TW202500582A - Methods of treating cancer with bispecific anti-cd22 x anti-cd28 molecules - Google Patents

Abstract

The present disclosure provides methods for treating, reducing the severity, or inhibiting the growth of cancer. The methods, in some embodiments, comprise administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody or antigen-binding fragment thereof that specifically binds CD22 and CD28, in combination with a bispecific antibody or antigen-binding fragment thereof that binds CD20 and CD3.

Description

Method for treating cancer using bispecific anti-CD22 x anti-CD28 molecules

The present disclosure provides a method for treating, reducing the severity of, or inhibiting the growth of cancer in an individual (e.g., a human) in need thereof, comprising administering to the individual a therapeutically effective amount of a combination of a bispecific antibody or an antigen-binding fragment thereof that specifically binds to CD22 and CD28 and a bispecific antibody or an antigen-binding fragment thereof that binds to CD20 and CD3.

CD28 is a type I transmembrane protein expressed on the surface of T cells with a single extracellular Ig-V-like domain assembled as a homodimer. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2) proteins and is activated by CD80 or CD86 expressed on antigen presenting cells (APCs). Binding of CD28 to CD80 or CD86 provides a co-stimulatory signal important for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation through CD28 also provides potent signals for the production of various interleukins. CD28 also enhances cell signaling, such as pathways controlled by the NFκB transcription factor after TCR activation. CD28 co-signaling is important for efficient T cell activation, such as T cell differentiation, proliferation, interleukin release, and cell death.

Anti-CD28 antibodies have been proposed for therapeutic purposes involving T cell activation. One specific anti-CD28 antibody, TGN1412 (anti-CD28 superagonist), was used in clinical trials in 2006. Six healthy volunteers were given a dose of 0.1 mg/kg of TGN1412 (anti-CD28 superagonist) intravenously. Within two hours, all six subjects developed a significant inflammatory response (cytokine storm), and all subjects developed multi-organ failure within sixteen hours. The subjects were treated with corticosteroids, and cytokine levels returned to normal levels within 2 to 3 days. The starting dose of 0.1 mg/kg in the Phase 1 study (associated with CRS) was based on the cynomolgus macaque "NOAEL" of 50 mg/kg, which is 500 times higher (Suntharalingam et al., Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412, NEJM 355:1018-1028 (2006)). Unfortunately, TGN1412 induced a cytokine storm, which was not predicted in the cynomolgus macaque toxicology studies or in the ex vivo human PBMC studies.

CD22 (also known as Siglec-2) is a member of the Siglec family that specifically recognizes α2,6 sialic acid and is a transmembrane protein that is preferentially expressed on B lymphocytes (B cells). CD22 has many intrinsic functions, including, for example, B cell homeostasis, B cell survival and migration, attenuation of TLR and CD40 signaling, and inhibition of B cell receptor (BCR) signaling by recruiting SH2 domain-containing phosphatases through phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region, as well as promoting adhesion between B cells and other cell types. CD22 is not present on the surface of B cells at early stages of development, nor is it expressed in stem cells. However, 60-70% of all B-cell lymphomas and leukemias express CD22.

Anti-CD22 antibodies have been studied for the treatment of B-cell lymphomas and leukemias. However, the monoclonal antibody Epratuzumab has had limited success. (Grant et al., (2013) Cancer 119(21): 10.1002/cncr.28299).

Bispecific antigen binding molecules that bind both CD28 and a target antigen (such as CD22) will be useful in therapeutic settings where specific targeting of cells expressing the target antigen and T cell-mediated killing of such cells are desired.

The present disclosure provides a method for treating a B cell proliferative disorder or malignancy (e.g., a cell malignancy expressing CD20) in an individual, the method comprising administering to the individual a therapeutically effective amount of a combination of a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or an antigen-binding fragment thereof, wherein the bispecific CD22×CD28 antibody or an antigen-binding fragment thereof comprises a first antigen-binding domain that binds cluster of differentiation factor 28 (CD28) and a second antigen-binding domain that binds cluster of differentiation factor 22 (CD22), and the bispecific CD3×CD20 antibody or an antigen-binding fragment thereof comprises a first antigen-binding domain that binds cluster of differentiation factor 3 (CD3) and a second antigen-binding domain that binds cluster of differentiation factor 20 (CD20), thereby treating the B cell proliferative disorder or malignancy (e.g., a malignancy of cells expressing CD20) in the individual.

In some embodiments, the B cell proliferative disorder is a B cell lymphoma. In some embodiments, the lymphoma is a B cell non-Hodgkin lymphoma (B-NHL). In some embodiments, the non-Hodgkin lymphoma is selected from the group consisting of diffuse large B cell lymphoma (DLBCL), marginal zone lymphoma (MZL), high grade B cell lymphoma, Burkitt's lymphoma, primary septal large B cell lymphoma, and follicular lymphoma.

In some embodiments, the method further comprises selecting an individual, wherein the individual has aggressive B-NHL.

In some embodiments, the individual meets or is selected based on at least one of the following criteria: has CD20+ aggressive B-NHL; has progressed after at least 2 lines of systemic therapy containing a CD20 inhibitor and an alkylating agent; has measurable disease on cross-sectional imaging; has adequate bone marrow and liver function; and/or has any of the following cancer types: DLBCL, primary lateral (thymic) large B-cell lymphoma, T-cell/tissue-rich large B-cell lymphoma, grade 3b follicular lymphoma, and high-grade B-cell lymphoma (HGBL) with or without MYC, BCL2, or BCL6 translocations.

In some embodiments, the individual has been treated with a prior therapy and has relapsed, or the condition has progressed during or after the prior therapy. In some embodiments, the individual has received CAR-T therapy.

In some embodiments, the individual has measurable CD20+ aggressive B-NHL that has progressed after ≥2 lines of systemic therapy containing at least a CD20 inhibitor and an alkylating agent.

In some embodiments, the CD20 inhibitor is an anti-CD20 antibody.

In some embodiments, the individual has been treated with CAR-T cell therapy.

In some embodiments, the individual has not been previously treated with a CD3×CD20 bispecific antibody.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg to about 400 mg.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg, 0.03 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in an amount of about 0.1 mg to about 400 mg.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in an amount of about 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg.

In some embodiments, the method comprises administering one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof in combination with one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof. In some embodiments, each of the one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is about 0.01 mg to about 400 mg. In some embodiments, each of the one or more doses is about 0.01 mg, 0.03 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg.

In some embodiments, each of the one or more doses of the bispecific CD3xCD20 antibody or antigen-binding fragment thereof is about 0.1 mg to about 400 mg.

In some embodiments, each of the one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is about 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg.

In some embodiments, one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered 1 day to 8 weeks after the previous dose.

In some embodiments, the one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are each administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, or once every six weeks.

In some embodiments, one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered once a week. In some embodiments, one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered once every two weeks.

In some embodiments, one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered once a week. In some embodiments, one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered once every two weeks.

In some embodiments, a dose of the bispecific CD3xCD20 antibody or antigen-binding fragment thereof is administered in a single administration, or is split and administered on two days no more than 3 days apart.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered intravenously. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered subcutaneously.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered on the same day.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered on different days. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered before or after the bispecific CD3×CD20 antibody or antigen-binding fragment thereof. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered one day before the bispecific CD3×CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the method comprises the following steps: (i) administering to the individual subcutaneously or intravenously a dose of 0.1 mg to 160 mg of the bispecific CD3×CD20 antibody or an antigen-binding fragment thereof weekly for a single treatment period, wherein the single treatment period is at least 2 weeks; and (ii) administering to the individual subcutaneously or intravenously a dose of 0.01 mg to 400 mg of the bispecific CD22×CD28 antibody or an antigen-binding fragment thereof weekly and administering to the individual intravenously or subcutaneously a dose of 80 mg to 160 mg per week. mg of the bispecific CD3×CD20 or antigen-binding fragment thereof for the duration of the combination therapy.

In some embodiments, the single therapy period is at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks.

In some embodiments, during the single treatment period the dose of the bispecific CD3xCD20 antibody or antigen-binding fragment thereof is split and administered on two different days no more than 3 days apart, or is administered in a single administration.

In some embodiments, the single treatment period in step (i) comprises administering an initial dose of the bispecific CD3×CD20 antibody and increasing the dose to a full dose by the end of the single treatment period. In some embodiments, the full dose of the bispecific CD3×CD20 in step (i) is 80 or 160 mg.

In some embodiments, the induction combination therapy period in step (ii) comprises: (a) administering an initial dose of the bispecific CD22×CD28 antibody, wherein the initial dose comprises 0.03 mg to 2 mg; (b) administering an intermediate dose of the bispecific CD22×CD28 antibody, wherein the intermediate dose comprises 0.1 mg to 20 mg; and (c) administering a full dose of the bispecific CD22×CD28, wherein the full dose comprises 0.3 mg to 160 mg.

In some embodiments, during step (ii), the bispecific CD3×CD20 antibody or antigen-binding fragment thereof, such as the bispecific CD22×CD28 antibody or antigen-binding fragment thereof, are administered on different days.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered one day after the bispecific CD22×CD28 antibody or antigen-binding fragment thereof.

In some embodiments, during step (ii), the bispecific CD3×CD20 antibody or antigen-binding fragment thereof and the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered on the same day.

In some embodiments, in step (ii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered in combination for at least 9 weeks. In some embodiments, in step (ii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered in combination for at least 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, or 15 weeks.

In some embodiments, the method further comprises: (iii) after step (ii), administering the combination of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and 160 mg or 320 mg of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof every two weeks or more for the duration of the combination therapy.

In some embodiments, in step (iii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered on the same day.

In some embodiments, in step (iii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered every two weeks or every four weeks.

In some embodiments, the method further comprises administering to the individual one or more additional agents to treat or prevent one or more symptoms of the adverse event.

In some embodiments, the bispecific antibodies are administered to the subject in combination with a second agent, wherein the second agent is selected from the group consisting of: dexamethasone, diphenhydramine, acetaminophen, steroids, antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), IL-6 antagonists, and IL-6R antagonists.

In some embodiments, the individual has stable disease, a partial response, or a complete response after at least one week of administration of a dose of about 0.01 mg to about 400 mg of the combination of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 antibody or antigen-binding fragment thereof.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises: i) a CD3 binding arm comprising: a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 5; and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6; and ii) a CD20 binding arm comprising: a heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 4; and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: The amino acid sequence of 6 amino acids contains three light chain complementation determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR).

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises: i) a CD3 binding arm comprising three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; HCDR2 comprises the amino acid sequence of SEQ ID NO: 11; HCDR3 comprises the amino acid sequence of SEQ ID NO: 12; LCDR1 comprises the amino acid sequence of SEQ ID NO: 13; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15; and ii) a CD20 binding arm comprising three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8; HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; LCDR1 comprises the amino acid sequence of SEQ ID NO: 13; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the HCVR of the CD3 binding arm comprises the amino acid sequence of SEQ ID NO: 5, the HCVR of the CD20 binding arm comprises the amino acid sequence of SEQ ID NO: 4, and the common LCVR comprises the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises: a heavy chain of a CD3 binding arm comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain of a CD20 binding arm comprising the amino acid sequence of SEQ ID NO: 1; and a common light chain comprising the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is odronextamab.

In some embodiments, the first antigen-binding domain that binds CD28 comprises: three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 20; and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the first antigen binding domain that binds CD28 comprises: a HCVR comprising the amino acid sequence of SEQ ID NO: 20; and a LCVR comprising the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the second antigen-binding domain that binds to CD22 comprises: three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 19; and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 22, HCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the second antigen binding domain that binds CD22 comprises: a HCVR comprising the amino acid sequence of SEQ ID NO: 19; and a LCVR comprising the amino acid sequence of SEQ ID NO: 21.

In some embodiments, (a) the first antigen-binding domain that binds to human CD28 comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21; and (b) the second antigen-binding domain that binds to CD22 comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 19; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21.

In some embodiments, the bispecific CD22×CD28 antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 17.

In some embodiments, the bispecific CD22×CD28 antibody comprises a second chain comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the bispecific CD22×CD28 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the bispecific CD22×CD28 antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 17; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16; and a common light chain comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the first antigen-binding domain comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the second antigen-binding domain comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 16; and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the bispecific CD22×CD28 antibody is REGN5837 or an antigen-binding fragment thereof.

Related applications

This application is related to and claims priority from U.S. Provisional Application No. 63/489,808 filed on March 13, 2023. The entire contents of the aforementioned application, including all drawings and sequence listings, are expressly incorporated herein by reference. Sequence Listing

This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on March 5, 2024, is named 118003-59220.XML, and is 39,470 bytes in size.

Before describing the present invention, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, because these methods and conditions can be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, because the scope of the present disclosure will be limited only by the scope of the attached patent application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, when used with reference to a particular stated value, the term "about" means that the value may vary from the stated value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety. Definitions

As used herein, the expression "CD28" refers to an antigen expressed as a co-stimulatory receptor on T cells. Human CD28 comprises the amino acid sequence shown in NCBI Accession No. NP_006130.1. Unless expressly specified as being from a non-human species, all references to proteins, polypeptides, and protein fragments herein are intended to refer to the human form of the respective protein, polypeptide, or protein fragment. Thus, unless specified as being from a non-human species, e.g., "mouse CD28," "monkey CD28," etc., the expression "CD28" means human CD28.

As used herein, "antibodies that bind to CD28" or "anti-CD28 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize monomeric CD28, as well as antibodies and antigen-binding fragments thereof that specifically recognize dimeric CD28. The antibodies and antigen-binding fragments disclosed herein can bind to soluble CD28 and/or CD28 expressed on the surface of cells. Soluble CD28 includes natural CD28 proteins that lack a transmembrane domain or are otherwise not associated with a cell membrane, as well as recombinant CD28 protein variants, such as monomeric and dimeric CD28 constructs.

As used herein, the expression "cell surface expressed CD28" means one or more CD28 proteins that are expressed on the surface of cells in vitro or in vivo such that at least a portion of the CD28 protein is exposed on the extracellular side of the cell membrane and accessible to the antigen binding portion of the antibody. "Cell surface expressed CD28" includes CD28 proteins contained in the context of functional T cell co-stimulatory receptors in the cell membrane. The expression "cell surface expressed CD28" includes CD28 proteins expressed on the cell surface as part of a homodimer. "Cell surface expressed CD28" may comprise or consist of CD28 proteins expressed on the surface of cells that normally express CD28 proteins. Alternatively, "cell surface-expressed CD28" may comprise or consist of a CD28 protein expressed on the surface of a cell that normally does not express human CD28 on its surface but has been artificially engineered to express CD28 on its surface.

As used herein, the expression "anti-CD28 antibody" includes monovalent antibodies with a single specificity, as well as bispecific antibodies comprising a first arm that binds CD28 and a second arm that binds a second (target) antigen, wherein the anti-CD28 arm comprises any of the HCVR/LCVR or CDR sequences shown in Table 1 herein. Examples of anti-CD28 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

Unless specified as being from a non-human species (e.g., "mouse CD22," "monkey CD22," etc.), the term "CD22" as used herein refers to the human CD22 protein. The human CD22 protein has the amino acid sequence shown in Accession No. CAA42006. The sequence of the recombinant human CD22 extracellular domain (D20-R687) with a myc hexameric histidine tag is shown in Accession No. NP_001762.2. hCD22 extracellular domain (D20-R687).hFc can also be purchased from R&D Systems Catalog No. 1968-SL-050.

As used herein, "antibodies that bind to CD22" or "anti-CD22 antibodies" include antibodies and antigen-binding fragments thereof that can bind to soluble CD22 and/or CD22 expressed on the surface of cells. Soluble CD22 includes natural CD22 proteins that lack a transmembrane domain or are otherwise not associated with a cell membrane, as well as recombinant CD22 protein variants, such as CD22 constructs.

