chimeric antigen receptor as used herein refers to an engineered polypeptide that includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen recognition domain comprises a VHH domain.

As used herein, the term substantially similar or substantially the same means a sufficiently high degree of similarity between two or more numerical values such that, from the point of view of one skilled in the art, such difference between the two or more values is low or non-biological and/or non-biological. or statistical significance in the context of the biological characteristic measured by the specified value. In some embodiments, two or more substantially identical values differ by no more than about any of 5, 10, 15, 20, 25, or 50%.

Variant polypeptide means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide after sequence alignment and the introduction of gaps as necessary to achieve the maximum percentage of sequence identity without taking into account any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the polypeptide. In some embodiments, the variant may have at least about 80% amino acid sequence identity. In some embodiments, the variant may have at least about 90% amino acid sequence identity. In some embodiments, the variant may have at least about 95% amino acid sequence identity to a native sequence polypeptide.

As used herein, percentage (%) amino acid sequence identity and homology to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after sequence alignment and insertion as appropriate omissions to achieve the maximum percentage of sequence identity without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer programs such as the BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) programs. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

An amino acid substitution may include, but is not limited to, replacing one amino acid in a polypeptide with another amino acid. Typical substitutions are shown in table. 1. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for the desired activity, such as maintaining/improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

Table 1

Original balance Illustrative Substitutions Ala (A) Vai; Leu; Not Arg(R) Lys; Gin; Asn

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Asn(N) Gin; His; Asp, Lys; Arg Asp (D) Glu; Asn Cys (C) Ser; Ala Gin (Q) Asn; Glu Glu (E) Asp; Gin Gly (G) Ala His(H) Asn; Gin; Lys; Arg He(I) Leu; Val; Met; Ala; Ph; norleucine Leu (L) norleucine; He; Val; Met; Ala; Phe Lys (K) Arg; Gin; Asn Met(M) Leu; Ph; Ile Phe (F) Trp; Leu; Val; lie; Ala; Tyr Pro (P) Ala Ser(S) Thr Thr (T) Val; Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Ph; Thr; Ser Val(V) lie; Leu; Met; Ph; Ala; norleucine

Amino acids can be grouped according to the following general side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues affecting chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions result in replacing a member of one of these classes with another class.

The term vector is used to describe a polynucleotide that can be constructed to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. The vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such such as antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidases). The term expression vector refers to a vector that is used to express a polypeptide of interest in a host cell.

Host cell refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic cells or eukaryotic cells. Examples of eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells such as yeast; plant cells; and insect cells. Non-limiting examples of mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell) and 293 and CHO cells, as well as derivatives thereof such as 293-6E cells, CHO-DG44, CHO-K1, CHO-S and CHO- D.S. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical (in morphology or complementary genomic DNA) to the original parent cell due to a single random or intentional mutation. Host cells include cells transfected in vivo with the polynucleotide(s) provided herein.

As used herein, the term isolated refers to a molecule that has been separated from at least some of the components with which it is normally found in nature or produced. For example, a polypeptide is said to be isolated when it is separated from at least

- 11 046221 some components of the cell in which it was obtained. When a polypeptide is secreted by a cell after expression, the physical separation of the supernatant containing the polypeptide from the cell producing it is considered release of the polypeptide. Similarly, a polynucleotide is said to be isolated if it is not part of a larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it normally occurs in nature, or is separated from at least some of its components the cell producing it, for example, in the case of an RNA polynucleotide. Thus, the DNA polynucleotide that is contained in the vector inside the host cell can be said to be isolated.

The terms individual and subject are used interchangeably herein to refer to an animal; for example, a mammal. In some embodiments, methods of treating mammals are provided, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sporting animals and domestic mammals. In some examples, the individual or subject refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, which indicates that the subject is identified as having the disorder being treated or at a significant risk of developing the disorder.

The term disease or disorder as used herein refers to a condition for which treatment is necessary and/or desirable.

The terms tumor cell, cancer cell, cancer, tumor and/or neoplasm, unless otherwise noted, are used interchangeably herein and refer to a cell (or cells) with uncontrolled growth and/or abnormal increase in cell survival and/or inhibition of apoptosis, interfering normal functioning of organs and systems of the body. This definition includes benign and malignant tumors, hematological cancers such as leukemias, lymphomas and multiple myelomas, polyps, hyperplasia, and latent tumors or micrometastases.

The terms cancer and tumor include solid and hematologic/lymphatic cancer, and also include malignant, premalignant, and benign growths such as dysplasia. Examples of cancer include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; cancer of the brain and central nervous system; mammary cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal carcinoma; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver carcinoma; hepatoma; intraepithelial neoplasm; kidney or kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer); melanoma; myeloma; neuroblastoma; cancer of the oral cavity (lips, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland carcinoma; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocyte NHL; moderate-grade/follicular NHL; moderate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small cell NHL; massive NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström's macroglobulinemia; acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL) , acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (eg, associated with brain tumors) and Meigs syndrome.

The term non-tumor cell or non-cancerous cell as used herein refers to normal cells or tissue. Examples of non-neoplastic cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, monocytes, macrophages, epithelial cells, fibroblasts, hepatocytes, renal interstitial cells, fibroblast-like synoviocytes , osteoblasts and cells located in the mammary gland, skeletal muscle, pancreas, stomach, ovary, small intestine, placenta, uterus, testes, kidneys, lungs, heart, brain, liver, prostate, colon, lymphoid organs, bone, and mesenchymal stem cells of bone origin. As used herein, the term peripherally located cell or tissue refers to non-tumor cells that are not located adjacent to tumor cells and/or in the tumor microenvironment.

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As used herein, the term cells or tissue in the tumor microenvironment refers to the cells, molecules, extracellular matrix and/or blood vessels that surround and/or nourish the tumor cell. Examples of cells or tissue in the tumor microenvironment include, but are not limited to, tumor vasculature; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; extracellular matrix (ECM) components; dendritic cells; antigen presenting cells; T cells; regulatory T cells (Treg cells); macrophages; neutrophils; myeloid-derived suppressor cells (MDSCs) and other immune cells located proximal to the tumor. Methods for identifying tumor cells and/or cells/tissues located in the tumor microenvironment are well known in the art, as disclosed herein below.

In some embodiments, an increase or decrease refers to a statistically significant increase or decrease, respectively. As will be appreciated by one skilled in the art, modulation may also involve changing (which may be either an increase or decrease) the affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, association into homomultimeric or heteromultimeric form or substrates; a change (which may be either an increase or a decrease) in the sensitivity of the target or antigen to one or more environmental conditions in which the target or antigen is present (for example, pH, ion strength, presence of associated factors, etc.); and/or cell proliferation or cytokine production compared to the same conditions but in the absence of the test agent. This can be determined by any suitable method and/or any suitable assay known or disclosed herein depending on the target involved.

As used herein, the term immune response refers to cellular and/or humoral immune responses that are sufficient to suppress or prevent the onset or alleviation of symptoms of a disease (eg, cancer or cancer metastases). The immune response can involve aspects of both the innate and adaptive immune systems.

As used herein, the term treatment is an approach to obtain beneficial or desired clinical results. As used herein, the term treatment includes any administration or use of a therapeutic agent to treat a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: relief of one or more symptoms, reduction in the extent of disease, prevention or delay of spread (e.g., metastasis, e.g., lung or lymph node metastasis) of disease, preventing or delaying the recurrence of a disease, delaying or slowing the progression of a disease, alleviating a disease state, suppressing a disease or its progression, suppressing or slowing a disease or its progression, arresting its development and remission (partial or complete). The term treatment also includes reducing the pathological consequences of a proliferative disease. The methods presented herein involve any one or more of these aspects of treatment. In keeping with the above, the term treatment does not require complete elimination of all aspects of the disorder.

Relief means a reduction or improvement of one or more symptoms compared to the absence of administration of the therapeutic agent. Relief also includes shortening or decreasing the duration of the symptom.

The term anticancer agent is used herein in its broadest sense to refer to agents that are used to treat one or more types of cancer. Examples of classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologic agents (such as cytokines, Fc-extracellular domain receptor fusion proteins, and antibodies), radiation therapy, CAR-T cell therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic agents. viruses.

The term biological sample means a quantity of a substance obtained from a living being or a previously living being. Such substances include, but are not limited to, blood (eg, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

The term control or reference in the context of an experiment or comparison refers to a composition that is known to not contain an analyte (negative control) or to contain an analyte (positive control). A positive control may include a known concentration of the analyte. A control or standard can also refer to a control agent that is known to lack the activity of the test agent, such as an antibody.

As used herein, the term disease retardation means delaying, containing, slowing, inhibiting, stabilizing, suppressing, and/or delaying the development of a disease (such as cancer). This delay may vary in length depending on the history

- 13 046221 disease and/or person to whom treatment is provided. As will be apparent to one skilled in the art, a sufficient or significant delay may actually involve prevention in the sense that the individual does not develop the disease. For example, late stage cancer, such as the development of metastases, may be delayed.

Prevention as used herein includes preventing the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with it. Unless otherwise specified, the terms reduce, inhibit or prevent do not mean or require complete prevention at all times, but only during the time period when the measurement occurs.

The therapeutically effective amount of a substance/molecule, agonist, or antagonist may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance/molecule, agonist, or antagonist to elicit a desired response in an individual. A therapeutically effective amount is also an amount at which the therapeutically beneficial effects outweigh any toxic or harmful effects of the substance/molecule, agonist or antagonist. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective in doses and for periods of time necessary to achieve the desired therapeutic and/or prophylactic result.