As used herein, the expression "anti-CD22 antibody" includes monovalent antibodies with a single specificity, as well as bispecific antibodies comprising a first arm that binds CD22 and a second arm that binds a second (target) antigen, wherein the anti-CD22 arm comprises any of the HCVR/LCVR or CDR sequences shown in Table 1 herein. Examples of anti-CD22 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

As used herein, the term "antibody" means any antigen-binding molecule or molecular complex comprising at least one complementary determining region (CDR) that specifically binds to or interacts with a specific antigen (e.g., CD28). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complement determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FRs of the anti-CD28 antibody and/or anti-CD22 antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

As used herein, the term "antibody" also includes antigen-binding fragments of intact antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and similar terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules using any suitable standard techniques, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and, optionally, constant domains of the antibody. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and/or constant domains into a suitable configuration, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains, are also encompassed by the expression "antigen-binding fragment" used herein.

The antigen-binding fragment of an antibody will generally include at least one variable domain. The variable domain may have any size or amino acid composition, and will generally include at least one CDR that is adjacent to or in the same frame as one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH domain and the VL domain may be positioned relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain monomeric VH and VL domains.

In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragment of an antibody disclosed herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable domains and constant domains, including any of the exemplary configurations listed above, the variable domains and constant domains may be directly linked to each other or may be linked by a complete or partial hinge region or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that produce a flexible or semi-flexible connection between adjacent variable domains and/or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragment may include any of the variable domain and constant domain configurations listed above that are non-covalently associated (e.g., by disulfide bonds) with each other and/or with one or more monomeric VH or VL domains as homodimers or heterodimers (or other polymers).

As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will generally comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different antigenic determinant on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be used in the context of antigen-binding fragments of the disclosed antibodies using routine techniques available in the art.

The antibodies of the present disclosure may act through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of cells expressing an antigen by an antibody of the present disclosure in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and thereby cause lysis of the target cells. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The homeostasis region of an antibody is critical for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether it is desired that the antibody mediate cytotoxicity.

In certain embodiments of the present disclosure, the anti-CD28 antibody and/or anti-CD22 antibody (monospecific or bispecific antibody) of the present disclosure is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, in CDRs and particularly in CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations induced by random or site-directed mutagenesis in vitro or introduced by in vivo in vivo somatic cell mutagenesis). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as mice) are transplanted onto human framework sequences.

In some embodiments, the antibodies disclosed herein are recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below); antibodies isolated from recombinant combinatorial human antibody libraries (described further below); antibodies isolated from animals (e.g., mice) transfected with human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, these recombinant human antibodies undergo in vitro mutation induction (or in vivo in vivo induction of somatic cell mutation when transgenic animals are transfected with human Ig sequences), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain structure of approximately 150-160 kDa, in which the dimer is held together by interchain heavy chain disulfide bonds. In the second form, the dimer is not linked by interchain disulfide bonds and forms a molecule of approximately 75-80 kDa composed of covalently coupled light and heavy chains (half-antibodies). These forms are extremely difficult to separate, even after affinity purification.

The frequency with which the second form appears in various intact IgG isotypes is due to, but not limited to, structural differences associated with the isotype of the antibody's hinge region. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using the human IgG1 hinge. The present disclosure encompasses antibodies having one or more mutations in the hinge, CH2, or CH3 region, which may be desirable, for example, in production to improve the yield of a desired antibody form.

The antibodies disclosed herein may be isolated antibodies. As used herein, "isolated antibodies" means antibodies that have been identified and separated and/or recovered from at least one component of their natural environment. For example, for the purposes of the present invention, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody." Isolated antibodies also include antibodies in situ in recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or separation step. According to certain embodiments, isolated antibodies may be substantially free of other cellular material and/or chemicals.

The anti-CD28 antibodies and/or anti-CD22 antibodies herein or their antigen binding domains may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the anti-CD28 antibodies and/or anti-CD22 antibodies or their antigen binding domains are derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences obtained, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding domains derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to the conservative amino acid substitutions of the corresponding germline residues (these sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain and light chain variable region sequences disclosed herein, a general person skilled in the art can easily generate a variety of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and/or CDR residues in the VH and/or VL domains are mutated back to the residues present in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues present within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues present within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and/or CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). In addition, the antibodies or antigen-binding domains thereof disclosed herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, such as where certain individual residues are mutated to corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. Once obtained, antibodies or antigen-binding fragments thereof containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies or antigen-binding fragments thereof obtained in this general manner are encompassed by the present disclosure.

The present disclosure also includes anti-CD28 antibodies and/or anti-CD22 antibodies and antigen-binding molecules comprising variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. Exemplary variants included within this scope of the present disclosure include variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-CD28 antibodies and antigen-binding molecules having HCVR, LCVR and/or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences shown in Table 1 herein.

The term "antigenic determinant" refers to an antigenic determinant that interacts with a specific antigen binding site (called a complementary site) in the variable region of an antibody molecule. A single antigen may have more than one antigenic determinant. Thus, different antibodies may bind to different regions on the antigen and may have different biological effects. Antigenic determinants may be conformational or linear. Conformational antigenic determinants are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear antigenic determinants are antigenic determinants produced by adjacent amino acid residues in a polypeptide chain. In certain instances, an antigenic determinant may include a carbohydrate, phosphoyl, or sulfonyl moiety on the antigen.

When referring to nucleic acids or fragments thereof, the term "substantial identity" or "substantially identical" indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity of at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any of the well-known sequence identity algorithms (such as FASTA, BLAST or Gap) discussed below. In certain instances, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence identical or substantially similar to that encoded by the reference nucleic acid molecule.

When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, and even better at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, the difference in the position of the inconsistent residues is a conservative amino acid substitution. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) via a side chain (R group). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids containing side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamine; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

The sequence similarity (also called sequence identity) of polypeptides is often measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different species of organisms) or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the optimal overlapping region between the query sequence and the search sequence and the percent sequence identity (Pearson (2000), supra). Another preferred algorithm when comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation that would benefit from treatment with the anti-CD28/anti-CD22 bispecific antigen binding molecules or methods of the present disclosure. Such disorders include chronic and acute disorders, including pathological conditions that predispose a mammal to the disorder in question. In one embodiment, the cell proliferative disorder is cancer, a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.

As used herein, "tumor" refers to all proliferative cell growth and proliferation (whether malignant or benign), and all precancerous and cancerous cells and tissues. As referred to herein, the terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive.

"B-cell proliferative disorders" include Hodgkin's lymphoma; non-Hodgkin's lymphoma (NHL), such as aggressive NHL, relapsed aggressive NHL, low-grade/follicular NHL, small lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, giant mass NHL, indolent NHL (including relapsed indolent NHL and rituximab-refractory indolent NHL), refractory NHL, refractory indolent NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia; lymphocyte predominant Hodgkin's disease (LPHD); small lymphocytic lymphoma (SLL); chronic lymphocytic leukemia (CLL); leukemias, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia; and other hematologic malignancies.

As used herein, the term "non-Hodgkin's lymphoma" or "NHL" refers to cancers of the lymphatic system other than Hodgkin's lymphoma. Hodgkin's lymphoma can be distinguished from non-Hodgkin's lymphoma by the presence of Reed-Sternberg cells in Hodgkin's lymphoma and their absence in non-Hodgkin's lymphoma. Examples of non-Hodgkin's lymphomas encompassed by the term as used herein include any non-Hodgkin's lymphoma identified by one skilled in the art (e.g., an oncologist or pathologist) according to a classification scheme known in the art, such as the Revised European-American Lymphoma (REAL) scheme described in Color Atlas of Clinical Hematology (3rd ed.), A. Victor Hoffbrand and John E. Pettit (Eds.) (Harcourt Publishers Ltd., 2000). See in particular the listings in Figures 11.57, 11.58, and 11.59. More specific examples include, but are not limited to, relapsed or refractory NHL, frontline low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, progenitor B-lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and/or lymphoplasma. Cytoid lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone MALT lymphoma, intranodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacytoma myeloma, low-grade/follicular lymphoma, intermediate-grade/follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), NHL relapsed after or refractory to autologous stem cell transplantation, primary septal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, massive NHL, Burkitt's lymphoma, progenitor (peripheral) large granulocytic lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin/cutaneous lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

As used herein, the expression "in combination with" means that a first therapeutic agent (e.g., a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof) is administered before, after, or simultaneously with a second therapeutic agent (e.g., a bispecific CD3×CD20 antibody or an antigen-binding fragment thereof). The term "in combination with" also includes sequential or simultaneous administration of a first therapeutic agent (e.g., a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof) and a second therapeutic agent (e.g., a bispecific CD3×CD20 antibody or an antigen-binding fragment thereof). Combination Therapy and Formulations

The present disclosure includes methods for treating, ameliorating or reducing the severity of at least one symptom or indication of cancer in an individual, or inhibiting the growth of cancer in an individual, comprising administering to an individual in need thereof a therapeutic composition comprising a combination of a multispecific (e.g., bispecific) antigen binding molecule that specifically binds to CD28 and CD22 and a bispecific antibody (e.g., onitumumab) that binds to CD3 and CD20. The therapeutic composition may comprise any of the multispecific antibodies or bispecific antigen binding molecules disclosed herein and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "individual in need thereof" means a human or non-human animal that exhibits one or more symptoms or markers of cancer (e.g., an individual that exhibits a tumor or suffers from any of the cancers mentioned herein below), or that would otherwise benefit from inhibition or reduction of CD22 activity or depletion of CD22+ cells.

The antibodies and bispecific antigen binding molecules of the present disclosure (and therapeutic compositions comprising the same) are particularly suitable for treating any disease or condition in which stimulation, activation and/or targeting of an immune response would be beneficial. Specifically, the anti-CD28/anti-CD22 bispecific antigen binding molecules of the present disclosure can be used to treat, prevent and/or ameliorate any disease or condition associated with or mediated by CD22 expression or activity or CD22+ cell proliferation. The mechanism of action for achieving the therapeutic methods of the present disclosure includes killing cells expressing CD22 in the presence of effector cells (e.g., T cells). Cells expressing CD22 that can be inhibited or killed using the bispecific antigen binding molecules of the present disclosure include, for example, cancerous B cells.

The antigen binding molecules disclosed herein can be used to treat primary and/or metastatic tumors arising in, for example, blood, bone marrow, lymph nodes (e.g., thymus, spleen), colon, liver, lung, breast, kidney cancer, central nervous system and bladder cancer. According to certain exemplary embodiments, the bispecific antigen binding molecules disclosed herein are used to treat B cell proliferative disorders.

In some embodiments, the B cell proliferative disorder is a B cell lymphoma, such as B cell non-Hodgkin lymphoma (B-NHL). In some embodiments, the B cell lymphoma is diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), high grade B cell lymphoma, Burkitt's lymphoma, primary septal large B cell lymphoma or follicular lymphoma.

The present disclosure also includes methods for treating residual cancer in an individual. As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in an individual after treatment with an anti-cancer therapy.

According to certain aspects, the disclosure provides methods for treating a disease or disorder associated with CD22 expression (e.g., a B cell proliferative disorder) comprising administering to a subject one or more bispecific antigen binding molecules described elsewhere herein after the subject has shown no response to other types of anti-cancer therapy.

For example, the disclosure includes a method for treating a B cell proliferative disorder comprising administering to an individual a combination of an anti-CD28/anti-CD22 bispecific antigen binding molecule and an anti-CD3/anti-CD20 bispecific antigen binding molecule 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year or more after the individual has received standard of care for individuals suffering from cancer (e.g., a B cell proliferative disorder such as DLBCL).

In certain embodiments, administration of a combination of a bispecific CD22×CD28 antibody or antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or antigen-binding fragment thereof results in increased cancer regression, tumor shrinkage and/or disappearance. In certain embodiments, administration of a bispecific CD22×CD28 or antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or antigen-binding fragment thereof results in a delay in tumor growth and progression, for example, tumor growth may be delayed by about 3 days, more than 3 days, about 7 days, more than 7 days, more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 1 year, more than 2 years, or more than 3 years compared to an untreated individual or an individual treated with either antibody as a monotherapy. In certain embodiments, administration of a bispecific CD22×CD28 or antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or antigen-binding fragment thereof prevents cancer recurrence and/or increases the duration of survival of an individual, for example, by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months compared to an untreated individual or an individual administered either antibody as a monotherapy.

In certain embodiments, administration of a bispecific CD22×CD28 or antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or antigen-binding fragment thereof increases the response and duration of the response in a subject, e.g., by more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 20%, more than 30%, more than 40%, or more than 50% compared to an untreated subject or a subject that has received either antibody as a monotherapy.

In certain embodiments, administration of bispecific CD22×CD28 or antigen-binding fragments thereof and bispecific CD3×CD20 antibodies or antigen-binding fragments thereof to an individual with cancer results in at least a 30% or greater reduction or decrease in tumor cells or tumor size (a "partial response"). In certain embodiments, administration of bispecific CD22×CD28 or antigen-binding fragments thereof and bispecific CD3×CD20 antibodies or antigen-binding fragments thereof to an individual with cancer results in complete disappearance of all evidence of tumor cells (a "complete response"). In certain embodiments, administration of bispecific CD22×CD28 or antigen-binding fragments thereof and bispecific CD3×CD20 antibodies or antigen-binding fragments thereof to an individual with cancer results in complete or partial disappearance of tumor cells/lesions (including new measurable lesions). Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.

In certain embodiments, administration of a bispecific CD22xCD28 or antigen-binding fragment thereof and a bispecific CD3xCD20 antibody or antigen-binding fragment thereof to an individual suffering from cancer results in a reduction or decrease in the size or number of lymphoma lesions.

In certain embodiments, administration of a bispecific CD22 x CD28 or antigen-binding fragment thereof and a bispecific CD3 x CD20 antibody or antigen-binding fragment thereof increases progression-free survival or overall survival.

In certain embodiments, the methods of the present disclosure comprise administering to an individual in need thereof a therapeutically effective amount of a combination of a bispecific CD22×CD28 or antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or antigen-binding fragment thereof, wherein administration of the combination results in an increase in overall survival (OS) or progression-free survival (PFS) of the patient compared to patients administered a standard of care (SOC) therapy (e.g., chemotherapy, surgery, or radiation). In certain embodiments, PFS is increased by at least one month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, or at least 3 years compared to patients administered any one or more SOC therapies. In certain embodiments, OS is increased by at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least one year, at least two years, or at least three years compared to patients administered any one or more SOC therapies. (i) Dosage and Timing

An "effective" or "therapeutically effective" dose of a bispecific CD22×CD28 antibody or antigen-binding fragment thereof (e.g., REGN5837) or a bispecific CD3×CD20 antibody or antigen-binding fragment thereof (e.g., onitumumab) for the treatment or prevention of cancer (e.g., cancer expressing CD22) is an amount of the antibody or antigen-binding fragment sufficient to reduce one or more signs and/or symptoms of the disease in the individual being treated, either by inducing regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms.

The dosage of the antigen binding molecule administered to an individual may vary depending on the individual's age and size, the target disease, the condition, the route of administration, and the like. The optimal dosage is usually calculated based on body weight or body surface area. The frequency and duration of treatment may be adjusted based on the severity of the condition.

In one embodiment, an antigen-binding molecule (e.g., a bispecific antigen-binding molecule that specifically binds CD22 and CD28) is administered to an individual in a weight-based dose. A "weight-based dose" (e.g., a dose in mg/kg) is a dose of an antibody or antigen-binding fragment thereof or a bispecific antigen-binding molecule that will vary according to the individual's weight.

In another embodiment, the antibody, antigen-binding fragment thereof, or bispecific antigen-binding molecule is administered to an individual in a fixed dose. "Fixed dose" (e.g., dose in mg) means that one dose of the antibody, antigen-binding fragment thereof, or bispecific antigen-binding molecule is used for all individuals, regardless of any particular individual-related factors, such as weight. In a specific embodiment, the fixed dose of the antibody, antigen-binding fragment thereof, or bispecific antigen-binding molecule disclosed herein is based on a predetermined weight or age.

In general, suitable dosages of the antigen-binding molecules of the present disclosure may be in the range of about 0.001 to about 200.0 mg per kg of recipient body weight, typically in the range of about 1 to 50 mg per kg of body weight. For example, the antibody or antigen-binding fragment thereof or bispecific antigen-binding molecule may be administered at about 0.1 mg/kg, about 0.2 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg per single dose. Values and ranges intermediate to the recited values are also intended to be part of the present disclosure.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg to about 400 mg. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg, 0.03 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg.