The terms pharmaceutical composition and pharmaceutical composition are used interchangeably and refer to a preparation that is in a form that provides effective biological activity of the active ingredient(s) and that does not contain additional components that are unacceptably toxic to the subject to whom the composition will be administered. Such compositions may be sterile.

A pharmaceutically acceptable carrier refers to a non-toxic solid, semi-solid or liquid excipient, diluent, encapsulating material, excipient or carrier commonly used in the art with a therapeutic agent, which together constitute a pharmaceutical composition for administration to a subject. The pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the composition used.

Administration in combination with one or more additional therapeutic agents includes simultaneous (parallel) and sequential administration in any order.

The term parallel is used herein to mean the administration of two or more therapeutic agents when the administrations are at least partially overlapping in time, or when the administration of one therapeutic agent occurs within a short period of time during the administration of another therapeutic agent, or when the therapeutic effects of both agents overlap. at least for some period of time.

The term sequentially is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or in which the therapeutic effects of the agents do not overlap.

As used herein, the term in combination with refers to the administration of one therapeutic agent in addition to another therapeutic agent. Thus, in combination with refers to the administration of one therapeutic agent before, during, or after the administration of another therapeutic agent to an individual.

The term package insert is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information regarding the indications, uses, dosage, routes of administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

An article of manufacture is any product (eg, package or container) or kit containing at least one reagent, such as a drug for treating a disease or disorder (eg, cancer), or a probe for the specific detection of a biomarker disclosed herein. In some embodiments, the product or kit is advertised, distributed, or sold as a unit to perform the methods presented herein.

The terms label and detectable label mean a moiety attached, for example, to an antibody or antigen to provide a detectable reaction (eg, binding) between members of a specific binding pair. The labeled member of a specific binding pair is called detection labeled. Thus, the term tagged binding protein refers to a protein with a tag included that allows identification of the binding protein. In some embodiments, the label is a detectable marker that can produce a visually or instrumentally detectable signal, for example, by incorporating a radiolabeled amino acid or attaching biotinyl moieties to the polypeptide that can

- 14 046221 be detected by labeled avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, 'In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, or ¹⁵³ Sm); chromogens, fluorescent tags (eg, FITC, rhodamine, lanthanide phosphors), enzymatic tags (eg, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predefined polypeptide epitopes recognized by the secondary reporter (eg, leucine zipper sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used for immunoassays include light-emitting moieties, such as acridinium compounds, and fluorescence-emitting moieties, such as fluorescein. In this case, the fragment itself may not be labeled with a detectable label, but may become detectable after reaction with another fragment.

Exemplary CD33-binding polypeptides.

Presented herein are CD33-binding polypeptides. In various embodiments, the CD33-binding polypeptides contain at least one VHH domain that binds to CD33. In some embodiments, the CD33 is human CD33. In some embodiments, the CD33-binding polypeptide blocks the binding of CD33 to sialic acid. In some embodiments, a CD33-binding polypeptide provided herein contains one, two, three, four, five, six, seven, or eight VHH domains that bind to CD33. In some embodiments, a CD33-binding polypeptide provided herein contains one, two, three, or four VHH domains that bind to CD33. CD33-binding polypeptides may contain one or more VHH domains that bind to one or more target proteins other than CD33. Such polypeptides may be referred to as polyspecific polypeptides.

In some embodiments, the CD33-binding polypeptide comprises at least one VHH domain that binds to CD33 and an Fc region. In some embodiments, a CD33 binding polypeptide provided herein comprises one, two, three, or four VHH domains and an Fc region. In some embodiments, the Fc region mediates dimerization of the CD33-binding polypeptide under physiological conditions to form a dimer that doubles the number of CD33 binding sites. For example, a CD33-binding polypeptide containing three VHH domains that bind CD33 and an Fc region is trivalent as a monomer, but under physiological conditions the Fc region can mediate dimerization such that the CD33-binding polypeptide exists in such conditions in the form of a hexavalent dimer.

In some embodiments, the CD33-binding polypeptide contains at least two VHH domains, where a first VHH domain binds to a first CD33 epitope and a second VHH domain binds to a second CD33 epitope. When a CD33-binding polypeptide contains a VHH domain that binds to a first CD33 epitope and a VHH domain that binds to a second CD33 epitope, the CD33-binding polypeptide may be referred to as bi-epitopic or bispecific. In some embodiments, the CD33-binding polypeptide contains at least two VHH domains, where the first VHH domain binds to CD33 and the second VHH domain binds to a non-CD33 antigen. Such polypeptides may be referred to as bispecific or polyspecific.

Non-limiting exemplary CD33-binding polypeptides are shown in table. 2. The sequences of the indicated single domain antibodies are shown in the table for some of the sequences presented in the present description. A polypeptide name beginning with hz indicates that it is a humanized version of the corresponding parent polypeptide.

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table 2

Polypeptides containing at least one VHH that binds to CD33

Name CDR VHH (or KP variant or SS variant) A07 SEQ ID NO: 3, 4 and 5 SEQ ID NO: 2 (or 123) 401-A9 SEQ GO NO: 7, 8 and 9 SEQ ID NO: 6 (or 131) B07 SEQ ID NO: 11, 12 and 13 SEQ ID NO: 10 (or 124) 1S7 SEQ ID NO: 15, 16 and 17 SEQ ID NO: 14 (or 125) 1E4 SEQ ID NO: 19, 20 and 21 SEQ ID NO: 18 (or 126) F02 SEQ ID NO: 23, 24 and 25 SEQ ID NO: 22 (or 127) 1G3 SEQ ID NO: 27, 28 and 29 SEQ ID NO: 26 (or 128) G11 SEQ ID NO: 31, 32 and 33 SEQ ID NO: 30 (or 129) 1H9 SEQ ID NO: 35, 36 and 37 SEQ ID NO: 34 (or 130) hzA07v4 SEQ ID NO: 47, 48 and 49 SEQ ID NO: 38 (or 114) hzB07v7 SEQ ID NO: 50, 51 and 52 SEQ ID NO: 39 (or 115) hzlC7vl SEQ ID NO: 53, 54 and 55 SEQ ID NO: 40 (or 116) hzlC7vll SEQ ID NO: 56, 57 and 58 SEQ ID NO: 41 (or 117) hzlE4v2 SEQ ID NO: 59, 60 and 61 SEQ ID NO: 42 (or 118) hzF02vl8 SEQ ID NO: 62, 63 and 64 SEQ ID NO: 43 (or 119) hzlG3v3 SEQ ID NO: 65, 66 and 67 SEQ ID NO: 44 (or 120) hzGl lv2 SEQ ID NO: 68, 69 and 70 SEQ ID NO: 45 (or 121) hzlH9v2 SEQ ID NO: 71, 72 and 73 SEQ ID NO: 46 (or 122)

CD33-binding polypeptides.

In various embodiments, the VHH domain that binds CD33 comprises a CDR1 sequence selected from SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 47, 50, 53, 56, 59, 62, 65, 68 and 71; CDR2 sequence selected from SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 48, 51, 54, 57, 60, 63, 66, 69 and 72; and a CDR3 sequence selected from SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 49, 52, 55, 58, 61, 64, 67, 70, and 73. In various embodiments the VHH domain that binds CD33 contains the sequences CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 3, 4 and 5; SEQ ID NO: 7, 8 and 9; SEQ ID NO: 11, 12 and 13; SEQ ID NO: 15, 16 and 17; SEQ ID NO: 19, 20 and 21; SEQ ID NO: 23, 24 and 25; SEQ ID NO: 27, 28 and 29; SEQ ID NO: 31, 32 and 33; SEQ ID NO: 35, 36 and 37; SEQ ID NO: 47, 48 and 49; SEQ ID NO: 50, 51 and 52; SEQ ID NO: 53, 54 and 55; SEQ ID NO: 56, 57 and 58; SEQ ID NO: 59, 60 and 61; SEQ ID NO: 62, 63 and 64; SEQ ID NO: 65, 66 and 67; SEQ ID NO: 68, 69 and 70; and SEQ ID NOs: 71, 72 and 73. In various embodiments, the VHH domain is humanized.

In some embodiments, the VHH domain that binds CD33 contains an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98%, at least 99% of the amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 39, 40, 41, 42, 43, 44, 45, 46, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, and 131. In some embodiments, a VHH domain that binds to CD33, contains an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 39, 40, 41, 42, 43, 44, 45, 46, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 and 131.

In various embodiments, the CD33-binding polypeptide contains one, two, three, or four VHH domains that bind to CD33.

In various embodiments, the CD33-binding polypeptide contains at least one VHH domain that binds to CD33 and at least one VHH domain that binds to a natural killer cell antigen or T-cell antigen. In some such embodiments, the CD33-binding polypeptide may be referred to as a multispecific antibody.

In some embodiments, the CD33 binding polypeptide comprises at least one VHH domain as defined herein fused to an Fc region. In some embodiments, the Fc region has a sequence selected from SEQ ID NO: 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 and 109.

In some embodiments, the VHH domain that binds CD33 may be

- 16 046221 manized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because humanized antibodies can reduce or eliminate the occurrence of an immune response in a person to non-human antibodies, which may induce an immune response to the therapeutic antibody and reduce the effectiveness of the therapeutic antibody. Typically, a humanized antibody contains one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody may also optionally contain at least a portion of the human constant region. In some embodiments, certain FR residues in the humanized antibody are replaced with corresponding residues of a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

A review of humanized antibodies and methods for their production is presented, for example, in Almagro and Fransson (2008), Front. Biosci., 13:1619-1633, they are also described, for example, in Riechmann et al. (1988), Nature, 332:323-329; Queen et al. (1989), Proc. Natl Acad. Sci., USA, 86:10029-10033; US patents No. 5821337, 7527791, 6982321 and 7087409; Kashmiri et al. (2005), Methods, 36:25-34; Padlan (1991), Mol. Immunol., 28:489-498 (describing surface restoration); Dall'Acqua et al. (2005), Methods, 36:43-60 (describing FR rearrangement); and Osbourn et al. (2005), Methods, 36:61-68; and Klimka et al., (2000), Br. J Cancer 83:252–260 (describing a guided selection approach for FR rearrangement).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the best-fit method (see, for example, Sims et al. (1993), J. Immunol., 151:2296); framework regions derived from the human antibody consensus sequence of a particular subset of heavy chain variable regions (see, for example, Carter et al. (1992), Proc. Natl. Acad. Sci., USA, 89:4285; and Presta et al. ( 1993), J. Immunol., 151:2623); mature (somatically mutated) human framework regions or human germline framework regions (see, for example, Almagro and Fransson (2008), Front. Biosci., 13:1619-1633); and framework regions obtained from FR screening libraries (see, for example, Baca et al. (1997), J. Biol. Chem., 272:10678-10684; and Rosok et al. (1996), J. Biol. Chem. ., 271:22611-22618). Typically, to obtain a humanized VHH, the FR regions in the VHH are replaced with human FR regions. In some embodiments, certain FR residues of human FR are replaced to improve one or more properties of the humanized VHH. VHH domains with such substituted residues are still referred to herein as humanized.

In various embodiments, the Fc region included in the CD33 binding polypeptide is a human Fc region or is derived from a human Fc region.

In some embodiments, the Fc region included in the CD33 binding polypeptide is derived from a human Fc region and contains a three amino acid deletion in the lower hinge corresponding to E233, L234, and L235 of IgG1, referred to herein as xELL Fc. xELL Fc polypeptides do not interact with FcyR and are thus referred to as effector silent or effector null, however, in some embodiments, xELL Fc regions bind to FcRn and therefore have increased half-life and transcytosis associated with FcRn-mediated recycling.

In some embodiments, the Fc region included in the CD33 binding polypeptide is derived from a human Fc region and contains the M252Y and M428V mutations, referred to herein as Fc-YV. In some embodiments, such mutations enhance FcRn binding at acidic endosomal pH (about 6.5) while losing detectable binding at neutral pH (about 7.2), resulting in enhanced FcRn-mediated recycling and increased half-life.

In some embodiments, the Fc region included in the CD33 binding polypeptide is derived from a human Fc region and contains heterodimerization mutations, referred to herein as knob and trench. In some embodiments, the Fc region of the protrusion contains the T366W mutation. In some embodiments, the Fc region depression contains mutations T366S, L368A, and Y407V. In some embodiments, the Fc regions used for heterodimerization contain additional mutations, such as the S354C mutation in the first member of the heterodimeric Fc pair, which forms an asymmetric disulfide with the corresponding Y349C mutation on the second member of the heterodimeric Fc pair. In some embodiments, one member of the heterodimeric Fc pair contains an H435R or H435K modification to prevent binding to protein A while maintaining binding to FcRn. In some embodiments, one member of the heterodimeric Fc pair contains an H435R or H435K modification, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the retained Fc region contains the H435R or H435K modification (in some cases where the H435R modification is referred to as recess-R), while the protrusion Fc region does not contain the modification. In some cases, the cavity-R mutation improves heterodimer purification compared to homodimeric cavity Fc regions that may be present.

- 17 046221

Non-limiting exemplary Fc regions that may be used in a CD33 binding polypeptide include Fc regions comprising the amino acid sequences of SEQ ID NOs: 74-109.

Chimeric receptors and engineered cells.

Provided herein are chimeric antigen receptors (CARs) having an extracellular domain containing one or more of the CD33 VHH binding domains described herein. The CAR constructs provided herein include an extracellular domain containing one or more CD33 VHH binding domains, a transmembrane domain, and an intracellular signaling region. One or more CD33-binding VHH domains that form the CAR antigen-binding unit bind or are capable of binding, i.e. target CD33 with sufficient affinity such that the CAR is useful in therapy for targeting a cell or tissue expressing CD33.

CARs are synthetic receptors typically containing an extracellular targeting/binding moiety that is associated with one or more signaling domains in a single fusion molecule that is expressed on the surface of a cell, such as a T cell. Thus, CARs combine antigen-specificity and T-cell activation properties into a single fusion molecule. First-generation CARs typically included the cytoplasmic region of the CD3zeta or Fc1 receptor γ chain as a signaling domain. First-generation CARs were tested in phase I clinical trials in patients with ovarian cancer, kidney cancer, lymphoma, and neuroblastoma, where they produced modest responses (reviewed by Sadelain et al., Curr. Opin. Immunol., 21(2):215-223 , 2009). Second-generation CARs that contain costimulatory molecule signaling domains such as CD28 and CD3zeta provide dual signaling to direct combined activating and costimulatory signals. Third generation CARs are more complex with three or more signaling domains (reviewed by Sadelain et al., Cancer Discovery (3), 388-398, 2013; and Dotti et al., Immuno. Rev., 257(1), 1-36 , 2014).

In some embodiments, the provided CAR comprises a CD33 VHH binding domain. In some embodiments, the CAR contains at least two VHH domains targeting one or more antigens. In one embodiment, the CAR antigen binding domain comprises two or at least two VHH CD33 binding domains, thereby providing a bivalent molecule. In one embodiment, the antigen binding domain comprises two or at least two CD33 VHH binding domains, but binds to different epitopes on CD33. In such cases, the antigen binding domain comprises a first CD33 binding VHH domain that binds to a first CD33 epitope and a second VHH domain that binds to a second CD33 epitope. Epitopes may overlap. Thus, in some embodiments, the antigen binding domain is biparatopic and the CAR is a biparatopic CAR. In another embodiment, the antigen binding domain comprises two CD33 VHH binding domains that bind to the same epitopes on CD33.

A CAR transmembrane domain, as defined herein, is a domain that typically crosses or is capable of crossing or passing through the plasma membrane and binds directly or indirectly (for example, through a spacer such as an immunoglobulin hinge sequence) to an extracellular antigen-binding domain and an endoplasmic region containing intracellular signaling domain. In one embodiment, the CAR transmembrane domain is the transmembrane region of a transmembrane protein (eg, type I transmembrane proteins), an artificial hydrophobic sequence, or combinations thereof. In one embodiment, the transmembrane domain includes a CD3zeta domain or a CD28 transmembrane domain. Those skilled in the art will recognize other transmembrane domains that may be used in connection with the CAR embodiments presented herein.

A CAR intracellular signaling region as defined herein comprises one or more intracellular signaling domains that convey a signal to a T cell upon engagement of a CAR antigen binding domain, for example upon binding of an antigen. In some embodiments, the intracellular region comprises an intracellular signaling domain that is or contains an ITAM signaling domain. Exemplary intracellular signaling domains include, for example, a signaling domain derived from the ζ chain of the T cell receptor complex or any of its homologues (e.g., η chain, FcsRIy and β chains, MB chain 1 (Iga), B29 (Ig) chain, etc.), human CD3 chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) etc.) and other molecules involved in T cell transduction, such as CD2, CD5, OX40 and CD28. In specific embodiments, the intracellular signaling region comprises an intracellular signaling domain derived from the zeta chain of human CD3.

In some embodiments, the CAR intracellular signaling region may further comprise an intracellular signaling domain derived from a co-stimulatory molecule. In such examples, such a signaling domain may enhance CAR-T cell activity, e.g.

- 18 046221 increasing the proliferation, survival and/or development of memory cells, after antigen-specific interaction, for example, compared to a CAR that contains only an ITAM containing a signaling domain, for example CD3 zeta. In some embodiments, the co-stimulatory domain is a functional signaling domain derived from a protein selected from: CD28, CD137 (4-IBB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1a /CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. In specific embodiments, the co-stimulatory signaling domain is derived from or derived from a human protein. In some aspects, the co-stimulatory signaling domain is derived from or derived from human CD28 or human CD137 (4-IBB).

In some embodiments, the co-stimulatory signaling domain is derived from CD28 or 41BB.

In specific embodiments, the CAR further comprises a hinge or spacer region that connects the extracellular antigen binding domain and the transmembrane domain. This hinge or spacer region can be used to achieve varying lengths and flexibility of the resulting CAR. Examples of a hinge or spacer region that can be used include, but are not limited to, Fc portions of antibodies or fragments or derivatives thereof, hinge regions of antibodies or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences such as peptide sequences, or combinations thereof. . Those skilled in the art will recognize other hinge or spacer regions that can be used. In one embodiment, the hinge is an IgG4 hinge or a CD8A hinge.

In some embodiments, the spacer and transmembrane domain are a hinge and transmembrane domain derived from CD8.

Also provided herein is an isolated nucleic acid construct comprising at least one nucleic acid encoding a CAR as provided herein. In some aspects, the construct is an expression vector for expressing a CAR in a cell. The expression vector may be a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). A number of viral systems have been developed to transfer genes into mammalian cells. For example, retroviruses such as adenoviral vectors are used. In one embodiment, a lentiviral vector is used.

In a further aspect, also provided is an isolated cell or population of cells containing one or more of the nucleic acid constructs described above. Also provided is an isolated cell or population of cells genetically modified to express the CAR described herein. Thus, provided herein are genetically engineered cells that contain, for example, stably express a CAR as provided herein. In one embodiment, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, hematopoietic stem cells, and/or pluripotent embryonic/induced stem cells. In some cases, the cell is a T cell, such as a CD4 and/or CD8 T cell. In some embodiments, the cells are autologous to the subject. For example, in some embodiments, T cells may be isolated from a patient (also referred to as primary T cells) for engineering, such as transfection or transduction with a CAR nucleic acid construct.