In some embodiments, the bispecific CD22×CD28 antibody is administered intravenously (IV). In some embodiments, the IV infusion is performed over about 1 hour, 2 hours, 3 hours, or 4 hours. In some embodiments, the bispecific CD22×CD28 antibody is administered subcutaneously.

In some embodiments of the present disclosure, the therapeutically effective dose of the bispecific CD22×CD28 antibody (eg, REGN5837) is 0.01-400 mg IV or SC every week, every two weeks, or every four weeks.

In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in an amount of about 0.1 mg to about 400 mg. In some embodiments, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in an amount of about 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg.

In some embodiments, the bispecific CD3×CD20 antibody is administered intravenously (IV). In some embodiments, the IV infusion is performed over about 1 hour, 2 hours, 3 hours, or 4 hours. In some embodiments, the bispecific CD3×CD20 antibody is administered subcutaneously.

In some embodiments of the present disclosure, the therapeutically effective dose of the bispecific CD3×CD20 antibody (e.g., onitumumab) is 0.1 to 320 mg IV or SC every week, every two weeks, or every four weeks.

In some embodiments, the bispecific CD22×CD28 antibody or antigen binding molecule is administered simultaneously with the bispecific CD3×CD20 antibody or antigen binding portion thereof. In some embodiments, the bispecific CD22×CD28 antibody or antigen binding molecule is administered on the same day as the bispecific CD3×CD20 antibody or antigen binding portion thereof. In some embodiments, the bispecific CD22×CD28 antibody or antigen binding molecule is administered before the bispecific CD3×CD20 antibody or antigen binding portion thereof, for example, 1 hour before, 2 hours before, 3 hours before, 4 hours before, 5 hours before, 6 hours before, 12 hours before, 1 day before, 2 days before, 3 days before, 4 days before, 5 days before, 6 days before, 7 days before, 8 days before, 9 days before, 10 days before, 11 days before, 12 days before, 13 days before, 14 days before, 15 days before, 16 days before, 17 days before, 18 days before, 19 days before, 20 days before, or 21 days before. In some embodiments, the bispecific CD3×CD20 antibody or antigen binding molecule is administered before the bispecific CD22×CD28 antibody or antigen binding portion thereof, for example, 1 hour before, 2 hours before, 3 hours before, 4 hours before, 5 hours before, 6 hours before, 12 hours before, 1 day before, 2 days before, 3 days before, 4 days before, 5 days before, 6 days before, 7 days before, 8 days before, 9 days before, 10 days before, 11 days before, 12 days before, 13 days before, 14 days before, 15 days before, 16 days before, 17 days before, 18 days before, 19 days before, 20 days before, or 21 days before.

According to certain embodiments of the present disclosure, multiple doses of antigen-binding molecules (e.g., bispecific antigen-binding molecules that specifically bind to CD22 and CD28) may be administered to an individual over a defined time course. Methods according to this scope of the present disclosure include sequentially administering multiple doses of the antigen-binding molecules of the present disclosure to an individual. As used herein, "sequential administration" means administering each dose of the antigen-binding molecule to an individual at different time points, such as on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising sequentially administering to an individual a single initial dose of an antigen-binding molecule, followed by one or more second doses of the antigen-binding molecule, and optionally one or more third doses of the antigen-binding molecule.

The terms "initial dose", "second dose" and "third dose" refer to the time sequence of administration of the antigen-binding molecules disclosed herein. Thus, the "initial dose" is the dose administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "second dose" (also referred to as the "intermediate dose") is the dose administered after the initial dose; and the "third dose" is the dose administered after the second dose. The initial dose, the second dose and the third dose may all contain the same amount of antigen-binding molecules, but may generally differ from one another in the frequency of administration. However, in certain embodiments, the amount of antigen-binding molecules contained in the initial dose, the second dose and/or the third dose differs from one another during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as a "loading dose," followed by less frequent subsequent doses (e.g., a "maintenance dose").

In an exemplary embodiment of the present disclosure, each second and/or third dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½ or more) weeks after the previous dose. As used herein, the phrase "previous dose" means the dose of antigen binding molecule administered to a subject in a sequence of multiple administrations prior to the administration of the next dose in the sequence, with no intervening doses in between.

Methods according to this scope of the present disclosure may include administering any number of second and/or third doses of an antigen binding molecule (e.g., an anti-CD28 antibody or a bispecific antigen binding molecule that specifically binds CD22 and CD28) to a subject. For example, in some embodiments, only a single second dose is administered to a subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to a subject. Similarly, in some embodiments, only a single third dose is administered to a subject. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to a subject.

In embodiments involving multiple second doses, each second dose can be administered at the same frequency as the other second doses. For example, each second dose can be administered to the individual 1 to 2 weeks after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered to the individual at the same frequency as the other third doses. For example, each third dose can be administered to the individual 2 to 4 weeks after the previous dose. Alternatively, the frequency of administering the second and/or third dose to the individual can vary during the course of the treatment regimen. The frequency of administration can also be adjusted by the physician according to the needs of the individual after clinical examination during the course of treatment.

In some embodiments, a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof and/or a bispecific CD3×CD20 antibody or an antigen-binding fragment thereof is administered in a small initial dose to observe the individual's tolerance to the antibody, and when the individual tolerates the administration, the amount of the antibody in one or more subsequent doses is increased to a therapeutically effective dose.

In some embodiments, a dose of a bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or a bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in a single administration. In some other embodiments, a dose of a bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or a bispecific CD3×CD20 antibody or antigen-binding fragment thereof is split and administered on two or more days. In some embodiments, the split doses are administered on two different days no more than 3 days apart.

In some embodiments, the treatment method comprises the following steps: (i) administering to the individual a dose of 0.1 mg to 160 mg of a bispecific CD3×CD20 antibody or an antigen-binding fragment thereof subcutaneously or intravenously weekly for a single treatment period, wherein the single treatment period is at least 2 weeks; and (ii) administering to the individual a dose of 0.01 mg to 400 mg of a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof subcutaneously or intravenously weekly and administering to the individual a dose of 80 mg to 160 mg of a bispecific CD3×CD20 or an antigen-binding fragment thereof intravenously or subcutaneously weekly for an induction combination therapy period.

In some embodiments, the single therapy period is at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks.

In some embodiments, the single treatment period in step (i) comprises increasing the dose of the bispecific CD3×CD20 from a lower initial dose to a therapeutically effective dose by the end of the single treatment period.

In some embodiments, the therapeutically effective amount of the bispecific CD3×CD20 in step (i) is 80 or 160 mg.

In some embodiments, inducing the combination therapy phase in step (ii) comprises increasing the dose of the bispecific CD22×CD28 from a lower initial dose to a therapeutically effective dose.

In some embodiments, during step (ii), the bispecific CD3×CD20 antibody or antigen-binding fragment thereof and the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered on different days (eg, one day after each other).

In some embodiments, during step (ii), the bispecific CD3×CD20 antibody or antigen-binding fragment thereof and the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered on the same day.

In some embodiments, in step (ii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered in combination for at least 9 weeks (e.g., at least 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks or 15 weeks).

In some embodiments, the method further comprises (iii): after step (ii), administering a combination of a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof and 180 mg or 320 mg of a bispecific CD3×CD20 or an antigen-binding fragment thereof every two weeks or more for the duration of the combination therapy.

In some embodiments, in step (iii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered on the same day. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered every two weeks or every four weeks. (ii) Route of Administration

The present disclosure provides methods for administering to an individual (e.g., a human suffering from cancer) the following alone or in combination with a bispecific CD3×CD20 antibody (e.g., onitumumab) or an antigen-binding fragment thereof: a bispecific CD22×CD28 antibody (e.g., REGN5837) or an antigen-binding fragment thereof, or any combination of an anti-CD22 HCVR paired with a HCVR from any CD28 antibody described herein, or a pharmaceutical composition thereof; such methods comprise, for example, introducing the antigen-binding protein or pharmaceutical composition into the body of an individual (e.g., a human) intravenously or subcutaneously. For example, the method comprises piercing the body of an individual with a needle of a syringe and injecting the antigen binding protein or pharmaceutical composition into the body of the individual, for example, into a vein, artery, skin, tumor, muscle tissue or subcutaneous tissue of the individual.

The mode of administration of the antibody or its pharmaceutical composition may vary. The routes of administration include parenteral, parenteral, oral, rectal, transmucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, intraocular, intravitreal, transdermal or intraarterial.

In some embodiments, the bispecific CD22×CD28 antibody or an antigen binding portion thereof is administered intravenously and the bispecific CD3×CD20 antibody or an antigen binding portion thereof is administered subcutaneously. In some embodiments, the bispecific CD22×CD28 antibody or an antigen binding portion thereof is administered subcutaneously and the CD3×CD20 antibody or an antigen binding portion thereof is administered intravenously. In some embodiments, the bispecific CD22×CD28 antibody or an antigen binding portion thereof is administered intravenously and the bispecific CD3×CD20 antibody or an antigen binding portion thereof is administered intravenously. In some embodiments, the bispecific CD22×CD28 antibody, or antigen binding portion thereof, is administered subcutaneously and the bispecific CD3×CD20, or antigen binding portion thereof, is administered subcutaneously.

In some other embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered to the subject for about 10 to 300, 20 to 240, 30 to 180, 45 to 150, or 60 to 120 minutes. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered to the subject for about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 120, 150, 180, 210, 240, 270, or 300 minutes.

In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered to the individual over a period of about 1 hour. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered to the individual over a period of about 2 hours. In some embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered to the individual over a period of about 4 hours.

The present disclosure also provides a container (eg, a plastic or glass vial or ampoule, for example, with a cap or a chromatography column, a hollow needle or a syringe), which contains the bispecific CD22×CD28 antigen binding protein or a pharmaceutical composition thereof of the present disclosure.

The present disclosure also provides an injection device, which includes one or more antigen-binding proteins (e.g., antibodies or antigen-binding fragments) that specifically bind to CD22 and CD28 (CD22×CD28) or pharmaceutical formulations thereof. The injection device can be packaged as a kit. The injection device is a device for introducing a substance into an individual's body via a non-parenteral route (e.g., intramuscularly, subcutaneously, or intravenously). For example, the injection device can be a syringe or an autoinjector (e.g., pre-filled with a pharmaceutical formulation), which, for example, includes a syringe or cylinder for containing a fluid to be injected (e.g., containing an antibody or fragment or a pharmaceutical formulation thereof); a needle for piercing the skin, blood vessels, or other tissues to inject the fluid; and a push rod for pushing the fluid out of the syringe and into the individual's body through the needle hole.

A prefilled syringe is a syringe that has been filled with a composition (e.g., a pharmaceutical composition comprising a multispecific antigen-binding protein and a pharmaceutically acceptable carrier) prior to sale or transfer to an end user (e.g., a physician or caregiver) who administers the composition to an individual.

The pharmaceutical composition is described in more detail below. (iii) Individual selection

In some embodiments, the methods described herein further comprise one or more steps of selecting individuals. Patients may be selected, for example, based on inclusion criteria, or may be excluded, for example, based on exclusion criteria. Inclusion and exclusion criteria are described in more detail in Example 2 below.

In some embodiments, the method comprises selecting an individual suffering from a disease or disorder associated with CD22 expression.

In some embodiments, the method comprises selecting an individual suffering from a B cell proliferative disorder.

In some embodiments, the method comprises selecting an individual suffering from lymphoma. In some embodiments, the method comprises selecting an individual suffering from B-cell non-Hodgkin lymphoma (B-NHL).

In some embodiments, the individual has an aggressive B-NHL. In some embodiments, the aggressive B-NHL is selected from the group consisting of DLBCL, primary septal (thymic) large B-cell lymphoma, T cell/tissue cell-rich large B-cell lymphoma, grade 3b follicular lymphoma, and high-grade B-cell lymphoma (HGBL) with and without MYC and BCL2 or BCL6 translocations.

In some embodiments, the individual meets or is selected based on at least one of the following criteria: (i) has CD20+ aggressive B-NHL; (ii) has disease progression after at least 2 lines of systemic therapy containing an anti-CD20 antibody and an alkylating agent; (iii) has measurable disease on cross-sectional imaging; (iv) has adequate bone marrow and liver function; and/or (v) has any of the following cancer types: DLBCL, primary lateral (thymic) large B-cell lymphoma, T-cell/tissue-rich large B-cell lymphoma, grade 3b follicular lymphoma, and high-grade B-cell lymphoma (HGBL).

In some embodiments, the individual has been treated with a prior therapy and has relapsed, or the disorder has progressed during or after a prior treatment.

In some embodiments, the individual has received CAR-T therapy. (iv) Adverse Events

As used herein, an "adverse event" is any unfavorable and unexpected sign (including abnormal laboratory findings), symptom, or disease that is associated in time with the use of an investigational drug, whether or not considered related to the investigational drug.

In some embodiments, the subject develops one or more mild symptoms of an adverse event following administration of the bispecific CD22×CD28 antibody alone or in combination with the bispecific CD3×CD20 antibody. In some embodiments, the one or more symptoms of the adverse event are symptoms of an infusion reaction (IR) or interleukin release syndrome (CRS), or a combination thereof.

In some embodiments, the symptoms of an infusion reaction are selected from the group consisting of persistent/severe cough, persistent chills/chills, rash, pruritus (itching), urticaria (hives, red bumps, wheals), diaphoresis (sweating), hypotension, dyspnea (shortness of breath), vomiting, and flushing.

In some embodiments, the symptoms of interleukin release syndrome are selected from the group consisting of fever, shortness of breath, headache, tachycardia, hypotension, rash and/or hypoxia.

In some embodiments, the subject receives one or more additional treatments to treat one or more symptoms of the adverse event.

In some embodiments, treatment with the bispecific CD22×CD28 antibody alone or in combination with a bispecific CD3× CD20 antibody is suspended when the subject develops one or more mild symptoms of an adverse event and is resumed when the one or more symptoms subside.

The present disclosure provides methods for treating cancer using antigen binding proteins that are multispecific (e.g., bispecific) and bind to at least CD28 and CD22, which are used in combination with bispecific anti-CD3 and anti-CD20 antibodies or antigen-binding fragments thereof (e.g., onitumumab).

Multispecific binding refers to binding to two or more different antigenic determinants (CD22 and CD28 or more) which may be located on the same or different antigens. Multispecificity includes bispecificity, trispecificity, and tetraspecificity. An antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody with a second binding specificity.

In certain embodiments, the multispecific antigen binding protein comprises a bispecific antigen binding protein. As used herein, the expression "bispecific antigen binding protein" means a protein, polypeptide or molecular complex (e.g., an antibody or antigen-binding fragment thereof) comprising at least a first antigen binding domain and a second antigen binding domain. Each antigen binding domain within a bispecific antigen binding molecule comprises at least one CDR that specifically binds to a specific antigen alone or in combination with one or more other CDRs and/or FRs. In the context of the present disclosure, the first antigen binding domain specifically binds to CD28, and the second antigen binding domain specifically binds to CD22.

According to certain exemplary embodiments, the present disclosure includes bispecific antigen binding molecules that specifically bind to CD28 and CD22. Such molecules may be referred to herein as, for example, "anti-CD28/anti-CD22", or "anti-CD28×CD22", or "CD28×CD22", or "anti-CD22/anti-CD28", or "anti-CD22×CD28", or "CD22×CD28" bispecific molecules, or "αCD22×αCD28", or "αCD28×αCD22", or other similar terms.

According to certain exemplary embodiments, a bispecific antigen binding molecule (e.g., a bispecific antibody) may have an effector arm and a targeting arm. The effector arm may be a first antigen binding domain (e.g., an anti-CD28 antibody) that binds to an antigen on an effector cell (e.g., a T cell). The targeting arm may be a second antigen binding domain (e.g., an anti-CD22 antibody) that binds to an antigen on a target cell (e.g., a tumor cell). According to certain exemplary embodiments, the effector arm binds to CD28 and the targeting arm binds to CD22. Bispecific anti-CD28/CD22 can provide a costimulatory signal to effector cells (e.g., T cells). The effector arm has no effect of stimulating T cells without aggregation. The effector arm alone has little effect in stimulating T cells unless combined with a targeting arm. Tumor targeting arms may have imperfect tumor specificity. The antigen (e.g., CD22) that is the target of the targeting arm may be expressed on a subset of tumor cells. The specificity of the tumor targeting arm may be increased by stacking with an anti-CD3 bispecific antigen binding molecule (e.g., an anti-CD3/CD20 bispecific antibody).