In an illustrative example, primary T cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with TCR/CD28 agonists, such as beads coated with anti-CD3 α anti-CD28 antibodies. After a 2- or 3-day activation process, the recombinant expression vector encoding the CAR can be stably introduced into primary T cells using standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies. Cells can be monitored for CAR expression, for example, by flow cytometry using an anti-epitope tag or antibodies that cross-react with the native parent molecule. T cells that express CAR can be enriched by sorting with tagged anti-epitope antibodies or can be enriched to achieve high or low levels of expression depending on the application.

CAR engineered T cells can be analyzed for appropriate function in a variety of ways. In some cases, in vitro cytotoxicity, proliferation, or cytokine (eg, IFN-gamma expression) assays may be used to assess the function of engineered T cells. Exemplary standard endpoints are the percentage of tumor line lysis, proliferation of engineered T cells, or expression of IFN-gamma protein in the culture supernatant. In some cases, the ability to stimulate T cell activation upon CAR stimulation, for example through an antigen, can be assessed, for example, by monitoring the expression of activation markers such as CD69, CD44 or CD62L, proliferation and/or cytokine production.

- 19 046221

Expression and production of polypeptides.

Presented are nucleic acid molecules containing polynucleotides that encode a CD33-binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of a CD33-binding polypeptide, which leader sequence is typically cleaved off and therefore is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence or may be another heterologous leader sequence.

Nucleic acid molecules can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.

Provided are vectors containing nucleic acids that encode the CD33-binding polypeptides described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector is selected that is optimized for expression of polypeptides in a desired cell type, such as CHO cells or CHO-derived cells, or NSO cells. Examples of such vectors are described, for example, in Running Deer et al., Biotechnol. Prog., 20:880-889 (2004).

In some embodiments, the CD33-binding polypeptide may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells and mammalian cells. Such expression can be carried out, for example, in accordance with procedures known in the art. Examples of eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S cells, DG44.Lec13 CHO cells and FUT8 CHO cells; PER.C6® cells (Crucell); and HCO cells. In some embodiments, CD33-binding polypeptides may be expressed in yeast. See, for example, US Patent Application Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modifications of the polypeptide. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids (such as vectors) into the desired host cell can be accomplished by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection etc. Non-limiting examples of methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells by any suitable method.

Also provided are host cells containing any of the nucleic acids or vectors disclosed herein. In some embodiments, a host cell is provided that expresses a CD33-binding polypeptide provided herein. CD33-binding polypeptides expressed in host cells can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind Fc regions. For example, a Protein A, Protein G, Protein A/G, or immobilized antibody column can be used to bind the Fc region and purify a CD33 binding polypeptide that contains the Fc region. For the purification of some polypeptides, such as antibodies, hydrophobic interactive chromatography, for example on a butyl or phenyl column, may also be suitable. Ion exchange chromatography (eg, anion exchange chromatography and/or cation exchange chromatography) may also be suitable for the purification of certain polypeptides, such as antibodies. Mixed chromatography (e.g., reverse phase/anion exchange, reverse phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.) may also be suitable for the purification of certain polypeptides, such as antibodies. .). Many methods for purifying polypeptides are known in the art.

In some embodiments, the CD33 binding polypeptide is produced in a cell-free system. Non-limiting examples of cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol., 498:229-44 (2009); Spirin, Trends Biotechnol., 22:538-45 (2004); Endo et al., Biotechnol. Adv., 21:695-713 (2003).

In some embodiments, CD33-binding polypeptides produced by the methods described above are provided. In some embodiments, the CD33-binding polypeptide is produced in a host cell. In some embodiments, the CD33-binding polypeptide is produced in a cell-free system. In some embodiments, the CD33-binding polypep

- 20 046221 tid is cleared. In some embodiments, a cell culture medium containing a CD33 binding polypeptide is provided.

In some embodiments, compositions are provided containing antibodies produced by the methods described above. In some embodiments, the composition comprises a CD33 binding polypeptide produced in a host cell. In some embodiments, the composition comprises a CD33 binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified CD33 binding polypeptide.

Exemplary methods of treating diseases using CD33-binding polypeptides.

In some embodiments, methods of treating a disease in an individual are provided, comprising administering a CD33 binding polypeptide or cells expressing a CD33 binding polypeptide. In some embodiments, methods of treating cancer in an individual are provided. In some embodiments, methods of treating CD33-expressing or CD33-positive cancer in an individual are provided. The method includes administering to an individual an effective amount of a CD33 binding polypeptide or cells expressing a CD33 binding polypeptide as provided herein. In some embodiments, the CD33 binding polypeptide blocks the binding of CD33 to sialic acid. In some embodiments, a CD33-binding polypeptide is used to transfer a cytotoxic agent into a CD33-expressing cell. In some such embodiments, the CD33 binding polypeptide comprises a binding domain that binds to a cytotoxic T cell or NK cell. In some such embodiments, the binding domain binds to CD3, T cell receptor (TCR)a, TCRe, CD28, CD16, CD32A, CD64, CD89, NKp46, or NKG2D. In some embodiments, the binding domain may be a VHH domain or an antibody binding domain comprising a heavy chain variable region and a light chain variable region, such as VH/VL, scFv, Fab fragment, etc.

In some embodiments, the CD33 binding polypeptide is coupled to a cytotoxic agent to form an immunoconjugate. Various cytotoxic agents used in immunoconjugates are known in the art and include, but are not limited to, calicheamicins, auristatins, dolastatins, tubulicins, maytansinoids, cryptophycins, duocarmycins, esperamycins, pyrrolobenzodiazepines, and enediyne antibiotics.

In some embodiments, the CD33 binding polypeptide is a chimeric antigen receptor expressed on a cytotoxic cell, such as a T cell (CAR-T) or NK cell (CAR-NK). Such treatments can be used in humans or animals. In some embodiments, methods of treating humans are provided.

Non-limiting exemplary cancers that can be treated with CD33-cbh-binding polypeptides or cells expressing CD33-binding polypeptides provided herein include, but are not limited to, lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade/follicular non-Hodgkin lymphoma (NHL); NHL from small lymphocytes (ML); moderate grade/follicular NHL; diffuse NHL of moderate severity; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; NHL from small non-divided cells with a high degree of malignancy; volumetric NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenström's macroglobulinemia; acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia. In some embodiments, the cancer is a CD33-expressing (ie, CD33-positive) cancer.

CD33-binding polypeptides or cells expressing CD33-binding polypeptides can be administered to subjects as needed. Determination of the frequency of administration can be made by those skilled in the art, such as a physician, taking into account the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the patient being treated, and the like. In some embodiments, an effective dose of CD33-binding polypeptides or cells expressing CD33-binding polypeptides is administered to a subject one or more times. In some embodiments, an effective dose of CD33-binding polypeptides or cells expressing CD33-binding polypeptides is administered to a subject daily, once a week, weekly, every two weeks, once a month, etc. An effective dose of CD33-binding polypeptides or cells expressing CD33-binding polypeptides is administered to a subject at least once. In some embodiments, an effective dose of the CD33 binding polypeptide or cells expressing the CD33 binding polypeptide may be administered multiple times, including multiple times over at least a month, at least six months, or at least a year.

In some embodiments, the pharmaceutical compositions are administered in an amount effective to treat (including prevent) cancer. The therapeutically effective amount will generally depend on the weight of the subject being treated, his or her physical or health status.

- 21 046221 vya, the extent of the condition to be treated, or the age of the subject to be treated. Typically, antibodies can be administered in amounts of from about 0.05 mg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 10 μg/kg body weight to about 100 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 50 μg/kg body weight to about 5 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 100 μg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.5 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.05 mg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of from about 0.05 mg/kg body weight to about 10 mg/kg body weight per dose. In some embodiments, the antibodies can be administered in an amount of about 5 mg/kg body weight or less, such as less than 4, less than 3, less than 2, or less than 1 mg/kg of antibody.

In some embodiments, CD33-binding polypeptides or cells expressing CD33-binding polypeptides can be administered in vivo by various routes, including, but not limited to, intravenous, intra-arterial, parenteral, intraperitoneal, or subcutaneous. A suitable composition and route of administration can be selected according to the intended use.

In some embodiments, therapeutic treatment using a CD33-binding polypeptide is achieved by targeting a cytotoxic agent to a CD33-expressing cell, such as a CD33-expressing cancer cell. In some such embodiments, the CD33 binding polypeptide is a chimeric antigen receptor expressed on a cytotoxic cell, such as a T cell or NK cell.

Pharmaceutical compositions.

In some embodiments, compositions containing CD33 binding polypeptides are formulated with a wide range of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparsions: Drugfacts Plus, 20th ed. (2003 ); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3 ed., Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers are available, which include carriers, adjuvants and diluents. In addition, various pharmaceutically acceptable excipients such as pH adjusters and buffering agents, tonicity agents, stabilizers, wetting agents and the like are also available. Non-limiting exemplary vehicles include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

In some embodiments, the pharmaceutical composition contains a CD33-binding polypeptide at a concentration of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, or 250 mg/ml .

Combination therapy.

The CD33-binding polypeptides or engineered cells of the present invention can be administered alone or in combination with other treatments, such as other anticancer agents. They may be provided before, substantially simultaneously with, or after other treatments (ie, simultaneously or sequentially). In some embodiments, a treatment method provided herein may further include administering: radiation therapy, chemotherapy, vaccination, targeted cancer therapy, CAR-T cell therapy, oncolytic viral therapy, cancer immunotherapy, cytokine therapy, surgical resection, chromatin modification, ablation, cryotherapy, antisense agent against a tumor target, siRNA agent against a tumor target, microRNA agent against a tumor target or anticancer/tumor agent or biological agent such as an antibody, cytokine or Fc-extracellular receptor domain fusion protein.