As used herein, the expression "antigen binding molecule" means a protein, polypeptide or molecular complex comprising or consisting of at least one complementary determining region (CDR), which specifically binds to a specific antigen alone or in combination with one or more other CDRs and/or framework regions (FRs). In certain embodiments, the antigen binding molecule is an antibody or an antibody fragment, as those terms are defined elsewhere herein.

As used herein, the expression "bispecific antigen-binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a specific antigen alone or in combination with one or more other CDRs and/or FRs. In the context of the present disclosure, the first antigen-binding domain specifically binds to a first antigen (e.g., CD28), and the second antigen-binding domain specifically binds to a second, different antigen (e.g., CD22).

In certain exemplary embodiments of the present disclosure, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated by the prefix "D1", and the CDRs of the second antigen-binding domain may be designated by the prefix "D2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as D2-HCDR1, D2-HCDR2, and D2-HCDR3.

The first antigen binding domain and the second antigen binding domain may be directly or indirectly connected to each other to form the bispecific antigen binding molecule disclosed herein. Alternatively, the first antigen binding domain and the second antigen binding domain may each be connected to a separate multimerization domain. The binding of one multimerization domain to another multimerization domain promotes the binding between the two antigen binding domains, thereby forming a bispecific antigen binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid that is capable of binding to a second multimerization domain having the same or similar structure or composition. For example, the multimerization domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is the Fc portion (comprising CH2-CH3 domains) of an immunoglobulin, such as the Fc domain of an IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and any isotype within each isotype group.

The bispecific antigen-binding molecules of the present disclosure will generally comprise two multimerization domains, such as two Fc domains, each of which is individually part of a separate antibody re-chain. The first and second multimerization domains may have the same IgG isotype, such as IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerization domains may have different IgG isotypes, such as IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence of 1 to about 200 amino acids long containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short peptide containing cysteine. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helical loop motif, or a coiled coil motif.

Any bispecific antibody format or technology can be used to prepare the bispecific antigen-binding molecules disclosed herein. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, e.g., scFv-based or bifunctional antibody bispecific formats, IgG-scFv fusions, bivariable domain (OVO)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEEO)body, leucine zipper, Duobody, IgG1/IgG2, dual-action Fab (OAF)-IgG, and Mab2 bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

In the context of the bispecific antigen-binding molecules of the present disclosure, a multimerization domain (e.g., an Fc domain) may comprise one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of the Fc domain. For example, the present disclosure includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain, wherein the one or more modifications cause the modified Fc domain to have an altered (e.g., enhanced or weakened) binding interaction between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a modification in the CH2 or CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., an endosome wherein the pH is in the range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, e.g., modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., LN/FIW or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/EID or T); or modifications at position 428 and/or 433 (e.g., UR/S/P/Q or K) and/or 434 (e.g., H/F or V); or modifications at position 250 and/or 428; or modifications at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V2591) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and/or 308 modifications (e.g., 308F or 308P).

The present disclosure also includes bispecific antigen-binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody without the amino acid difference. In one embodiment, the first Ig CH3 domain binds to Protein A, and the second Ig CH3 domain contains a mutation that reduces or eliminates Protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). Other modifications that may be present in the second CH3 include: D16E, L18M, N44S, K52N, V57M and V821 in the case of IgG1 antibodies (according to IMGT; D356E, L358M, N384S, K392N, V397M and V4221 according to EU); N44S, K52N and V821 in the case of IgG2 antibodies (IMGT; N384S, K392N and V4221 according to EU); and Q15R, N44S, K52N, V57M, R69K, E79Q and V821 in the case of IgG4 antibodies. (According to IMGT; according to EU: Q355R, N384S, K392N, V397M, R409K, E419Q and V4221).

In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include part or all of the CH2 sequence derived from the CH2 region of human IgG1, human IgG2, or human IgG4, and part or all of the CH3 sequence derived from human IgG1, human IgG2, or human IgG4. A chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may include a combination of an "upper hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4 and a "lower hinge" sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4. A specific example of a chimeric Fc domain that may be included in any antigen-binding molecule described herein comprises, from N-terminus to C-terminus: [IgG4 CH1] - [IgG4 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any antigen-binding molecule described herein comprises, from N-terminus to C-terminus: [IgG1 CH1] - [IgG1 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any antigen-binding molecule of the present disclosure are described in WO2014/022540A1, the entire contents of which are incorporated herein by reference. Chimeric Fc domains and variants thereof having these general structural arrangements may have altered Fc receptor binding, which in turn affects Fc effector functions.

According to certain exemplary embodiments of the present disclosure, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, a light chain variable region and/or a complementation determining region (CDR) comprising any amino acid sequence of the bispecific CD22×CD28 antibody described in U.S. Patent Publication No. 11,396,544. In certain exemplary embodiments, a bispecific CD22×CD28 antibody or antigen-binding fragment thereof that can be used in the context of the disclosed methods comprises: (a) a first antigen-binding arm that binds to CD28, comprising: a heavy chain variable region (CD28-HCVR) comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain complementary determining region (CD28-HCDR1, CD28-HCDR2, and CD28-HCDR3), and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 21, a light chain complementary determining region (CD28-LCDR1, CD28-LCDR2, and CD28-LCDR3); and (b) a second antigen-binding arm that binds to CD22, comprising: a heavy chain CDR of a HCVR (CD22-HCVR) comprising the amino acid sequence of SEQ ID NO: 19. (CD22-HCDR1, CD22-HCDR2 and CD22-HCDR3), and light chain CDRs (CD22-LCDR1, CD22-LCDR2 and CD22-LCDR3) of a LCVR (CD22-LCVR) comprising the amino acid sequence of SEQ ID NO: 21.

According to certain embodiments, CD28-HCDR1 comprises the amino acid sequence of SEQ ID NO: 25; CD28-HCDR2 comprises the amino acid sequence of SEQ ID NO: 26; CD28-HCDR3 comprises the amino acid sequence of SEQ ID NO: 27; CD28-LCDR1 comprises the amino acid sequence of SEQ ID NO: 28; CD28-LCDR2 comprises the amino acid sequence of SEQ ID NO: 29; CD28-LCDR3 comprises the amino acid sequence of SEQ ID NO: 30; CD22-HCDR1 comprises the amino acid sequence of SEQ ID NO: 22, CD28-HCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and CD28-HCDR3 comprises the amino acid sequence of SEQ ID NO: 24, and CD22-LCDR1 comprises the amino acid sequence of SEQ ID NO: 28; CD22-LCDR2 comprises the amino acid sequence of SEQ ID NO: The amino acid sequence of CD22-LCDR3 is as follows:

In yet other embodiments, the bispecific CD22×CD28 antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding arm comprising: a HCVR comprising SEQ ID NO: 20 (CD28-HCVR) and a LCVR comprising SEQ ID NO: 21 (CD28-LCVR); and (b) a second antigen-binding arm comprising: a HCVR comprising SEQ ID NO: 19 (CD22-HCVR) and a LCVR comprising SEQ ID NO: 21 (CD22-LCVR).

In certain exemplary embodiments, the bispecific CD22×CD28 antibody comprises: a CD28 binding arm comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18; and a CD22 binding arm comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

An exemplary CD22×CD28 bispecific antibody for use in the disclosed methods comprises: a HCVR of a CD28 binding arm comprising the amino acid sequence of SEQ ID NO: 20; a HCVR of a CD22 binding arm comprising the amino acid sequence of SEQ ID NO: 19; and a common LCVR comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the CD22×CD28 bispecific antibody is REGN5837 or an antigen-binding fragment thereof. (i) Sequence variants

The antibodies and bispecific antigen-binding molecules disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains are derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences obtained, for example, from public antibody sequence databases. The antigen-binding molecules disclosed herein may include antigen-binding fragments derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or are mutated to the corresponding residues of another human germline sequence, or are mutated to the conservative amino acid substitutions of the corresponding germline residues (these sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain and light chain variable region sequences disclosed herein, a general person skilled in the art can easily generate a variety of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and/or CDR residues in the VH and/or VL domains are mutated back to the residues present in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues present within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues present within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and/or CDR residues are mutated to one or more corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen binding domain was originally derived). In addition, the antigen binding domain may contain any combination of two or more germline mutations in the framework and/or CDR regions, such as where certain individual residues are mutated to corresponding residues of a particular germline sequence, while certain other residues that are different from the original germline sequence are retained or mutated to corresponding residues of a different germline sequence. Once obtained, antigen binding domains containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen binding molecules comprising one or more antigen binding domains obtained in this general manner are encompassed by the present disclosure.

The disclosure also includes antigen binding molecules wherein one or both antigen binding domains comprise a variant of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the disclosure includes antigen binding molecules comprising an antigen binding domain comprising a HCVR, LCVR and/or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) via a side chain (R group). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. Examples of groups of amino acids containing side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamine; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamine-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

The disclosure also includes antigen-binding molecules comprising an antigen-binding domain comprising HCVR, LCVR and/or CDR amino acid sequences substantially identical to any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. When referring to amino acid sequences, the term "substantial identity" or "substantially identical" means that the two amino acid sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, the difference in the position of the inconsistent residue is a conservative amino acid substitution. In cases where the difference between two or more amino acid sequences is a conservative substitution, the percentage of sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.

The sequence similarity (also called sequence identity) of polypeptides is often measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different species of organisms) or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Peptide sequences can also be compared using FASTA (a program in GCG version 6.1) using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping region between the query and search sequences and percent sequence identity (Pearson (2000), supra). Another preferred algorithm when comparing the disclosed sequences to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402. (ii) pH- dependent binding

The present disclosure includes anti-CD28/anti-CD22 bispecific antigen binding molecules having pH-dependent binding properties. For example, the binding of the anti-CD28 antibodies of the present disclosure to CD28 may be reduced at acidic pH compared to neutral pH. Alternatively, the binding of the anti-CD22 antibodies of the present disclosure to CD22 may be enhanced at acidic pH compared to neutral pH. The expression "acidic pH" includes pH values below about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or lower. As used herein, the expression "neutral pH" means a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

In certain instances, "reduced binding at acidic pH compared to neutral pH" is expressed in terms of the ratio of the KD value for binding of an antibody to its antigen at acidic pH to the KD value for binding of the antibody to its antigen at neutral pH (or vice versa). For example, for the purposes of this disclosure, an antibody or antigen-binding fragment thereof may be considered to have "reduced binding to CD28 at acidic pH compared to neutral pH" if the antibody or antigen-binding fragment thereof exhibits an acidic/neutral KD ratio of about 3.0 or greater. In certain exemplary embodiments, the acidic/neutral KD ratio of the antibodies or antigen-binding fragments of the present disclosure may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies that have reduced (or enhanced) binding to a specific antigen at acidic pH compared to neutral pH. In addition, modifications to the antigen-binding domain at the amino acid level can generate antibodies with pH-dependent properties. For example, by replacing one or more amino acids in the antigen-binding domain (e.g., within the CDR) with a histidine residue, an antibody can be obtained that has reduced antigen binding at acidic pH relative to neutral pH. (iii) Antibodies comprising Fc variants

According to certain embodiments of the present disclosure, anti-CD28/anti-CD22 bispecific antigen binding molecules are provided, comprising an Fc domain comprising one or more mutations that enhance or reduce binding of the antibody to an FcRn receptor at, for example, an acidic pH compared to a neutral pH. For example, the present disclosure includes antibodies and antigen binding molecules comprising mutations in the CH2 or CH3 region of the Fc domain, wherein the one or more mutations increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., an endosome wherein the pH is in the range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or modifications at positions 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or modifications at positions 250 and/or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and/or 308 modifications (e.g., 308F or 308P).

For example, the disclosure includes anti-CD28/anti-CD22 bispecific antigen-binding molecules comprising an Fc domain comprising one or more mutation pairs or sets selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are encompassed within the scope of the disclosure. Bispecific CD3×CD20 Antibodies and Antigen-Binding Fragments Thereof

According to certain exemplary embodiments of the present disclosure, the methods comprise administering a therapeutically effective amount of a bispecific antigen-binding molecule or an antigen-binding fragment thereof that binds CD3 and CD20 in combination with a bispecific CD22×CD28 antigen-binding molecule or an antigen-binding fragment thereof.

CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the dimerization of two of four different chains: epsilon, zeta, delta, and gamma. CD3 dimerization arrangements include gamma/epsilon, delta/epsilon, and zeta/zeta. As used herein, molecules that "bind to CD3" include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The bispecific CD3×CD20 antibodies and antigen-binding fragments used in the disclosed methods can bind to soluble CD3 and/or CD3 expressed on the surface of cells. Soluble CD3 includes native CD3 proteins that lack a transmembrane domain or are otherwise not associated with the cell membrane, as well as recombinant CD3 protein variants, such as monomeric and dimeric CD3 constructs.

CD20 is a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. CD20 is considered a B cell tumor-associated antigen because more than 95% of B cell non-Hodgkin lymphomas (NHL) and other B cell malignancies express CD20, but it is not present on pro-B cells, dendritic cells, and plasma cells.

As used in the context of the present disclosure, antibodies that “specifically bind” CD3 or CD20 include antibodies or antigen-binding fragments thereof that bind CD3 or CD20, or a portion thereof, with a KD of less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM as measured in a surface plasmon resonance assay. However, isolated antibodies or antigen-binding fragments that specifically bind human CD3 or CD20 may cross-react with other antigens, such as CD3 or CD20 molecules from other (non-human) species.

According to certain exemplary embodiments of the present disclosure, the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR), a light chain variable region (LCVR) and/or a complementation determining region (CDR) comprising any amino acid sequence of the anti-CD3 antibody and anti-CD20 described in U.S. Patent No. 9,657,102.

In certain exemplary embodiments, the CD3 binding arm of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof that can be used in the context of the disclosed methods comprises: a heavy chain complementation determining region (HCDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 5; and a light chain complementation determining region (LCDR) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6.

According to certain embodiments, the CD3 binding arm of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; HCDR2 comprises the amino acid sequence of SEQ ID NO: 11; HCDR3 comprises the amino acid sequence of SEQ ID NO: 12; LCDR1 comprises the amino acid sequence of SEQ ID NO: 13; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15.

In yet other embodiments, the CD3 binding arm of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises: a HCVR comprising SEQ ID NO: 5; and a LCVR comprising SEQ ID NO: 6. In certain embodiments, the methods of the disclosure comprise the use of a bispecific CD3×CD20 antibody, wherein the CD3 binding arm comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD3 binding arm comprises a light chain comprising an amino acid sequence of SEQ ID NO: 3.

In certain exemplary embodiments, the CD20 binding arm of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof that can be used in the context of the disclosed methods comprises: a heavy chain complementation determining region (HCDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 4; and a light chain complementation determining region (LCDR) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6.

According to certain embodiments, the CD20-binding arm of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8; HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; LCDR1 comprises the amino acid sequence of SEQ ID NO: 13; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15.

In yet other embodiments, the CD20 binding arm of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises: a HCVR comprising SEQ ID NO: 4; and a LCVR comprising SEQ ID NO: 6. In certain embodiments, the methods of the disclosure comprise the use of a bispecific CD3×CD20 antibody, wherein the CD20 binding arm comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD20 binding arm comprises a light chain comprising an amino acid sequence of SEQ ID NO: 3.

An exemplary antibody comprising: a HCVR comprising a CD3 binding arm having an amino acid sequence of SEQ ID NO: 5; a HCVR comprising a CD20 binding arm having an amino acid sequence of SEQ ID NO: 4; and a common LCVR comprising an amino acid sequence of SEQ ID NO: 6 is the antibody known as onitumumab (also known as REGN1979).

According to certain exemplary embodiments, the methods of the present disclosure include the use of onitumumab or a bioequivalent thereof. As used herein, the term "bioequivalent" refers to an antigen binding protein (e.g., an antibody or an antigen binding fragment thereof) that binds to CD3 and CD20, which is a pharmaceutical equivalent or pharmaceutical substitute that has no significant difference in absorption rate and/or extent from onitumumab when administered at the same molar dose (single dose or multiple doses) under similar experimental conditions. In the context of the present disclosure, the term refers to an antigen binding protein that binds to CD3 and CD20 that has no clinically significant difference from onitumumab in terms of safety, purity and/or efficacy. Bioequivalent

The disclosure encompasses antigen binding molecules with amino acid sequences that are different from the amino acid sequences of the described antibodies but retain the ability to bind CD28 and CD22 or to bind CD3 and CD20. When compared to the parent sequence, these variant molecules include one or more amino acid additions, deletions or substitutions, but exhibit biological activities that are substantially equivalent to the biological activities of the described antigen binding molecules. Similarly, the DNA sequences encoding antigen binding molecules disclosed herein encompass sequences of antigen binding molecules that include one or more nucleotide additions, deletions or substitutions compared to the disclosed sequences, but encode antigen binding molecules that are substantially biologically equivalent to the antigen binding molecules described in the disclosure. Examples of these variant amino acids and DNA sequences are discussed above.

The present disclosure includes antigen binding molecules that are bioequivalent to any of the exemplary antigen binding molecules described herein. For example, if two antigen binding proteins or antibodies are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in absorption rate and extent when administered at the same molar dose (single dose or multiple doses) under similar experimental conditions, they are considered bioequivalent. If some antibodies are equivalent in terms of absorption extent but not in terms of absorption rate, they are considered equivalents or pharmaceutical substitutes and can also be considered bioequivalent because such differences in absorption rate are intentional and reflected in the labeling, are not essential to achieve effective body drug concentrations, such as during long-term use, and are considered medically insignificant for the specific drug under study.

In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in safety, purity, and potency.

In one embodiment, two antigen binding proteins are bioequivalent if a subject can switch between the reference product and the biological product one or more times without an expected increase in the risk of adverse reactions, including clinically significant changes in immunogenicity or reduction in effectiveness, compared to continued treatment without such a switch.

In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action for one or more conditions of use, to the extent such mechanisms are known.

Bioequivalence can be demonstrated by both in vivo and in vitro methods. Bioequivalence measurements include, for example: (a) in vivo tests in humans or other mammals in which the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids is measured over time; (b) in vitro tests that are correlated with and reasonably predictive of the in vivo bioavailability data in humans; (c) in vivo tests in humans or other mammals in which appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) rigorously controlled clinical trials to determine the safety, efficacy or bioavailability or bioequivalence of the antibody.

Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not essential for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or inappropriate intramolecular disulfide bridges upon renaturation. In other cases, bioequivalent antibodies can include the exemplary bispecific antigen-binding molecules described herein that include amino acid changes that alter the glycosylation characteristics of the antibody, such as mutations that eliminate or remove glycosylation. Therapeutic Formulations and Administration

The present disclosure provides pharmaceutical compositions comprising the antigen binding molecules of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated with suitable carriers, excipients and other agents that provide improved transfer, delivery, tolerance and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

The dosage of the antigen binding molecule administered to an individual may vary depending on the age and size of the individual, the target disease, the condition, the route of administration, and the like. The preferred dosage is usually calculated based on body weight or body surface area. When the bispecific antigen binding molecules of the present disclosure are used for therapeutic purposes in adult individuals, it may be advantageous to administer the bispecific antigen binding molecules of the present disclosure intravenously in a single dose of about 0.01 to 400 mg. The frequency and duration of treatment may be adjusted according to the severity of the condition.

Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local.

The pharmaceutical compositions disclosed herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices are readily applicable to delivering the pharmaceutical compositions disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. In practice, the disposable pen delivery device is pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition in the reservoir is used up, the entire device is discarded.

A variety of reusable pen and autoinjector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPENT™ (Owen Mumford, Inc., Woodstock, UK), DISETRONICT™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICL1K™ (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices useful for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, the SOLOSTART™ pen (Sanofi-Aventis), FLEXPENT™ (Novo Nordisk), and KWIKPENT™ (Eli Lilly), SURECLlCK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name a few.

In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (Eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the general review of Langer, 1990, Science 249:1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. Such injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying the above-described antibody or its salt in a sterile aqueous medium or oily medium known for injection. The injection thus prepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a dosage form in a unit dosage suitable for matching the dosage of the active ingredient. Such dosage forms in a unit dosage include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained in each dosage form in a unit dosage is usually about 5 to about 500 mg; in particular, for the injection form, it is preferably about 5 to about 100 mg of the aforementioned antibody, and for other dosage forms, it is preferably about 10 to about 250 mg of the aforementioned antibody. Additional Combination Therapy

The present disclosure includes methods for treating, ameliorating or reducing the severity of at least one symptom or indication of cancer in a subject, or inhibiting the growth of cancer in a subject, comprising administering to a subject in need thereof a therapeutic composition comprising a multispecific (e.g., bispecific) antigen binding molecule that specifically binds CD28 and CD22, alone as a single therapy, or together with a bispecific antibody that binds CD3 and CD20 (e.g., onitumumab) as a combination therapy, and optionally with one or more therapeutic agents (e.g., at least a third therapeutic agent or therapy).

Exemplary third therapeutic agents or therapies that can be combined or administered in combination with the antigen-binding molecules of the present disclosure include, for example, surgery; chemotherapy; radiation therapy; checkpoint inhibitors targeting PD-1 (e.g., anti-PD-1 antibodies, such as pembrolizumab, nivolumab, or cemiplimab), CTLA-4, LAG3, TIM3, and others; co-stimulatory agonist bivalent antibodies targeting, for example, GITR, OX40, 4-1BB, and others; CD3× bispecific antibodies (see, for example, US9,657,102, WO2017/053856A1, WO2014/047231A1, WO2018/067331A1, and WO2018/0 58001A1); other antibodies targeting CD22×CD3, CD22×CD28 or targeting CD20×CD3; other co-stimulatory CD28×bispecific antibodies; oncolytic viruses; cancer vaccines; tamoxifen; aromatase inhibitors; interleukin inhibitors, including small molecule interleukin inhibitors; and antibodies that bind to interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors. The anti-CD28/anti-CD22 antigen binding molecules disclosed herein (e.g., pharmaceutical compositions comprising the anti-CD28/anti-CD22 bispecific antigen binding molecules disclosed herein) can also be administered as part of a treatment regimen comprising one or more therapeutic combinations selected from the following: "ICE": isocyclic phosphamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and "ESHAP": ethioposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ). The anti-CD28/anti-CD22 antigen binding molecules disclosed herein can also be administered in combination with any antigen binding molecule mentioned herein and an inhibitor of one or more of the following: VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvlll, cMet, IGF1 R, B-raf, PDGFR-o, PDGFR-I3, FOLH1, PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin or any of the aforementioned interleukins, wherein the inhibitor is an aptamer, an antisense molecule, a ribonuclease, siRNA, a peptibody, a nanobody or an antibody fragment (e.g., a Fab fragment; a F(ab')2 fragment; a Fd fragment; a Fv fragment; a scFv; a dAb fragment; or other engineered molecules, such as bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, minibodies and minimal recognition units). The anti-CD28/anti-CD22 antigen binding molecules disclosed herein can also be administered in combination with antiviral agents, antibiotics, analgesics, corticosteroids and/or NSAIDs and/or co-formulated. The antigen binding molecules disclosed herein may also be administered as part of a treatment regimen that also includes radiation therapy and/or conventional chemotherapy, or treatment with biologics, including treatment with checkpoint inhibitors or other bispecific antibodies.

The present disclosure includes compositions and therapeutic formulations comprising any antigen binding molecule described herein in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylphosphamide, triethylsulfonamide; phosphamides and trihydroxymethylmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, isocyclophosphamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, mustard; nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); antibiotics (e.g., aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, actinomycin D dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methylamine pterin, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-hydroxypurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; glycoside; aminoacetyl propionic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSK™; razoxane; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxotere™; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; hydroxypurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; VP-16; isocyclic phosphamide; mitomycin C; mitoxantrone; vincristine; vinorelbine lbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and any pharmaceutically acceptable salts, acids or derivatives thereof. Also included within this definition are antihormonal agents used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

The additional therapeutically active component may be administered prior to, simultaneously with, or shortly after administration of the antigen binding molecules of the disclosure; (for purposes of the disclosure, such administration regimens are considered administration of a "combination" of an antigen binding molecule and an additional therapeutically active component).

The present disclosure includes pharmaceutical compositions in which the antigen binding molecules of the present disclosure are co-formulated with one or more additional therapeutically active ingredients described elsewhere herein .

The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions disclosed herein and are not intended to limit the scope of what the inventors regard as their disclosure. Example 1 : Bispecific CD22×CD28 and CD3×CD20 Antibodies

Bispecific CD22×CD28 antibodies are described in WO 2020/132066, the entire contents of which are expressly incorporated herein by reference. An exemplary bispecific CD22×CD28 antibody used in the following examples is REGN5837.

Table 1 lists the amino acid sequence identifiers of the heavy chain and light chain variable regions and CDRs of REGN5837. Table 1 : Amino acid sequence of REGN5837 Anti- CD22 antigen binding domain Anti- CD28 antigen binding domain Common light chain variable region HCVR HCDR1 HCDR2 HCDR3 HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 19 twenty two twenty three twenty four 20 25 26 27 twenty one 28 29 30

Sequence of the heavy chain of the CD22 binding arm of REGN5837: HCVR: EVQLVQSGAEVKKPGESLKISCKGSGYNFATYWIAWVRQMPGKGLELMGIIYPGDSETTYNPSFQGQVTISADKSISNAYLQWSSLKASDTAMYYCARVGGYCSGTSCHNWFDPWGLGTLVTVSS (SEQ ID NO: 19) HCDR1: GYNFATYW (SEQ ID NO: 22) HCDR2: IYPGDSET (SEQ ID NO: 23) HCDR3: ARVGGYCSGTSCHNWFDP (SEQ ID NO: 24) HC：EVQLVQSGAEVKKPGESLKISCKGSGYNFATYWIAWVRQMPGKGLELMGIIYPGDSETTYNPSFQGQVTISADKSISNAYLQWSSLKASDTAMYYCARVGGYCSGTSCHN WFDPWGLGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 16)

Sequence of the heavy chain of the CD28 binding arm of REGN5837: HCVR: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 20) HCDR1: GGSISSYY (SEQ ID NO: 25) HCDR2: IYYSGIT (SEQ ID NO: 26) HCDR3: ARWGVRRDYYYYGMDV (SEQ ID NO: 27) HCVR: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGITHYNPSLKSRVTISVDTSKIQFSLKLSSVTAADTAVYYCARWGVRRDYYYYG MDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 17)

Sequence of the common light chain of the CD22 and CD28 binding arms of REGN5837: LCVR: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 21) LCDR1: QSVSSSY (SEQ ID NO: 28) LCDR2: GAS (SEQ ID NO: 29) LCDR3: QQYGSSPWT (SEQ ID NO: 30) LC: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 18)

Bispecific CD3×CD20 antibodies are described in US 9,657,102, the entire contents of which are expressly incorporated herein by reference. An exemplary bispecific CD3×CD20 antibody used in the following examples is REGN1979.

Table 2 lists the amino acid sequence identifiers of the heavy chain and light chain variable regions and CDRs of REGN1979. Table 2 : Amino acid sequence of REGN1979 Anti- CD3 antigen binding domain Anti- CD20 antigen binding domain Common light chain variable region HCVR HCDR1 HCDR2 HCDR3 HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 5 10 11 12 4 7 8 9 6 13 14 15

Sequence of the heavy chain of the CD3 binding arm of REGN1979: HCVR: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDVWGQGTTVTVAS (SEQ ID NO: 5) HCDR1: GFTFDDYT (SEQ ID NO: 10) HCDR2: ISWNSGSI (SEQ ID NO: 11) HCDR3: AKDNSGYGHYYYGMDV (SEQ ID NO: 12) HC: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGM DVWGQGTTVTVASASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSLGK (SEQ ID NO: 2)

Sequence of the heavy chain of the CD20 binding arm of REGN1979: HCVR: EVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSS (SEQ ID NO: 4) HCDR1: GFTFNDYA (SEQ ID NO: 7) HCDR2: ISWNSDSI (SEQ ID NO: 8) HCDR3: AKDNHYGSGSYYYYQYGMDV (SEQ ID NO: 9) HC: EVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQ YGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1)

Sequence of the common light chain of the CD3 and CD20 binding arms of REGN1979: LCVR: EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIKR (SEQ ID NO: 6) LCDR1: QSVSSN (SEQ ID NO: 13) LCDR2: GAS (SEQ ID NO: 14) LCDR3: QHYINWPLT (SEQ ID NO: 15) LC: EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3) Example 2 : Clinical Evaluation of Combination Treatment of REGN5837 and REGN1979

This example describes a phase 1 clinical study of the combination of REGN5837 (a CD22×CD28 co-stimulatory bispecific antibody) and REGN1979 (onitumumab) in individuals with relapsed or refractory aggressive B-cell non-Hodgkin's lymphoma.

The primary objectives of the study are to evaluate the safety, tolerability, and dose-limiting toxicities (DLTs) of the combination of REGN5837 and onitumumab in individuals with relapsed or refractory aggressive B-NHL and to determine the recommended phase 2 dose (RP2D) regimen (defined as the maximum tolerated dose (MTD) regimen or a lower dose regimen).

The secondary objectives of the study are to: (i) characterize the pharmacokinetics (PK) of REGN5837 when administered in combination with REGN1979 (onitumumab); (ii) evaluate the PK of REGN1979 (onitumumab) when administered in combination with REGN5837; (iii) evaluate the immunogenicity of REGN5837 and onitumumab; and (iv) evaluate the preliminary antitumor activity of the combination of REGN5837 and REGN1979 (onitumumab) in individuals with relapsed or refractory aggressive B-NHL.

The exploratory objectives of the study are to: (i) evaluate the association between clinical efficacy and safety and systemic immune activation biomarkers (serum interleukin levels, T cell counts and activation markers); (ii) evaluate the association between disease response and/or relapse and changes from baseline in counts and phenotypes of tumor-infiltrating T cells and tumor B cell target antigens (CD20, CD22); (iii) evaluate the association of molecular minimal residual disease (MRD) status with progression-free survival (PFS) and overall survival (OS) in individuals with a clinical complete response (CR); and (iv) evaluate the association between REGN5837 and REGN1979. (onitumumab) combination exposure, antitumor activity, and other biomarkers (pharmacodynamic, predictive, and prognostic) potentially associated with safety; and (v) assess the relationship between pharmacodynamics, drug concentrations, and clinical safety and efficacy measures. Study Design

The purpose of this Phase 1 open-label FIH study is to evaluate the safety, PK and pharmacodynamic properties and preliminary clinical activity of the combination of anti-CD22 × anti-CD28 bispecific antibody REGN5837 and anti-CD20 × anti-CD3 bispecific antibody onitumumab (REGN1979) in individuals with relapsed or refractory aggressive B-NHL (excluding mantle cell lymphoma (MCL)) (hereinafter referred to as "aggressive B-NHL"). The study is divided into two parts, dose escalation and dose expansion.

To reduce the likelihood and severity of interleukin release and reduce the risk of TLS, REGN1979 (onitumumab) will be introduced as monotherapy from Cycle 1 Day 1 to Cycle 2 Day 8. REGN5837 will be initiated on Cycle 2 Day 15. REGN5837 will be initiated after a single infusion of onitumab at a QW dose in the previous week without signs or symptoms of interleukin release syndrome (CRS), infusion-related reactions (IRR), or tumor lysis syndrome (TLS). The sequential introduction of REGN1979 (onitumumab) and REGN5837 allows individuals who experience CRS, IRR, or TLS on onitumumab monotherapy to recover before receiving REGN5837. The dose of onitumab will be increased in a stepwise manner until Day 1 of Cycle 2, with an initial dose on Days 1 and 2 of Cycle 1, followed by an intermediate dose on Days 8 and 9 of Cycle 1, a second intermediate dose on Days 15 and 16 of Cycle 1, and a full cycle dose on Day 1 and thereafter of Cycle 2. In ongoing studies of onitumab, no severe CRS has been observed in individuals with diffuse large B-cell lymphoma (DLBCL) once individuals have tolerated 2 full nominal doses of onitumab.