In some embodiments, a CD33 binding polypeptide provided herein is administered concomitantly with one or more chemotherapeutic agents, CAR-T cell (chimeric antigen receptor T-cell) therapy, oncolytic viral therapy, cytokine therapy, and/or agents targeting other checkpoint molecules such as VISTA, gpNMB, B7H4, HHLA2, CD73, CTLA4, TIGIT, etc.

In some embodiments, the CD33 binding polypeptide or engineered cells of the present invention are used in combination with other antineoplastic agents, such as anti-IBD-2 antibodies, anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor ), HER1/EGFR inhibitor (eg, erlotinib (TARCEVA®), platelet-derived growth factor inhibitors (eg, GLEEVEC® (Imatinib Mesylate)), COX-2 inhibitor (eg, celecoxib), interferons, CTLA4 inhibitors (eg, anti-CTLA

- 22 046221 ipilimumab antibody (YERVOY®)), PD-1 inhibitors (eg anti-PDl antibody, BMS-936558), PDL1 inhibitors (eg anti-PDLl antibody, MPDL3280A), PDL2 inhibitors (eg anti-PDL2 antibody), cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4, or VEGF receptor(s) , TRAIL/Apo2, and other bioactive and organic chemical agents, etc.

In some embodiments, a CD33-binding polypeptide or engineered cell provided herein is administered concomitantly with PD-1/or PD-L1 therapy. Examples of PD-1/PD-L1 therapies include nivolumab (BMS); pidilizumab (CureTech, CT-011), pembrolizumab (Merck); durvalumab (Medimmune/AstraZeneca); atezolizumab (Genentech/Roche); avelumab (Pfizer); AMP-224 (Amplimmune); BMS-936559; AMP-514 (Amplimmune); MDX-1105 (Merck); TCP-042 (Tesaro/AnaptysBio, ANB-011); STI-A1010 (Sorrento Therapeutics); STI-A1110 (Sorrento Therapeutics); and other agents that are directed against programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1).

In some embodiments, a CD33 binding polypeptide or engineered cell of the present invention may be used in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbochon, meturedopa and uredopa; ethylene imines and methyl melamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethylol melamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including a synthetic analogue of topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (eg calicheamicin, especially calicheamicin gammaP and calicheamicin omegaP (see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dinemycin, including dinemycin A, bisphosphonates; such as clodronate; esperamycin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, outramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, deto rubicin, 6-diazo- 5-oxo-E-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nagalamycin, olivomycin s, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that suppress adrenal function, such as aminoglutethimide, mitotane, trilostane; a folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquon; elfornitine; elliptinium acetate; epothilone; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenomet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurine A, roridin A and anhydrine); urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ non-creamaphore formulation containing albumin-bound nanoparticle paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine GEMZAR®; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP16;); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including irinotecan regimen with 5-FU and leucovorin); topoisomerase inhibitors RFS 2000; difluoromethy

- 23 046221 lornithine (DFMO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin regimen (FOLFOX); inhibitors of PKC-alpha, Raf, HRas, EGFR (eg, erlotinib (TARCEVA®)) and VEGF-A that reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Additional non-limiting illustrative chemotherapeutic agents include antihormonal agents that act by regulating or inhibiting the action of hormones on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4- hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone and toremifene FARESTON®; aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestane, fadrozole, RIVISOR® vorozole, FEMARA® letrozole and ARIMIDEX ® anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as troxacitabine (cytosine analogue of 1,3-dioxolane nucleoside); antisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor (eg ANGIOZYME® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; PROLEUKIN® (aldesleukin) rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® GnRH agonist; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

In some embodiments, the CD33 binding polypeptide and the additional agent form a single therapeutic composition, and the CD33 binding polypeptide and the additional agent are administered simultaneously. Alternatively, the CD33 binding polypeptide or engineered cell and the additional agent are separated from each other, for example, each formulated as a separate therapeutic composition, and the CD33 binding polypeptide or engineered cell and the additional agent are administered simultaneously, or the CD33 binding polypeptide or engineered cell and the additional agent the agent is administered at different times according to the treatment regimen. For example, the CD33-binding polypeptide or engineered cell is administered before administration of the additional agent, the CD33-binding polypeptide or engineered cell is administered after administration of the additional agent, or the CD33-binding polypeptide or engineered cell and the additional agent are administered alternately. The CD33-binding polypeptide and the additional agent can be administered in single or multiple doses.

In some embodiments, the CD33-binding polypeptide or engineered cell and the additional agent(s) are administered simultaneously. For example, the CD33 binding polypeptide and the additional agent(s) may be formulated as a single composition or may be administered as two or more separate compositions. In some embodiments, the CD33 binding polypeptide or engineered cell and the additional agent(s) are administered sequentially, or the CD33 binding polypeptide or engineered cell and the additional agent are administered at different times according to the treatment regimen.

Non-limiting illustrative methods of diagnosis and treatment.

In some embodiments, the methods presented herein are useful for evaluating a subject and/or a sample obtained from a subject (eg, a cancer patient). In some embodiments, the assessment is one or more of the following: diagnosis, prognosis, and/or response to treatment.

In some embodiments, the methods provided herein include assessing the presence, absence, or level of a protein. In some embodiments, the methods provided herein include assessing the presence, absence, or expression level of a nucleic acid. The compositions presented herein can be used for these measurements. For example, in some embodiments, the methods provided herein include contacting a sample of a tumor or cells cultured from a tumor with a therapeutic agent provided herein.

In some embodiments, the assessment allows treatment to be directed (including treatment with antibodies provided herein). In some embodiments, the assessment allows the use or avoidance of adjuvant therapy after resection to be determined. Adjuvant therapy, also called adjuvant treatment, is treatment that is given in addition to primary, primary, or initial treatment. By way of non-limiting example, adjuvant therapy may be an additional treatment typically given after surgery that has removed all detectable disease but remains at statistical risk of recurrence due to occult disease. In some embodiments, the polypeptides are used as adjuvant therapy in the treatment of cancer. In some embodiments, the polypeptides are used as the only adjuvant therapy in the treatment of cancer. IN

- 24 046221 In some embodiments, the polypeptides provided herein are not used as adjuvant therapy in the treatment of cancer. For example, if a patient's response to an antibody described herein is unlikely or minimal, then in the interests of quality of life and to avoid unnecessary toxicity from ineffective chemotherapy drugs, treatment may be withheld. In such cases, palliative care can be used.

In some embodiments, the polypeptides are administered as neoadjuvant therapy before resection. In some embodiments, neoadjuvant therapy refers to therapy aimed at shrinking and/or downgrading the tumor before any surgery. In some embodiments, neoadjuvant therapy refers to chemotherapy given to cancer patients before surgery. In some embodiments, neoadjuvant therapy means that the antibody is administered to cancer patients prior to surgery. Types of cancer for which neoadjuvant chemotherapy is usually prescribed include, for example, breast, colon, ovarian, cervical, bladder and lung cancer. In some embodiments, the polypeptides are used as neoadjuvant therapy in the treatment of cancer. In some embodiments, such application is performed prior to resection.

In some embodiments, the tumor microenvironment considered in the methods presented herein is one or more of: the tumor vasculature; tumor-infiltrating lymphocytes; fibroblastic reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; extracellular matrix (ECM) components; dendritic cells; antigen presenting cells; T cells; regulatory T cells; macrophages; other lymphoid cells; neutrophils; and other immune cells located proximal to the tumor.

Sets.

Also provided are articles and kits that include any of the CD33-binding polypeptides described herein and suitable packaging. In some embodiments, the invention includes a kit with (i) a CD33 binding polypeptide and (ii) instructions for using the kit to administer the CD33 binding polypeptide to an individual.

Suitable packaging for the compositions provided herein is known in the art and includes, for example, vials (eg, sealed vials), vials, ampoules, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), and the like. These products can be additionally sterilized and/or sealed. Unit dosage forms containing the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in one or more unit doses and may be further sterilized and sealed. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (eg, on a piece of paper included in the kit), but machine-readable instructions (eg, instructions on a magnetic or optical storage disk) are also possible. Instructions related to the use of antibodies typically include information on dosage, dosing schedule, and route of administration for targeted treatment or industrial use. The kit may further comprise a description of selecting a suitable individual or treatment.

Containers may be unit doses, bulk units (eg, multi-dose units), or subunit units. For example, kits may also be provided that contain sufficient doses of the molecules provided herein to provide effective treatment to an individual over an extended period, for example, for about 1, 2, 3, 4, 6, 8, 3, 4, 5. 6, 7, 8, 9 months or more. The kits may also include multiple unit doses of the molecules and instructions for use, and may be packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kit includes a dry (eg, lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a generally stable aqueous suspension of the antibody.

Examples

The examples discussed below are intended solely to illustrate the invention and should not be construed as limiting the invention in any way. The examples are not intended to demonstrate that the experiments below are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g. quantity, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example 1. Ahtu-CD33 single domain antibodies.

Single domain antibodies targeting human CD33 were generated by immunizing llamas and

- 25 046221 alpaca recombinant version of the extracellular domain of human CD33.

After generating specific anti-CD33 antibody titers, llama/alpaca peripheral blood mononuclear cells (PBMCs) were isolated from 500 ml of immunized animal blood and total mRNA was isolated using the Qiagen RNeasy Maxi Kit and then transformed into first-strand cDNA using Thermo Superscript reverse transcriptase IV and oligo-dT priming. VHH sequences were specifically amplified by PCR using cDNA as a template and cloned into a vector into yeast cells as VHH-Fc-AGA2 fusion proteins for display on the yeast surface.