To further reduce the risk of severe CRS, IRR, or TLS when REGN5837 is introduced in the presence of onitumab, REGN5837 is administered using an escalating dosing schedule, following the same approach as the onitumab escalation dosing, with a lower initial dose on Day 15 of Cycle 2, an intermediate dose on Day 1 of Cycle 3, and a full cycle dose on Day 8 of Cycle 3.

Weekly dosing of REGN5837 and onitumumab will continue until Day 8 of Cycle 6, after which dosing will switch to a Q2W schedule. The QW dosing period will be the induction period (Cycles 1 to 6), followed by Q2W dosing, which will be the maintenance period. Subjects who achieve and maintain a complete response (CR) for 9 months will transition to Q4W dosing. Prior to switching from Q2W dosing to Q4W dosing, subjects must have received at least 3 doses of the previous dose on the Q2W dosing schedule.

The dose escalation part of the study will follow the Bayesian Optimal Interval Design (BOIN) to evaluate the safety of the combination of REGN5837 and onitumumab and select the recommended phase 2 dose (RP2D) of the combination of REGN5837 and onitumumab. The DLT observation period will be 35 days from the start of REGN5837 administration (usually from day 15 of cycle 2 to day 7 of cycle 4) and will consist of at least 3 full doses of the combination of REGN5837 and onitumumab.

For the dose expansion portion, there will be 2 expansion cohorts: A and B. Cohort A consists of individuals with aggressive B-NHL who have not received prior chimeric antigen receptor T cell (CAR-T) therapy. Cohort B consists of individuals with aggressive B-NHL who have progressed after failure of CAR-T therapy. Individuals enrolled in the expansion cohorts will receive the combination treatment at RP2D. Initial antitumor activity will be explored, and safety and tolerability, PK properties, and biomarker responses will be further characterized. Study Duration

After a 28-day screening period, study treatment will consist of six 21-day cycles of inducement dosing consisting of a weekly regimen of onitumumab monotherapy from Day 1 of Cycle 1 to Day 8 of Cycle 2 followed by REGN5837 plus onitumumab combination therapy from Day 15 of Cycle 2 to Day 8 of Cycle 6.

The combination of REGN5837 and onitumumab will be dosed every 2 weeks (Q2W) during the maintenance period following a 28-day cycle schedule and beginning 2 weeks after the last induction of a weekly (QW) dose. The combination of REGN5837 and onitumab will continue on the Q2W schedule until disease progression or discontinuation of treatment for other regimen-defined reasons. Subjects who achieve and maintain a complete response (CR) for at least 9 months will be switched to a every 4-week (Q4W) schedule of REGN5837 and onitumab until disease progression or discontinuation of treatment for other regimen-defined reasons. Prior to switching from Q2W dosing to Q4W dosing, subjects must have received at least 3 designated doses of the previous dose on the Q2W dosing schedule.

Safety follow-up: The safety follow-up period consisted of three Q4W safety follow-up visits, including a safety follow-up visit 4 weeks after the last dose (1st safety follow-up visit), a safety follow-up visit 8 weeks after the last dose (2nd safety follow-up visit), and a safety follow-up visit 12 weeks after the last dose (3rd safety follow-up visit). Safety follow-up continued until all 3 visits were completed, or until non-protocol anti-lymphoma therapy was started, or the individual withdrew consent, whichever came first.

Extended Follow-up: Extended follow-up is available for subjects who discontinue study drug for any reason other than disease progression, initiation of non-protocol anti-lymphoma therapy, withdrawal of consent, or death. Disease response will be assessed until the earlier of disease progression, death, initiation of non-protocol anti-lymphoma therapy, or the subject withdraws consent for disease status follow-up.

Survival follow-up: After the safety follow-up period and the extended follow-up period (if applicable), all study subjects will be followed up for survival at Q12W intervals until death, loss to follow-up, withdrawal of consent for follow-up by the subject, or termination of the study by the sponsor, whichever comes first. Survival follow-up status can be determined at the outpatient clinic visit or remotely by the study center (e.g., by telephone).

The end of study time for each individual will be when the individual stops participating in the study until the end of the extended follow-up and before the survival follow-up. All individuals will continue to be followed until death, loss to follow-up, withdrawal of consent for follow-up, or termination of the study, whichever comes first.

The study and all follow-up visits will end when all study subjects discontinue study or the study is closed, whichever comes first.

Onitumab monotherapy ( Cycle 1 Day 1 to Cycle 2 Day 8 ) : The study center should ensure that each subject receives 2 doses of anti-IL6 therapy (e.g., tocilizumab) prior to administration of any study medication. Onitumab will be infused weekly as monotherapy from Cycle 1 Day 1 to Cycle 2 Day 8 of study treatment. For the initial dose (0.7 mg, split into 0.2 mg/0.5 mg), intermediate dose 1 (4 mg, given as 2 mg/2 mg divided infusions), and intermediate dose 2 (20 mg, given as 10 mg/10 mg divided infusions), onitumumab will be administered as divided infusions, preferably on consecutive days no more than 3 days apart, to improve tolerability and reduce CRS, IRR, and TLS. For example, onitumumab dosing days will be Cycle 1 Day 1 and Cycle 1 Day 2 for the initial dose, Cycle 1 Day 8 and Cycle 1 Day 9 for the first intermediate dose, and Cycle 1 Day 15 and Cycle 1 Day 16 for the second intermediate dose. Administer divided doses over 4 hours each day for 2 days. The initial dose and two intermediate doses should always be split, even if treatment delays result in dosing beyond Day 15 of Cycle 1. On Day 1 of Cycle 2, a full dose of 160 mg (Note: 80 mg QW in DL1) will be administered as a single infusion over 4 hours. On Day 8 of Cycle 2, another full dose of onitumumab will be administered. If the first full QW dose of onitumumab is tolerated as a single infusion, subsequent doses (usually Day 8 of Cycle 2 and thereafter) may be administered as a single infusion over 1 to 4 hours, depending on prior tolerability. To be able to continue combination therapy, the most recent onitumumab dose must have been tolerated as a single infusion without any grade of CRS, IRR, or TLS.

Individuals should be hospitalized during the onitumumab infusion and for at least 24 hours after the end of the infusion (including days of divided dosing) until a complete undivided dose has been administered and no CRS occurs. Each infusion should be administered over 4 hours.

REGN5837/ Onetumab Dosing Weekly (Cycle 2 Day 15 to Cycle 6 Day 8): Start REGN5837 on Cycle 2 Day 15. REGN5837 will also be administered in an escalating dosing format. All doses of REGN5837 will be administered as a single infusion. For all dose levels, REGN5837 dosing will only be initiated if the individual has demonstrated tolerance to 2 full QW doses of Onetumab as defined for each individual dose level (DL) (Table 4). In addition, before an individual can start REGN5837, the most recent QW dose of Onetumab monotherapy must have been administered as a single infusion without the occurrence of any grade of CRS, IRR, or TLS. Considering the potential for exacerbated CRS risk with combination therapy of REGN5837 and onitumumab, individuals who experience grade 3 CRS during the monotherapy run-in period will not be eligible for REGN5837 but may continue single-agent onitumumab for the duration of the study.

On Day 15 of Cycle 2 of study treatment, REGN5837 will be introduced at a low initial dose, followed by a QW dose of onitumab on Day 16. On Day 1 of Cycle 3, REGN5837 will be increased to an intermediate dose. On Day 8 of Cycle 3, REGN5837 will be increased to a full dose. When combination therapy begins, REGN5837 will be administered during Cycles 2 and 3 the day before onitumab administration. This staggered dosing schedule will include step-escalation dosing of REGN5837 on Day 15 of Cycle 2, Day 1 of Cycle 3, and Day 8 of Cycle 3 plus a second full combination dose on Day 15 of Cycle 3. If staggered dosing is well tolerated without any Grade 2 or higher CRS, same-day dosing of REGN5837 and onitumumab will begin on Day 1 of Cycle 4. However, if Grade 2 or higher CRS occurs with staggered dosing, same-day dosing of the combination will be delayed until two full combination doses of REGN5837 and onitumumab have been tolerated without any such events. Once initiated, same-day dosing of REGN5837 and onitumumab will continue for the remainder of study treatment. During same-day dosing of combination therapy, REGN5837 will always be administered first, followed by onitumumab, which will be initiated within 60 minutes of the completion of the REGN5837 infusion.

For doses <1 mg, the duration of the REGN5837 infusion is 1 hour and REGN5837 will be administered by syringe pump. For doses ≥1 mg throughout the staggered dosing period and the Cycle 4 Day 1 dose, the duration of the REGN5837 infusion is 2 hours by IV pump. If the individual does not experience IRR, CRS, or TLS, the duration of the REGN5837 infusion may be reduced to 1 hour starting on Cycle 4 Day 8. The duration of the infusion may be extended based on clinical judgment. The timing of onitumumab infusion during the REGN5837 escalation period and the second QW full dose (Day 15 of Cycle 2, Day 1 of Cycle 3, Day 8 of Cycle 3, and Day 15 of Cycle 3) will be consistent with the infusion time on Day 8 of Cycle 2.

Once combination therapy is initiated, individuals should be hospitalized to receive infusions of REGN5837 and onitumumab until at least 48 hours after completion of the REGN5837 infusion (24 hours after completion of the onitumumab infusion). The observation period in the protocol will vary based on the following rationale: the risk of CRS is highest within the first 24 hours after completion of the bispecific antibody infusion. During the hospitalization period, individuals should be monitored for IRR, CRS, and TLS during and after each infusion according to institutional observation guidelines. Monitoring will include, but is not limited to: vital signs (including temperature, blood pressure, and oxygen saturation); clinical and laboratory (at least serum chemistry) assessments.

Hospitalization for combination therapy will continue until two full doses of the combination therapy have been administered without CRS. Beginning on Cycle 4 Day 1, hospitalization is not required as long as the individual receives and tolerates a full dose of REGN5837 in combination with a full dose of onitumumab. However, if an individual experiences Grade 2 or higher CRS on or after Cycle 3 Day 8, they must be monitored in the hospital for at least 48 hours after the completion of the REGN5837 infusion until they tolerate both study drugs without an event of Grade 2 or higher CRS.

Beginning on Day 1 of Cycle 4 or when hospitalization is no longer required (whichever is later), for outpatient infusion visits during the first 4 weeks, individuals were required to be observed for at least 2 hours after the end of the last infusion; clinical status assessments, including vital sign checks, were required at least hourly. Individuals were required to be called on the day after the first 4 outpatient infusion visits for safety assessments.

If an individual is unable to start combination treatment with REGN5837 on Day 15 of Cycle 3 due to the emergence or persistence of a safety event, and criteria for permanent discontinuation of onitumumab have not been met, onitumumab monotherapy may be continued at the indicated dose. In such cases, no attempt to introduce REGN5837 will be made, and individuals in the dose escalation group may be substituted for DLT evaluation.

During the REGN5837/onitumumab induction dosing period, subjects will receive 12 weekly doses of the REGN5837 and onitumab combination therapy. Assuming REGN5837 is introduced on Day 15 of Cycle 2, this means that subjects will receive a total of 17 doses of onitumab after the REGN5837/onitumab weekly dosing period is completed. If dosing is interrupted, the REGN5837/onitumab weekly dosing period will be extended until the subject receives 12 doses of the REGN5837 and onitumab combination.

Pre-treatment therapy : Figure 3 and the following text illustrate the pre-treatment medication requirements before the step-up dosing of REGN5837 is completed.

The following premedication is indicated for initial onitumumab monotherapy, from the initial dose to the first single infusion of a full weekly (QW) dose. If an individual experiences an IRR and/or CRS of any grade on the first full QW dose, premedication is continued until the full QW dose is tolerated without experiencing an IRR and/or CRS. a) 12 to 24 hours prior to the scheduled start of the first divided infusion or first QW full-dose single infusion and 12 to 24 hours prior to the scheduled start of the second divided infusion when each onitumumab dose is administered on non-consecutive days: i. Dexamethasone 10 mg PO or equivalent steroid* b) Medication prior to each onitumumab divided infusion day and QW full-dose single infusion day: i. Dexamethasone 20 mg IV 1 to 3 hours prior to the start of infusion ii. Diphenhydramine 25 mg IV or PO 30 to 60 minutes prior to infusion (may be substituted with another equivalent antihistamine) iii. Acetaminophen 650 mg 30 to 60 minutes prior to infusion PO, except in individuals who have received acetaminophen within 4 hours prior to infusion of onitumumab or who are allergic to acetaminophen c) Within 24 (±4) hours after completion of the second divided infusion or first full QW bolus infusion: i. Dexamethasone 10 mg PO or equivalent steroid*

The following premedication is applicable to the onitumumab treatment day of Day 8 of Cycle 2 ( or the second full dose of onitumumab), provided that the individual has not experienced any grade of IRR and/or CRS during the previous 4 weeks of onitumumab monotherapy: Premedication prior to the single infusion day of onitumumab : i. Dexamethasone 10 mg IV 1 to 3 hours before the start of the infusion on the day of treatment ii. Diphenhydramine 25 mg IV or PO (can be substituted with another equivalent antihistamine) 30 to 60 minutes before the infusion iii. Acetaminophen 650 mg PO 30 to 60 minutes before the infusion, except in the following circumstances: the individual has received acetaminophen within 4 hours before the onitumumab infusion or is allergic to acetaminophen

The following pre-driver medications were applied to the combination of REGN5837 and onitumumab from Cycle 2 Day 15 to Cycle 3 Day 8 ( Step -wise escalation of the combination of REGN5837 and onitumumab) (Figure 3). If an individual experienced any grade of IRR and/or CRS on the first full QW dose of REGN5837, pre-driver medications were continued until the full QW dose was tolerated without experiencing IRR and/or CRS. a) 12 to 24 hours before the scheduled start of REGN5837 infusion: i. Dexamethasone 10 mg PO or equivalent steroid* b) Medication prior to each REGN5837/onitumumab combination therapy day: ii. Dexamethasone 10 mg IV 1 to 3 hours before starting REGN5837 and onitumumab infusion on the day of treatment iii. Diphenhydramine 25 mg IV or PO (may be substituted with another equivalent antihistamine) 30 to 60 minutes before REGN5837 and onitumumab infusion iv. Acetaminophen 650 mg 30 to 60 minutes before REGN5837 and onitumumab infusion PO, except in individuals who have received acetaminophen within 4 hours prior to infusion of REGN5837 or onitumumab or who are allergic to acetaminophen c) Within 24 (±4) hours after completion of onitumumab infusion: i. Dexamethasone 10 mg PO or steroid equivalent* *Steroid equivalents to dexamethasone 10 mg include prednisone/prednisolone 60 mg or methylprednisolone 50 mg (PO dose only).

The following prodromal medications are for the combination of REGN5837 and onitumumab during Days 15 and 16 of Cycle 3 or for the second complete combination dose of REGN5837 and onitumumab without any grade of IRR and/or CRS, whichever occurs later: Prodromal medications for each infusion day: i. Dexamethasone 10 mg IV 1 to 3 hours before the start of REGN5837 and onitumumab infusion on the day of treatment ii. Diphenhydramine 25 mg IV or PO (may be substituted with another equivalent antihistamine) 30 to 60 minutes before REGN5837 and onitumumab infusion iii. Acetaminophen 650 mg 30 to 60 minutes before REGN5837 and onitumumab infusion PO, except in individuals who have received acetaminophen within 4 hours prior to infusion of REGN5837 or onitumumab or who are allergic to acetaminophen

For subsequent dosing of the combination of REGN5837 and onitumumab: a) If the individual continues to adequately tolerate the combination of REGN5837 and onitumumab without experiencing any grade of IRR and/or CRS while taking the dexamethasone doses described above, then discontinuation of corticosteroids (along with diphenhydramine and acetaminophen) is encouraged for subsequent dosing of the combination of REGN5837 and onitumumab. b) If continued corticosteroid administration is necessary, dexamethasone may be substituted with another intermediate- or long-acting corticosteroid (e.g., methylprednisolone).