Yeast clone libraries displaying VHH-Fc-AGA2 fusion proteins were enriched using recombinant forms of ECD CD33 by magnetic bead isolation followed by fluorescence-activated cell sorting (FACS). Sorted yeast cells were plated and isolated colonies were collected into 96-well blocks and grown in a medium that switched expression from VHH-Fc being displayed on the surface to being secreted into the medium. Supernatants from 96-well yeast secretions were applied to transiently transfected 293F cells (CD33 positive) or untransfected 293F cells (CD33 negative), washed, treated with fluorophore-labeled secondary antibodies to human IgG1 Fc, and analyzed by flow cytometry in a 96-well tablet.

Nucleic acid sequences encoding VHH that bound to CD33-positive cells rather than CD33-negative cells were cloned in frame with the human Fc coding region into mammalian expression vectors and expressed by transient transfection in HEK293 Freestyle cells (cells 293F) or CHO cells using polyethylenimine. The supernatant was collected after 3-7 days, the secreted recombinant protein was purified by protein A chromatography, and the concentration was calculated from the absorbance at 280 nm and the extinction coefficient.

Single domain antibody (sdAb) epitopes containing VHH domains that bound CD33 were compared using biolayer interferometry. 7 μg/ml AviTag™-histidine-tagged human CD33 was immobilized onto streptavidin-coated capture sensors. 100 nM of sdAb alone was then loaded onto the CD33 antigen and allowed to reach equilibrium. The sensors were then transferred to 100 nM of the second sdAb. An increase in the assay signal indicated binding, indicating that the second sdAb targets a different epitope than the first sdAb.

Camel-derived VHH CD33 was humanized using the human germline VH3-23 as a scaffold. Camelite residues that affect solubility, specificity, stability, and/or affinity were left unmodified. In addition, where possible and as needed, amino acid sequences that had potential to exhibit activity were changed to reduce the risk of this event. In addition, all humanized variants contained the Leu11Glu modification (L11E), as described in US 20160207981.

The results show that one epitope is found among the humanized versions of 1E4, 1H9, 1G3 and 1C7 sdAbs. As shown in FIG. 1, hz1E4v2, hz1H9v2, hz1G3v3 and hz1C7v1 share a common epitope.

Example 2. Binding of polypeptides to CD33.

The binding of sdAb to human CD33 was assessed by flow cytometry. Each sdAb contained a VHH domain, as shown in Table. 3 below, and the xELL Fc region of human IgG1 in which the amino acids Glu233, Leu234 and Leu235 have been deleted according to EU numbering (SEQ ID NO: 75). MOLM-13 cells were transiently transfected with a plasmid encoding a sequence containing CD33M (SEQ ID NO: 112) or a plasmid encoding a sequence containing a truncated version of CD33m (SEQ ID NO: 113). The sequence encoded by the CD33M plasmid included a leader sequence, a complete extracellular domain, a transmembrane domain, and a cytoplasmic domain. The sequence encoded by the CD33m plasmid included a leader sequence, a membrane-proximal IgC2 domain, and a transmembrane domain. Untransfected HEK 293 cells were used as CD33-negative cells. Each cell type was seeded at 30,000 cells/well in FACS buffer (PBS, 1% BSA, 0.1% NaN ₃ , pH 7.4) in a 96-well round bottom plate. SdAb was diluted in FACS buffer by a 3-fold 11-point serial dilution. Dilutions of sdAb were added to the seeded cells, and the assay plates were incubated for 30 min at 4°C. After washing twice in 150 μl of FACS buffer, the cells in each well were resuspended in 100 μl of Alexa Fluor 647-conjugated secondary anti-human IgG antibody diluted 1:2000 in FACS buffer and incubated at 4°C for 30 min. Cells were washed twice, then bound antibody was detected by flow cytometry.

Flow cytometry analysis was performed on an Intellicyte iQue Plus, and fluorescence values were plotted as median fluorescence intensity. Apparent affinity (K _d , nM) was determined by nonlinear single-site binding regression using PRISM graphics software.

As seen in FIG. 2A-2M, the sdAbs tested showed binding to CD33 but did not bind to untransfected cells not expressing CD33. Apparent binding affinity

- 26 046221 is given in table. 3 below.

Table 3

sdAb CD33 isoform Apparent Ka (nM) Domain SEQ ID NO VHH 1E4 CD33M 0.05 18 hzlE4v2 CD33M 0.32 42 1H9 CD33M 0.14 34 hzlH9v2 CD33M 0.27 46 1G3 CD33M 0.49 26 hzlG3v3 CD33M 1.60 44 1C7 CD33M 6.64 14 hzlC7vl CD33M 6.25 40 hzlC7vll CD33M 5.45 41 A07 CD33M 53.50 2 A07 CD33m 19.90 2 hzA07v4 CD33M 56.61 38 hzA07v4 CD33m 11.76 38 F02 CD33M 15.25 22 F02 CD33m 5.15 22 hzF02v!8 CD33M 14.83 43 hzF02vl8 CD33m 9.13 43 hzB07v7 CD33M 0.75 39 Gil CD33M 1.1 thirty hzGllv2 CD33M 2.67 45

The invention may be embodied in other specific forms without departing from its essence or essential characteristics. Accordingly, the foregoing embodiments should be considered in all respects as illustrative and not limiting of the invention. The scope of the invention is thus defined by the appended claims and not by the preceding description, and therefore all changes covered by the meaning and range of equivalence of the claims are included within the scope of the invention.

Table of some sequences

SEQ ID NO Description Subsequence 1 Human CD33; mature form DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHG YWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRN NCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTD LTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAA PT SLGPRTTHS S VLIITPRPQDHGTNLTC QVKFAGAGVTTER TIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAG VTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASP KHQKKSKLHGPTETS SC SGAAPTVEMDEELHYASLNFHGM NPSKDTSTEYSEVRTQ 2 A07 QVQLVQSGGGLVQAGGSLTLSCAASRSSGIDVMGWYRQAP

- 27 046221

GKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQ MKNLKPEDTAVYYCNAHSFLDLVGAWGQGTQVTV 123 A07KP QVQLVQSGGGLVQAGGSLTLSCAASRSSGIDVMGWYRQAP GKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQ MKNLKPEDTAVYYCNAHSFLDLVGAWGQGTQVTVKP 3 A07 CDR1 RSSGIDVMG 4 A07 CDR2 EISGVGDTN 5 A07 CDR3 HSFLDLVGA 6 401-A9 QVQLQQSGGGLVQAGGSLRLSCAAPGSINVMEWYRQA PGKERDLVAGIT SDGDTNYVD S VPGRFT IT RD NTM RT VDLQ MNNLKADDTAVYYCRARDWGSLTDYWGQGTQVTV 131 401-A9KP QVQLQQSGGGLVQAGGSLRLSCAAPGSINVMEWYRQA PGKERDLVAGIT SDGDTNYVD S VPGRFTITRDNTMRT VDLQ MNNLKADDTAVYYCRARDWGSLTDYWGQGTQVTVKP 7 401-A9 CDR1 GSINSINVME 8 401-A9 CDR2 GITSDGDTN 9 401-A9 CDR3 RDWGSLTDY 10 B07 EVQLVQSGGGLVQTGGSLRLSCGASGRTISDYVVGWFRQA PGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQ VDSLRPEDTAVYFCAALQNDVRNNHSPTSYDYWGQGTQV TV 124 B07KP EVQLVQSGGGLVQTGGSLRLSCGASGRTISDYVVGWFRQA PGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQ VD SLRPEDTAVYFC AALQND VRNNHSPT S YD YWGQGTQV TVKP eleven B07 CDR1 GRTISDYVVG 12 B07 CDR2 AISRYGTTY 13 B07 CDR3 LQNDVRNNHSPTSYDY 14 1C7 QVQLQQSGGGLVQAGGSLRLSCAASGRTFSGYIMGWFRQV PGKERELVARISGNNLSTEYGGSVKGRFTISRDSAKETMYL QMNSLKPEDTAIYYCAAEYDYSSGDFVYWGQGTQVTV 125 1C7KP QVQLQQSGGGLVQAGGSLRLSCAASGRTFSGYIMGWFRQV PGKERELVARISGNNLSTEYGGSVKGRFTISRDSAKETMYL QMNSLKPEDTAIYYCAAEYDYSSGDFVYWGQGTQVTVKP 15 1C7 CDR1 GRTFSGYIMG 16 1C7 CDR2 RISGNNLSTE 17 1C7 CDR3 EYDYSSGDFVY 18 1E4 QVQLQQSGGGSVQAGGSLRLSCVASGSGFSASLMSWHRQ APGSQRDLVASITRDGRANYVDSVKDRFTISRDNAKNTAYL QMDSLKPEDTAAYYCHAYSFDYPIRSYWGQGTQVTV 126 1E4KP QVQLQQSGGGSVQAGGSLRLSCVASGSGFSASLMSWHRQ APGSQRDLVASITRDGRANYVDSVKDRFTISRDNAKNTAYL QMDSLKPEDTAAYYCHAYSFDYPIRSYWGQGTQVTVKP 19 1E4 CDR1 GSGFSASLMS 20 1E4 CDR2 SITRDGRAN 21 1E4 CDR3 YSFDYPIRSY 22 F02 QVQLVQSGGGLVQAGGSLRLSCAASGSISSYNVVGWYRQL SGNERGGRTMVAQINAYGDTNYANAVVGRFTISRDDAKNT VYLHMSNLKPEDTGVYYCNGQRMLENYTYRDQSWGQGT