If an individual experiences any grade of CRS or IRR at any point during treatment, subsequent dosing also requires premedication with acetaminophen/antihistamine.

Additional prophylactic medication with antihistamines, acetamide, and/or corticosteroid equivalents may also be considered.

If 2 individuals experience ≥ Grade 3 CRS events, the oral dexamethasone (or equivalent) dose will be increased to 20 mg around the dosing date for that group. Maintenance dosing period

REGN5837/ Onetumab Q2W and Q4W Dosing : After completing the REGN5837/Onetumab weekly induction dosing period, subjects will enter the Q2W treatment period and receive the designated QW REGN5837 dose and 320 mg of onitumab Q2W (or for DL1, 160 mg of onitumab Q2W). During the Q2W treatment period, REGN5837 should be administered on the same day as onitumab (or on 2 consecutive days if necessary for scheduling reasons). The combination of REGN5837 and onitumab will continue on a Q2W schedule until disease progression or treatment is discontinued for other protocol-defined reasons. However, if the subject achieves a CR lasting at least 9 months, the subject will be switched to a Q4W dosing schedule, receiving QW doses of REGN5837 and 320 mg of onitumumab (note: 160 mg for DL1). Prior to switching from Q2W to Q4W dosing, the subject must have received at least 3 doses of the designated REGN5837/onitumumab dose on the Q2W dosing schedule.

Safety of Q4W dosing will be evaluated after the first 16 subjects receive 2 combined doses at this frequency. •      If 4 or fewer subjects experience ≥ Grade 2 CRS events, subsequent subjects continue Q4W dosing •      If 5 or more subjects experience ≥ Grade 2 CRS events, all subjects stop Q4W dosing and continue Q2W dosing

This rule is based on the lower limit of the one-sided 80% confidence interval. If the lower limit of the one-sided 80% confidence interval for the ≥ Grade 2 CRS rate is > 20%, the switch from Q2W to Q4W will be suspended. After the switch from Q2W to Q4W dosing, subjects who experience ≥ Grade 2 CRS after the first dose of either study drug will be included in the analysis. Dose Escalation and Study Groups

The dose escalation rule will be based on a Bayesian optimal interval design (BOIN) with a target dose-limiting toxicity (DLT) rate of 28 % . This target DLT rate corresponds to a descending margin similar to a 3+3 design. All dose levels will enroll at least 3 subjects. The decision rule for dose level j is summarized as follows: • If the observed DLT rate is ≤ 0.221, escalate to dose level j+1; the observed DLT rate is calculated as the number of subjects with DLT in the dose level divided by the number of subjects evaluable for DLT. • If the observed DLT rate is ≥ 0.334, descend to dose level j-1; 3 additional subjects will be added to dose level j-1. • If 0.221 < observed DLT rate < 0.334, dose level j is maintained and 3 additional subjects are added for further evaluation. • If the number of subjects with DLT among the evaluable subjects reaches a boundary defined as the posterior probability ≥ 0.95 that the true DLT rate is higher than the target DLT rate, dose level j and higher dose levels will be eliminated from the trial. If the first dose level is eliminated, the trial will be terminated. Posterior probabilities can be estimated based on a β-binomial model assuming a non-informative prior distribution of β(1,1).

The decision rule will be repeated until the pre-specified maximum sample size of 54 subjects is exhausted, at which point the MTD is selected as the dose whose isosmolar estimate of the probability of DLT is closest to the target DLT rate of 28%.

It should be noted that the actual number of evaluable subjects in each group can vary. As long as there is at least one evaluable subject in a dose level (except for DL1 and DL2, where at least 3 DLT evaluable subjects will be required), the decision rule during the study applies and varies depending on the number of evaluable subjects at each dose level and the number of subjects with DLTs observed. In Table 3, detailed decision rules for various numbers of DLT subjects and evaluable subjects are provided. Table 3 : Dose escalation / decrease rule for current dose level J , target DLT rate 28% Boundary Number of evaluable individuals treated at current dose level J 1 2 3 4 5 6 7 8 9 10 Incremental, if the number of DLT individuals ≤ 0 0 0 0 1 1 1 1 1 2 Decrease, if the number of DLT individuals ≥ 1 1 2 2 2 3 3 3 4 4 Cancel*, if the number of DLT individuals ≥ NA NA 3 3 4 4 4 5 5 6 Boundary Number of evaluable individuals treated at current dose level J 11 12 13 14 15 16 17 18 19 20 Incremental, if the number of DLT individuals ≤ 2 2 2 3 3 3 3 3 4 4 Decrease, if the number of DLT individuals ≥ 4 5 5 5 6 6 6 7 7 7 Cancel*, if the number of DLT individuals ≥ 6 6 7 7 8 8 8 9 9 9 *Dose levels j and above will be removed from the trial and the trial will be terminated when the first dose level is removed.

The operating characteristics based on 1000 simulations for the true toxicity probability (0.05, 0.15, 0.28, 0.45, 0.6) are: • Percentage (%) selected for each dose level: 1.2, 27.3, 50.6, 18.4, 2.5 • Number of subjects treated at each dose level: 3.954, 6.921, 6.933, 2.817, 0.375 • Number of subjects observed with toxicity at each dose level: 0.177, 1.002, 1.910, 1.274, 0.226 • Average number of subjects with toxicity: 4.589 • Average number of subjects: 21 • Percentage of subjects stopped early due to toxicity: 0.0% • Overdose risk (>60% of subjects received treatment beyond the MTD): 0.0% • Overdose risk (>80% of subjects received treatment beyond the MTD): 0.0% Table 4 : Dose escalation schedule Dosage level Onitumumab (mg) REGN5837 (mg)** Cycle 1 Cycles 2 to 6 Cycle ≥7 Cycle 2 Cycle 3 Cycle 3 Day 1/Day 2 Day 8/Day 9 Day 15/Day 16 Day 1/Day 8/Day 15*** Day 1 to Day 15 Day 15 Day 1 Day 8 Initial dose Intermediate dose 1 Intermediate dose 2 Full/QW dose Q2W Dosage Initial dose* Intermediate dose* Full/QW dose DL 1 0.2/0.5 2/2 10/10 80 80 80 160 0.03 0.1 0.3 DL 2 0.2/0.5 2/2 10/10 160 160 160 320 0.03 0.1 0.3 DL 3 0.2/0.5 2/2 10/10 160 160 160 320 0.1 0.3 1 DL 4 0.2/0.5 2/2 10/10 160 160 160 320 0.3 1 3 DL 5 0.2/0.5 2/2 10/10 160 160 160 320 0.5 3 10 DL 6 0.2/0.5 2/2 10/10 160 160 160 320 0.75 5 20 DL 7 0.2/0.5 2/2 10/10 160 160 160 320 1 10 40 DL 8 0.2/0.5 2/2 10/10 160 160 160 320 1.5 20 80 DL9 0.2/0.5 2/2 10/10 160 160 160 320 2 20 160 *Based on tolerability, dose may be fixed at a lower level**REGN5837 initiated on or after Cycle 2 Day 15 and continued with onitumumab throughout disease progression***For combination therapy during Cycles 2 and 3, onitumab administration will be staggered by at least 24 hours after REGN5837 dose; no REGN5837 and onitumumab dose on Cycle 6 Day 15

If intolerance is determined for the initial (Cycle 2 Day 15) dose of REGN5837 in the dose escalation group, the initial dose for subsequent groups will be reduced to the highest tolerated initial dose of REGN5837. Similarly, if continued escalation of the intermediate (Cycle 3 Day 1) dose of REGN5837 is intolerant, the Cycle 3 Day 1 dose will be fixed at the highest tolerated intermediate dose.

If the initial and/or intermediate doses of REGN5837 are fixed, the purpose of the DLT window will be to evaluate the safety of the combination of a full dose of REGN5837 and onitumumab. If an individual experiences any toxicity requiring discontinuation of the drug before the first full dose of the combination of REGN5837 and onitumumab, the individual will be considered DLT unevaluable. Adverse events that occur before the first full dose of the combination of REGN5837 and onitumumab will inform the overall safety profile of the combination of REGN5837 and onitumumab, and will affect dose escalation according to the dose escalation limit rules (outlined below). Individuals who are considered DLT unevaluable may be replaced.

Dosing of REGN5837 follows rule-based dose escalation guidelines and is between the values in Table 4, but not exceeding the pre-specified maximum dose value, which may be explored based on observed safety events. Dose levels are defined by the Cycle 3 Day 8 dose. Safety events that occur at the designated dose of REGN5837 on Cycle 2 Day 15 that was tolerated as the Cycle 3 Day 1 dose at the previous dose level will be discussed by the SSET and a decision may be made to reduce the Cycle 2 Day 15 dose. The Cycle 2 Day 15 dose may be fixed at a lower dose to allow for continued dose escalation on Cycle 3 Day 1. Similarly, safety events occurring at the designated dose of REGN5837 on Cycle 3 Day 1 that was tolerated at the previous dose level as the Cycle 3 Day 8 dose will be evaluated, and a decision may be made to reduce the Cycle 3 Day 1 dose. The Cycle 3 Day 1 dose may be fixed at a lower dose to allow for continued dose escalation on Cycle 3 Day 8. In all cases, modifications were only applicable to implement more conservative dose escalation between groups (e.g., a more gradual increase between 2 consecutive DLs, or splitting the initial or subsequent dose into 2 doses).

Safety of at least 3 DLT-evaluable individuals in each group will be evaluated prior to escalation to dose level 3. Escalation to DL3 will occur only if the safety profiles of DL1 and DL2, including the frequency and severity of CRS/IRR events on combination therapy, are comparable. However, if a higher proportion of individuals in DL2 experience Grade 2 or higher CRS/IRR events on combination therapy compared to DL1, dose escalation of REGN5837 from DL3 will be conducted in combination with onitumumab 80 mg QW. Preliminary pharmacodynamic data from DL1 and DL2 may also be evaluated when available but will not be included in this decision.

The following dose increment limits for REGN5837 will also be implemented: a) If any subject in a given group experiences a Grade ≥2 AE (except AEs clearly attributable to underlying disease or external causes) during the DLT period, the maximum dose increment for REGN5837 will be limited to 100%, including the step-up dose and the complete treatment dose of the next dosing group. b) If 2 or more subjects in a given group experience a Grade ≥2 AE (except AEs clearly attributable to underlying disease or external causes) during the DLT period, the maximum dose increment for REGN5837 will be limited to 50%, including the step-up dose and the complete treatment dose of the next dosing group. c) If any subject in a given group experiences a DLT during the DLT period, the maximum dose increment of REGN5837 will be limited to 50%, including the step-up dose and the full treatment dose of the next dosing group. d) If 2 or more subjects in a given group experience a DLT during the DLT period, the maximum dose increment of REGN5837 will be limited to 30%, including the step-up dose and the full treatment dose of the next dosing group.

The following AEs are commonly observed with onitumumab monotherapy and may be present at a low grade when REGN5837 dosing is introduced: asthenia, cytopenia, clinically significant electrolyte disturbances, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, diarrhea, rash, headache. For these AEs, the dose increment limit guidance numbers (a) and (b) above require the presence of Grade 3 or higher events, rather than Grade 2 or higher events. In addition, the rules (a) and (b) listed above do not apply to infections other than Grade 4 or elevated liver enzymes and bilirubin that resolve within 72 hours.

Intra-individual dose escalation will not be permitted (Table 4). Dose-Limiting Toxicity (DLT)

A DLT will be defined as any toxicity listed below unless the event is clearly attributable to the underlying disease or an external cause (including concomitant medications).

The grades of these toxicities were defined according to the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, with the exception of interleukin-release syndrome (CRS) and immune-effector cell-associated neurotoxicity syndrome (ICANS), which were defined according to the American Society for Transplantation and Cellular Therapy (ASTCT) criteria.

DLTs are defined as follows: Non-hematologic toxicities : • Any Grade 5 toxicity • Seizure of any grade • Grade 4 alanine transaminase/aspartate transaminase (ALT/AST) values persisting for more than 3 consecutive days • Any other ≥Grade 3 non-hematologic toxicity, except the following: o Alopecia o Nausea, vomiting, fatigue, or diarrhea persisting for less than 72 hours with supportive care measures prescribed by the treating physician o Grade 3 tumor lysis syndrome (TLS) o Grade 3 infusion-related reaction (IRR) or CRS responsive to medical management and acute effects that resolve to Grade 1 or baseline within 72 hours. NOTE: Associated laboratory abnormalities may remain at the same grade for 7 days if there is no evidence of ongoing organ damage. o Isolated laboratory abnormalities without clinical symptoms for the following: ≤Grade 2 alkaline phosphatase, gamma glutamyl transferase, amylase, lipase, INR, activated partial thromboplastin time, hypertriglyceridemia, electrolyte abnormalities Hematologic toxicities : • Any Grade 5 hematologic toxicity • Grade 4 neutropenia persisting for more than 7 days despite administration of granulocyte colony-stimulating factor (G CSF) • Grade 4 febrile neutropenia • Grade 4 thrombocytopenia persisting for more than 7 days • Grade 3 thrombocytopenia associated with ≥Grade 2 bleeding (excluding Grade 2 epistaxis) • Neutropenia grade 3 or higher with documented infection

Treatment-emergent adverse events (TEAEs) that appear to meet the definition of a DLT will be evaluated. Situations in which two full doses of REGN5837 in combination with onitumumab cannot be administered within 35 days due to study drug toxicity will also be discussed. The final decision on whether a TEAE meets the definition of a DLT will be based on a careful review of all relevant data.

Individuals who do not meet the protocol-defined permanent stopping criteria may continue treatment only if it is determined that resumption of combination study treatment following the development of a DLT is in the best interest of the individual.

A total of up to 37 subjects will be enrolled in the dose expansion phase at the RP2D determined in the dose escalation portion of the study, divided into 2 expansion groups: A and B. Groups A and B will enroll subjects with aggressive B-NHL who have not received prior CAR-T therapy and who have progressed after failure of prior CAR-T therapy, respectively, and in whom safety and efficacy can be evaluated. Analyses of these subjects will be combined with subjects treated at the Phase 2 recommended dose (RP2D) in the dose escalation portion, for a total of 20 subjects treated at the RP2D in each group.

Subjects enrolled in the dose expansion cohort will receive an RP2D to further evaluate preliminary anti-tumor activity, safety, PK properties, and biomarker changes associated with the REGN5837 and onitumumab combination therapy.

Individuals in the dose expansion group who have received at least 1 dose of REGN5837 will not be replaced.

The study population will consist of individuals with aggressive B-NHL lymphoma according to WHO criteria who have progressed after at least 2 lines of therapy including an anti-CD20 antibody and an alkylating agent.