- 28 046221

QVTV 127 F02KP QVQLVQSGGGLVQAGGSLRLSCAASGSISSYNVVGWYRQL SGNERGGRTMVAQINAYGDTNYANAVVGRFTISRDDAKNT VYLHMSNLKPEDTGVYYCNGQRMLENYTYRDQSWGQGT QVTVKP 23 F02 CDR1 GSISSYNVVG 24 F02 CDR2 QINAYGDTN 25 F02 CDR3 QRMLENYTYRDQS 26 1G3 EVQLQQSGGGEVQPGGSLRLSCEASGFTFSSYAMGWFRQA PGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYL QMS SLRAEDTAVYYC AAHRGGGVIDYWGQGTQVTV 128 1G3 KP EVQLQQSGGGEVQPGGSLRLSCEASGFTFSSYAMGWFRQA PGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYL QMS SLRAEDTAVYYC AAHRGGGVIDYWGQGTQVTVKP 27 1G3 CDR1 GFTFSSYAMG 28 1G3 CDR2 AITTSGDTTY 29 1G3 CDR3 HRGGVIDY thirty Gil EVQLVQSGGGSVQVGGSLRLSCAASGSTLNIDHIGWYRQA PGKERELVGVIS SGAGPNYAD S VKGRFTISRDNAKNT VYLQ MNSLKPEDTAVYNCNAWIDYGSGLPQNYWGQGTQVTV 129 Gil KP EVQLVQSGGGSVQVGGSLRLSCAASGSTLNIDHIGWYRQA PGKERELVGVISSGAGPNYADSVKGRFTISRDNAKNTVYLQ MNSLKPEDTAVYNCNAWIDYGSGLPQNYWGQGTQVTVKP 31 Gil CDR1 GSTLNIDHIG 32 Gil CDR2 VISSGAGPN 33 Gil CDR3 WIDYGSGLLPQNY 34 1H9 EVQLQQSGGAVVQPGGSLRLSCAASGSIFSISIMGWYRQAP GKERELVASTTSSGTTNYVDSVKGRFTASRDNAKNTVYLQ MNSLKPDDTAIYHCHAYIATTTDRGYRGYWGQGTQVTV 130 1H9KP EVQLQQSGGAVVQPGGSLRLSCAASGSIFSISIMGWYRQAP GKERELVASTTSSGTTNYVDSVKGRFTASRDNAKNTVYLQ MNSLKPDDTAIYHCHAYIATTTDRGYRGYWGQGTQVTVKP 35 1H9 CDR1 GSIFSISIMG 36 1H9 CDR2 STTSSGTTN 37 1H9 CDR3 YIATTTDRGYRGY 38 hzA07v4 EVQLVESGGGEVQPGGSLRLSCAASRSSGIDVMGWYRQAP GKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQ MSSLRAEDTAVYYCNAHSFLDLVGAWGQGTLVTV 114 hzA07v4 KP EVQLVESGGGEVQPGGSLRLSCAASRSSGIDVMGWYRQAP GKERELVAEISGVGDTNYAASLADRFTVSRDNAKNTVYLQ MSSLRAEDTAVYYCNAHSFLDLVGAWGQGTLVTVKP 39 hzB07v7 EVQLVESGGGEVQPGGSLRLSCAASGRTISDYWGWFRQA PGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQ VDSLRAEDTAVYYCAALQNDVRNNHSPTSYDYWGQGTLV TV 115 hzB07v7 KP EVQLVESGGGEVQPGGSTRLSCAASGRT1SDYWGWFRQA PGKERAFVAAISRYGTTYYAASVQGRFTISRDNPRNTVYLQ VDSLRAEDTAVYYCAALQNDVRNNHSPTSYDYWGQGTLV TVKP

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40 hzlC7vl EVQLVESGGGEVQPGGSLRLSC A A SGRTF SGYIMGWFRQ A PGKERELVARISGNNLSTEYAESVKGRFTISRDNAKNTLYLQ MS SLRAEDTAVYYCAAEYDYS SGDF VYWGQGTLVTV 116 hzlC7vl KP EVQLVESGGGEVQPGGSLRLSCAASGRTFSGYIMGWFRQA PGKERELVAR1SGNNLSTEYAESVKGRFT1SRDNAKNTLYLQ MS SLRAEDTAVYYCAAEYDYS SGDF VYWGQGTLVTVKP 41 hzlC7vll EVQLVESGGGEVQPGGSLRLSCAASGRTFSGYIMGWFRQA PGKERELVARISGNNLATEYAESVKGRFTISRDNAKNTLYLQ MS SLRAEDTAVYYCAAEYDYS SGDF VYWGQGTLVTV 117 hzlC7vll KP EVQLVESGGGEVQPGGSLRLSC A A SGRTF SGYIMGWFRQ A PGKERELVARISGNNLATEYAESVKGRFTISRDNAKNTLYLQ MS SLRAEDTAVYYCAAEYDYS SGDF VYWGQGTLVTVKP 42 hzlE4v2 EVQLVESGGGEVQPGGSLRLSCAASGSGFSASLMSWHRQA PGKQRDLVASITRDGRANYVESVKGRFTISRDNAKNTLYLQ MS SLRAEDTAVYYCHAYSFDYPIRS YWGQGTLVTV 118 hzlE4v2 KP EVQLVESGGGEVQPGGSLRLSCAASGSGFSASLMSWHRQA PGKQRDLVASITRDGRANYVESVKGRFTISRDNAKNTLYLQ MSSLRAEDTAVYYCHAYSFDYPIRSYWGQGTLVTVKP 43 hzF02v!8 EVQLVESGGGEVQPGGSLRLSCAASGSTSSYNVMGWYRQA PGKQRELVAQINAYGDTNYANAWGRFTISRDNAKNTVYL QMS SLR AEDTAV Y YCNGQRMLEN YT YRDQ SWGQGTLVT V 119 hzF02vl8KP EVQLVESGGGEVQPGGSLRLSCAASGSISSYNVMGWYRQA PGKQRELVAQINAYGDTNYANAWGRFTISRDNAKNTVYL QMS SLRAEDTAVYYCNGQRMLENYT YRDQ SWGQGTLVT V KP 44 hzlG3v3 EVQLVESGGGEVQPGGSLRLSCAASGFTFSSYAMGWFRQA PGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYL QMS SLRAEDTAVYYC AAHRGGGVID YWGQGTLVTV 120 hzlG3v3 EVQLVESGGGEVQPGGSLRLSCAASGFTFSSYAMGWFRQA PGKGREWVAAITTSGDTTYYAESVKGRFTISRDNAKNTVYL QMS SLRAEDTAVYYC AAHRGGGVID YWGQGTLVTVKP 45 hzGllv2 EVQLVESGGGEVQPGGSLRLSCAASGSTLNIDHIGWYRQAP GKERELVGVISSGAGPNYAESVKGRFTISRDNAKNTVYLQM SSLRAEDTAVYYCNAWIDYGSGLPQNYWGQGTLVTV 121 hzGllv2KP EVQLVESGGGEVQPGGSLRLSCAASGSTLNIDHIGWYRQAP GKERELVGVISSGAGPNYAESVKGRFTISRDNAKNTVYLQM SSLRAEDTAVYYCNAWIDYGSGLPQNYWGQGTLVTVKP 46 hzlH9v2 EVQLVESGGGEVQPGGSLRLSCAASGSIFSISIMGWYRQAP GKERELVASTTSSGTTNYVESVKGRFTISRDNAKNTLYLQM SSLRAEDTAVYYCHAYIATTTDRGYRGYWGQGTLVTV 122 hzlH9v2 KP EVQLVESGGGEVQPGGSLRLSCAASGSIFSISIMGWYRQAP GKERELVASTTSSGTTNYVESVKGRFTISRDNAKNTLYLQM SSLRAEDTAVYYCHAYIATTTDRGYRGYWGQGTLVTVKP 47 hzA07v4 CDR1 RSSGIDVMG 48 hzA07v4 CDR2 EISGVGDTN 49 hzA07v4 CDR3 HSFLDLVGA 50 hzB07v7 CDR1 GRTISDYVVG

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51 hzB07v7 CDR2 AISRYGTTY 52 hzB07v7 CDR3 LQNDVRNNHSPTSYDY 53 hzlC7vl CDR1 GRTFSGYIMG 54 hzlC7vl CDR2 RISGNNLSTE 55 hzlC7vl CDR3 EYDYSSGDFVY 56 hzlC7vll CDR1 GRTFSGYIMG 57 hzlC7vll CDR2 RISGNNLATE 58 hzlC7vll CDR3 EYDYSSGDFVY 59 hzlE4v2 CDR1 GSGFSASLMS 60 hzlE4v2 CDR2 SITRDGRAN 61 hzlE4v2 CDR3 YSFDYPIRSY 62 hzF02vl8 CDR1 GSISSYNVMG 63 hzF02vl8 CDR2 QINAYGDTN 64 hzF02vl8 CDR3 QRMLENYTYRDQS 65 hzlG3v3 CDR1 GFTFSSYAMG 66 hzlG3v3 CDR2 AITTSGDTTY 67 hzlG3v3 CDR3 HRGGVIDY 68 hzGllv2 CDR1 GSTLNIDHIG 69 hzGllv2 CDR2 VISSGAGPN 70 hzGllv2 CDR3 WIDYGSGLLPQNY 71 hzlH9v2 CDR1 GSIFSISIMG 72 hzlH9v2 CDR2 STTSSGTTN 73 hzlH9v2 CDR3 YIATTTDRGYRGY 74 Human Fc region IgGl DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVF

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SCSVMHEALHNHYTQKSLSLSPGK 75 xELL Fchuman area IgGl DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYI<TTPPVLDSDGSFF LYSI<LTVDI<SRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 76 M252Y and M428V (YV) S354C T366W lug Fc section DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVVHE ALHNHYTQKS LSLSPGK 77 M252Y, M428V, H435R(YVR) T366S, L368A, Y407V cavity Fc section DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVVHE ALHNRYTQKS LSLSPGK 78 H435RxELL Fc-section DKTHTC PPCPAPGGPS VFLFPPKPKD TLM1SRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 79 M252Y and M428V (YV) xELL Fcsection DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 80 M252Y and M428L (YL) xELL Fcsection DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 81 M252Y, M428L, H435R (YLR) xELL Fcsection DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 82 M252Y, M428V, DKTHTC PPCPAPGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT

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H435R(YVR) xELL Fcsection KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 83 S354C T366W xELL Fc-section projection DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 84 H435RS354C T366W xELL Fc-section projection DKTHTC PPCPAPGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 85 M252Y and M428V (YV) S354C T366W xELL Fc-section projection DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VVHEALHNHYTQKSL SLSPGK 86 M252Y and M428L (YL) S354C T366W xELL Fc-section projection DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VLHEALHNHYTQK SLSLSPGK 87 M252Y, M428L, H435R (YLR) S354C T366W xELL lug Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCS VLHEALHNRYTQKSLSLSPGK 88 M252Y, M428V, H435R(YVR) S354C T366W xELL lug Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNG QPENN YKTTPP VLD SDGSFFLY SKLT VDK SRW QQGN VF S C S VVHEALHNRYTQKSLSLSPGK 89 T366S, L368A, Y407V xELL cavity of the Fc region DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPP VLD SDG SFFLV SKLT VDKSRWQQGNVF SC S VMHEALHNHYTQK SLSLSPGK 90 H435R, T366S, L368A, DKTHTCPPCPAPGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

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Y407V depression xELL Fc-section EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDTAVEWESNG QPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCS VMHEALHNRYTQKSLSLSPGK 91 M252Y and M428V (YV) T366S, L368A, Y407V xELL cavity Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFSCS VVHEALHNHYTQKSL SLSPGK 92 M252Y and M428L (YL) T366S, L368A, Y407V xELL cavity Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVF SC S VLHEALHNHYTQKSLSLSPGK 93 M252Y, M428L, H435R (YLR) T366S, L368A, Y407V xELL cavity Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCS VLHEALHNRYTQKSLSLSPGK 94 M252Y, M428V, H435R(YVR) T366S, L368A, Y407V xELL cavity Fc-section DKTHTCPPCPAPGGPSVFLFPPKPKDTLYISRTPEVTCVVVD VSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCS VVHEALHNRYTQK SLSLSPGK 95 H435RFcsection DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 96 M252Y and M428V (YV) Fc-section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNHYTQK SLSLSPGK 97 M252Y and M428L (YL) Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD

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SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNHYTQK SLSLSPGK 98 M252Y, M428L, H435R (YLR) Fc region DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVLH EALHNRYTQK SLSLSPGK 99 M252Y, M428V, H435R (YVR) Fc-section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLYISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVVH EALHNRYTQK SLSLSPGK 100 S354C T366W lug Fc section DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMTSRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 101 H435R S354C T366W Fc section lug DKTHTCPPCP APELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPCRDELT KNQVSLWCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLSLSPGK 102 M252Y and M428L (YL) S354C T366W lug Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVF SC S VLHEALHNHYTQKSLSLSPGK 103 M252Y, M428L, H435R (YLR) S354C T366W Fc section lug DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVF SCSVLHEALHNRYTQKSLSLSPGK 104 M252Y, M428V, H435R(YVR) S354C T366W Fc section projection DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVF SC S VVHEALHNRYTQKSLSLSPGK 105 T366S, L368A, Y407V depression Fc- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES

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Claims (33)

plot NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVF SC S VMHEALHNHYTQKSLSLSPGK 106 H435R, T366S, L368A, Y407V cavity Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAP1EKTISKAKG QPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVF SC S VMHEALHNRYTQKSLSLSPGK 107 M252Y and M428V (YV) T366S, L368A, Y407V cavity Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSD1AVEWESN GQPENNYKTTPPVLDSDGSFFLVSK LTVDKSRWQQGNVFS C S VVHEALHNHYTQKSLSLSPGK 108 M252Y and M428L (YL) T366S, L368A, Y407V depression of Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFS C S VLHEALHNHYTQKSLSLSPGK 109 M252Y, M428L, H435R (YLR) T366S, L368A, Y407V cavity Fc section DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFS C S VLHEALHNRYTQKSLSLSPGK BY CD33 ECD DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHG YWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRN NCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTD LTHRPKILIPGTLEPGHSKNLTC S VSWACEQGTPPIF SWLS AA PT SLGPRTTHS S VLIITPRPQDHG TNLTC QVKFAGAGVTTER TIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH 111 CD33 ECD- AviTag ^1M -His DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHG YWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRN NCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTD LTHRPKILIPGTLEPGHSKNLTC S VSWACEQGTPPIF SW LS AA PT SLGPRTTHS S VLIITPRPQDHGTNLTC QVKFAGAGVTTER TIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGGTGGS GLNDIFEAQKIEWHEHHHHHH 112 CD33M DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHG YWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRN NCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTD LTHRPKILIPGTLEPGHSKNLTC S VSWACEQGTPPIF SWLS AA PT SLGPRTTHS S VLIITPRPQDHG TNLTC QVKFAGAGVTTER TIQLNVTYVPQNPTTGIFPGDGSGKQETRAGWHGAIGGAG VTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTT 113 CD33m DLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSA APTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTE RTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH CLAIM 1. A polypeptide containing at least one VHH domain that binds to CD33, where at least one VHH domain contains a CDR1 containing the amino acid sequence of SEQ ID NO: 47; CDR2 containing the amino acid sequence SEQ ID NO: 48; and CDR3 containing the amino acid sequence of SEQ ID NO: 49. 2. The polypeptide of claim 1, wherein at least one VHH domain is humanized. 3. The polypeptide of claim 1 or 2, wherein at least one VHH domain contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 38 or 114. 4. The polypeptide according to claim 1 or 2, wherein at least one VHH domain contains the amino acid sequence of SEQ ID NO: 38 or 114. - 36 046221 5. A polypeptide according to any one of claims 1 to 4, containing two VHH domains. 6. A polypeptide according to any one of claims 1 to 4, containing three VHH domains. 7. A polypeptide according to any one of claims 1 to 6, comprising at least one binding domain that binds to an antigen other than CD33. 8. The polypeptide of claim 7, comprising at least one binding domain that binds CD3, T cell receptor (TCR)a, TCRe, CD28, CD16, CD32A, CD64, CD89, NKp46 or NKG2D. 9. The polypeptide of claim 7 or 8, wherein each VHH domain binds to CD33. 10. The polypeptide of claim 9, wherein each VHH domain contains the same amino acid sequences CDR1, CDR2 and CDR3. 11. The polypeptide of claim 9, wherein each VHH domain contains the same VHH sequence. 12. A polypeptide according to any one of claims 1 to 4, containing one VHH domain. 13. A polypeptide according to any one of claims 1 to 12, containing an Fc region. 14. The polypeptide according to claim 13, wherein the Fc region comprises an amino acid sequence selected from SEQ ID NO: 74-109. 15. The polypeptide according to claim 13 or 14, which forms a dimer under physiological conditions. 16. The polypeptide according to any one of claims 1 to 15, wherein CD33 is human CD33. 17. The polypeptide of claim 16, wherein human CD33 comprises the sequence SEQ ID NO: 1. 18. An immunoconjugate containing a polypeptide according to any one of claims 1 to 17 and a cytotoxic agent. 19. The immunoconjugate of claim 18, wherein the cytotoxic agent is selected from calicheamicin, auristatin, dolastatin, tubulicin, maytansinoid, cryptophycin, duocarmycin, esperamycin, pyrrolobenzodiazepine and enediyne antibiotic. 20. A pharmaceutical composition containing a polypeptide according to any one of claims 1 to 19 or an immunoconjugate according to claim 18 or 19 and a pharmaceutically acceptable carrier. 21. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 17. 22. A vector containing the nucleic acid according to claim 21. 23. A host cell containing the nucleic acid of claim 21 or the vector of claim 22. 24. A host cell that expresses the polypeptide according to any one of claims 1 to 17. 25. A method for producing a polypeptide according to any one of claims 1 to 17, comprising incubating a host cell according to claim 23 or 24 under conditions suitable for expression of the polypeptide. 26. The method of claim 25, further comprising isolating the polypeptide. 27. A method of treating cancer, comprising administering to a subject suffering from cancer a pharmaceutically effective amount of a polypeptide according to any one of claims 1 to 17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20. 28. The method according to claim 27, where the cancer is selected from lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade/follicular non-Hodgkin's lymphoma (NHL); NHL from small lymphocytes (ML); moderate malignancy/follicular NHL; diffuse NHL of moderate malignancy; immunoblastic NHL of high grade; high-grade lymphoblastic NHL; NHL from small non-divided cells with a high degree of malignancy; volumetric NHL; mantle cell lymphomas; AIDS-related lymphoma; Waldenström's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; and chronic myeloblastic leukemia. 29. The method of claim 27 or 28, wherein the cancer is acute myeloid leukemia (AML). 30. The method according to any one of claims 27-29, further comprising the administration of an additional therapeutic agent. 31. The method of claim 30, wherein the additional therapeutic agent is an anticancer agent. 32. The method of claim 31, wherein the anticancer agent is selected from a chemotherapeutic agent, an anticancer biological agent, radiation therapy, CAR-T cell therapy, and an oncolytic virus. 33. The method according to any one of claims 27 to 32, wherein the cancer is a CD33-expressing cancer. -