Individuals had to meet the following criteria to be eligible for study inclusion: 1. Age 18 years or older 2. Documented CD20 ⁺ aggressive B-NHL with disease progression after at least 2 lines of systemic therapy containing anti-CD20 antibodies and alkylating agents. Lymphoma subtypes were based on the World Health Organization (WHO) classification. Eligible subtypes included: DLBCL, primary lateral (thymic) large B-cell lymphoma, T-cell/tissue-rich large B-cell lymphoma, grade 3b follicular lymphoma, and high-grade B-cell lymphoma (HGBL) with and without MYC and BCL2 or BCL6 translocations. Subjects must require systemic therapy for lymphoma at study entry, at the discretion of the investigator. NOTES: • Subjects who have relapsed after the most recent prior line of therapy or who have had disease progression during or after prior therapy are eligible. • Subjects who have received CAR-T therapy are eligible. Ongoing studies have shown that onitumumab is tolerated by and provides efficacy in these individuals. In the dose expansion phase, subjects who have not received CAR-T and those following CAR-T failure are evaluated separately in Cohorts A and B, respectively. • DLBCL transformed from lower-grade tumors (e.g., FL or CLL) may be included. Subjects who have transformed to DLBCL from prior CLL may only be included if a leukemic CLL component is not present. For individuals with transformed DLBCL, prior systemic therapy for lower-grade tumors will not be considered prior lines of therapy for eligibility purposes. 3. Measurable disease on cross-sectional imaging (defined as at least 1 bidimensionally measurable lymph node lesion with a greatest transverse diameter (GTD) ≥1.5 cm, regardless of short-axis diameter) as documented by diagnostic imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) 4. Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1 5. Adequate bone marrow function as documented by: a. Platelet count ≥50 × 109/L. Subjects may not have received platelet transfusion therapy within 7 days prior to the first dose of onitumab to meet platelet eligibility criteriab. Hemoglobin ≥9.0 g/dL; transfusions are permitted per protocol to meet this criterion.c. Absolute neutrophil count (ANC) ≥1.0 × 109/L. Subjects may not have received G CSF within 2 days prior to the first dose of onitumab to meet ANC eligibility criteriaNote: Subjects with bone marrow involvement or splenic sequestration should meet the following hematologic parameters: – Platelet count ≥25 × 109/L. Individuals may not have received platelet transfusion therapy within 3 days prior to the first dose of onitumumab in order to meet platelet eligibility criteria – hemoglobin ≥7.0 g/dL – absolute neutrophil count (ANC) ≥ 0.5 × 109/L. Individuals may not receive G-CSF within 2 days prior to the first dose of onitumumab in order to meet ANC eligibility criteria 6. Adequate liver function: a. Total bilirubin ≤1.5 × upper limit of normal (ULN) (≤3 × ULN if due to hepatic lymphoma infiltration) b. Alanine transaminase (ALT) and aspartate transaminase (AST) ≤3 × ULN (≤5 × ULN if due to hepatic lymphoma infiltration) c. Alkaline phosphatase (ALP) ≤2.5 × ULN (≤5 × ULN if due to hepatic lymphoma infiltration) Note: * AST >3 × ULN and/or ALT >3 × ULN is considered adequate for ANC eligibility regardless of the presence of hepatic lymphoma infiltration Subjects with ULN and total bilirubin >1.5 x ULN will be excluded *Subjects with known Gilbert syndrome will be excluded if total bilirubin value is >4 x ULN of the local general population7. Creatinine clearance calculated by Cockcroft-Gault formula ≥50 mL/min: Note: Subjects with calculated creatinine clearance <50 mL/min may be considered for inclusion if the measured creatinine clearance (based on 24-hour urine collection or other reliable method) is ≥50 mL/min8. Subjects should be willing to undergo mandatory tumor biopsy during the dose expansion phase of the study if the investigator believes that the subject has accessible lesions that can be biopsied without significant risk to the subject. If such lesions are not present at screening, archival tissue samples up to 6 months old (and not undergoing interventional treatment) may be considered acceptable for the individual's research eligibility (after approval by the Medical Monitor). 9. Able to understand the purpose and risks of the research and provide signed and dated informed consent and authorize the use of protected health information (in accordance with national and local privacy laws). 10. Willing and able to comply with clinical interviews and research-related procedures. 11. Provide informed consent signed by the research subject or a legally acceptable representative. Exclusion Criteria

Subjects meeting any of the following criteria will be excluded from the study: 1. Prior treatment: • Prior allogeneic stem cell transplantation or solid organ transplantation • Subjects previously treated with anti-CD20 × anti-CD3 bispecific antibodies (such as onitumumab) 2. Diagnosed with mantle cell lymphoma (MCL) 3. Primary central nervous system (CNS) lymphoma or known involvement of non-primary CNS lymphoma (even if treated to complete remission). In addition to mandatory head CT or MRI, suspected CNS lymphoma should be evaluated by lumbar puncture as needed. 4. Any systemic anti-lymphoma therapy within 5 half-lives or 14 days before the first dose of study drug (whichever is closer) 5. Standard radiation therapy within 14 days before the first dose of study drug. Note: Palliative radiation therapy for symptomatic lymph nodes/lesions is allowed, provided that one or more lesions or nodes irradiated are not target lesions for tumor assessment 6. Continuing systemic corticosteroid treatment with more than 10 mg of prazolone or corticosteroid equivalents per day within 72 hours of starting onitumab 7. Complications: a. History of neurodegenerative conditions or CNS movement disorders. Subjects with a history of epileptic seizures within 12 months prior to study enrollment were excluded. b. Another malignant tumor within the past 5 years, except for any localized tumor (e.g., non-melanoma skin cancer or cervical carcinoma in situ) that was effectively treated with clear local control (with or without continued adjuvant hormone therapy). c. Cardiac ejection fraction <40% on cardiac ultrasound (ECHO) or multi-gated acquisition (MUGA) scan. d. Other significant concomitant diseases or medical conditions that the investigator believes may interfere with the conduct of the study or place the subject at significant risk, including (but not limited to) significant cardiovascular disease (e.g., New York Heart Association Class III or IV heart disease, myocardial infarction within the previous 6 months, unstable arrhythmia, or unstable angina), pulmonary disease (e.g., history of obstructive pulmonary disease and symptomatic bronchospasm), gastrointestinal disease, liver disease, renal disease, endocrine disease, hematological disease, autoimmune disease, psychiatric disease, or nervous system disease. 8. Infection: a. Any infection requiring hospitalization or treatment with IV anti-infective agents within 2 weeks after the first dose of study drug b. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B (HBV), or hepatitis C (HCV); or other uncontrolled infection – Individuals with controlled HIV infection are allowed (spontaneously or on a stable antiviral regimen, undetectable viral load and CD4 count above 350 cells/μL) – Individuals with controlled hepatitis B infection (HepBsAg+) are allowed (serum hepatitis B virus DNA polymerase chain reaction [PCR] below the limit of detection and receiving antiviral therapy for hepatitis B) – Individuals with controlled hepatitis C virus antibody positive (HCV Ab+) infection are allowed (spontaneously or in response to a previous successful course of anti-HCV therapy, undetectable HCV RNA by PCR) 9. Cytomegalovirus infection as indicated by detectable levels of CMV on peripheral blood PCR analysis. Individuals who demonstrate detectable levels of CMV at screening will need to be treated with appropriate antiviral therapy and have at least 2 undetectable levels of CMV demonstrated by PCR analysis (at least 7 days apart) before reconsideration for eligibility. 10. Hypersensitivity/allergic reactions: known hypersensitivity to allopurinol and rasburicase 11. History of severe allergic reactions to compounds with similar chemical or biological composition to the study drug or formulation 12. Vaccination with a replication-competent vector within 28 days prior to the first dose of study drug 13. Members of the clinical center research team or their immediate family members, unless approved in advance by the Sponsor. 14. Women of childbearing potential (WOCBP) with a positive serum β-hCG pregnancy test. 15. Pregnant or lactating women. 16. Women* and men of childbearing potential who are unwilling to use highly effective contraceptive measures before the initial dose/first treatment, during the study, and for at least 6 months after the last dose. Sperm donation is prohibited during the study and within 6 months after the last dose of study drug. Highly effective contraceptive measures include: a. Stable use of combined (estrogen and progesterone) hormonal contraception (oral, vaginal, transdermal) or progesterone-only hormonal contraception (oral, injectable, implantable) with ovulation suppression started 2 or more menstrual cycles prior to screening b. Intrauterine device (IUD); intrauterine hormone-releasing system (IUS) c. Bilateral tubal ligation d. Partner vasectomy† (with the restriction that the vasectomized male partner is the study participant’s only sexual partner and the partner has been medically evaluated for the success of the surgical procedure). e. And/or abstinence‡,§. f. Male study participants with WOCBP partners are required to use condoms unless they are vasectomized† or abstinent. ‡, § * Women of childbearing potential are defined as women who are capable of reproduction from menarche until after menopause, unless they are permanently infertile. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. The postmenopausal state is defined as the absence of menstruation for 12 consecutive months without other possible medical reasons. High follicle-stimulating hormone (FSH) levels in the postmenopausal range can be used to confirm the postmenopausal state in women who are not using hormonal contraception or hormone replacement therapy. However, a single FSH measurement is not sufficient to confirm the presence of the postmenopausal state in the absence of 12 months of amenorrhea. The above definitions are based on the Clinical Trial Facilitation Group (CTFG) guidelines. Women with a documented hysterectomy do not need pregnancy testing or contraception. † Vasectomized partners or vasectomized study participants must have received a medical evaluation of the success of the procedure. ‡ Abstinence is considered effective only when it is defined as avoiding heterosexual intercourse for the entire risk period associated with the study drug. The reliability of abstinence needs to be evaluated based on the duration of the clinical trial and the individual's preferences and habitual lifestyle. § Periodic abstinence (calendar method, symptom-temperature method, postovulation method), extracorporeal ejaculation (intercourse interruption), spermicides only, and the lactational amenorrhoea method (LAM) are not acceptable methods of contraception. Female and male condoms should not be used together. 17. Individuals who have been committed to an institution pursuant to an order issued by a judicial or administrative body. Research Treatment

REGN5837 and onitumumab (REGN1979) will be provided as liquids in sterile single-use vials for administration by IV infusion.

A pharmacist or other qualified individual will be assigned at each center to prepare REGN5837 and onitumumab for administration. The dose administered will be a fixed dose and will not be determined by the individual's weight or body surface area.

During the initial 5 weeks of study treatment, subjects will receive onitumumab administered weekly as monotherapy. The stepwise escalation consists of an initial dose of 0.7 mg, followed by a first intermediate dose of 4 mg and a second intermediate dose of 20 mg. The initial dose and two intermediate doses are always divided into 2 separate infusions, with the initial dose divided into 0.2 mg/0.5 mg, the first intermediate dose divided into 2 mg per infusion, and the second intermediate dose divided into 10 mg per infusion. Each divided dose is administered over 4 hours each day for 2 days, preferably consecutively but not more than 3 days apart (e.g., the initial dose is administered on Day 1 of Cycle 1 and Day 2 of Cycle 1, the first intermediate dose is administered on Day 8 of Cycle 1 and Day 9 of Cycle 1, and the second intermediate dose is administered on Day 15 of Cycle 1 and Day 16 of Cycle 1); these step-up doses should be separated even if treatment delays result in dosing beyond Day 15 of Cycle 1. On Day 1 of Cycle 2, a full dose of 160 mg (Note: 80 mg QW in DL1) will be administered. On Day 8 of Cycle 2, another full dose of onitumumab will be administered. The first QW dose will be administered as a single infusion on Day 1 of Cycle 2 and continue thereafter.

REGN5837 will be initiated on Cycle 2 Day 15 to allow individuals to tolerate onitumumab monotherapy prior to starting the combination therapy. REGN5837 will also be initially administered as a step-up dosing regimen. All doses of REGN5837 will be administered as a single infusion. On Cycle 2 Day 15 of study treatment, REGN5837 will be introduced at a lower initial dose in combination with 160 mg (Note: 80 mg in DL1) onitumab. On Cycle 3 Day 1, REGN5837 will be given at an intermediate dose. On Cycle 3 Day 8, REGN5837 will be stepped up to the full QW dose.

Safety data from DL1 and DL2 will be evaluated prior to escalation to DL3. Escalation to DL3 will occur only if the safety profiles of DL1 and DL2 are comparable. However, if the safety profile of DL2 is deemed inferior, escalation of REGN5837 from DL3 will be performed in combination with onitumumab 80 mg QW.

During the REGN5837/onitumumab induction phase, subjects will receive 12 doses of the combination of REGN5837 and onitumumab. Upon completion of the REGN5837/onitumumab induction phase, subjects will have received a total of 17 doses of onitumumab.

After completing the REGN5837/onitumumab induction phase, subjects will enter a Q2W maintenance phase where they will receive their assigned QW REGN5837 dose and 320 mg of onitumumab (160 mg for DL1).

The primary endpoints are: •       The incidence of DLT from the first dose of the combination of REGN5837 and onitumumab to the end of the DLT observation period •       The incidence and severity of treatment-emergent adverse events (TEAEs) and adverse events of special interest (AESIs) during the treatment period of the combination of REGN5837 and onitumumab

Secondary endpoints were: •      Serum concentrations of REGN5837 and onitumumab •      Immunogenicity measured by anti-drug antibodies (ADA) of REGN5837 and onitumumab •      Overall response rate (ORR), which is the proportion of patients who achieved a best overall response (CR) or PR according to the Lugano Classification for malignant lymphoma response as assessed by the investigator during or after study treatment •      Complete response (CR) rate, which is the proportion of patients who achieved a best overall response (CR) according to the Lugano Classification as assessed by the investigator during or after study treatment •     Progression-free survival (PFS) by Lugano classification as assessed by the investigator, determined as the time from the start of study treatment until the first day of disease progression or death from any cause, whichever occurs first. •      Overall survival (OS), measured from the start of study treatment until death from any cause •      Duration of response (DOR) by Lugano classification as assessed by the investigator, determined as the time from the date of the first documented CR or PR until the first day of disease progression or death from any cause, whichever occurs first

Exploratory endpoints are: • Changes in absolute numbers of peripheral T cells, including activation and proliferation phenotypes of T cell subsets measured by multiparameter flow cytometry, and changes in serum interleukin levels • Comparison of responses at baseline (according to the Lugano classification) and changes in lymph node T cell density and expression of immune markers such as CD28, 41BB, programmed death receptor-1 (PD-1), Lag3, GzmB, IFN-γ, Ki67, and B cell markers (CD20, CD22) during treatment/relapse, as measured by multiplex IHC and/or RNA Scope • Proportion of molecular MRD-negative individuals achieving clinical complete response by next-generation sequencing and correlation with PFS and OS • Procedures and assessments to assess the relationship between clinical drug concentrations and pharmacodynamic, safety and efficacy measures

Data will be summarized by dose level, disease subtype, and prior treatment group. Demographics and baseline characteristics will be summarized descriptively. Safety observations and outcome measures will be summarized, including drug exposure, adverse events, laboratory data, vital signs, and European Oncology Cooperative Group performance status.

The following procedures and assessments will be used to evaluate treatment • Screening/Baseline Only Procedures • Safety Procedures • Immunological Safety Assessments • Laboratory Testing • Efficacy Procedures • Drug Concentrations and Measurements • Immunogenicity Measurements and Samples • Pharmacodynamic and Exploratory Biomarkers • Future Biomedical Studies (optional) • Pharmacogenomic Analysis (optional) Statistical Plan and Analysis

This section provides the basis for the Statistical Analysis Plan (SAP) for the study. The SAP will be revised before the end of the study to accommodate changes to the clinical study protocol and to accommodate unexpected problems during the conduct of the study and data that may affect the planned analysis.

The plan is to recruit up to 91 individuals in total.

Dose escalation portion: Assuming an average of 6 subjects per dose level across 9 dose levels, up to 54 subjects will be enrolled in the dose escalation portion. The actual sample size of these dose escalation groups will depend on the number of subjects with DLTs observed and the number of dose levels implemented.

Dose Expansion Part: There will be 2 dose expansion cohorts of individuals with aggressive B-NHL: 1 cohort for individuals who have not received prior CAR T therapy, and 1 cohort for individuals whose disease has progressed after failure of prior CAR-T therapy. In each cohort, 20 individuals are required, including 3 individuals treated at RP2D in the dose escalation part. Therefore, up to a total of 37 individuals will be enrolled in the dose expansion part.

The sample size of 20 subjects in each group was determined to further explore the safety of the combination of REGN5837 and onitumumab in subjects treated at RP2D and to better understand its preliminary anti-tumor activity.

Preliminary Assessment of Antitumor Activity: Twenty subjects treated at RP2D in the dose escalation and expansion portions will provide a preliminary assessment of antitumor activity.

Efficacy Analysis Set: The Full Analysis Set (FAS) includes all subjects who received any study drug. Efficacy endpoints will be analyzed using the FAS.

Safety Analysis Set: The Safety Analysis Set (SAF) includes all subjects who received any study drug and will be based on the treatment received. Treatment compliance/dosing and all clinical safety variables will be analyzed using the SAF.

Pharmacokinetic Analysis Set: The PK analysis set included all subjects who received at least one dose of study drug and had at least one non-missing drug concentration result after the first dose of study drug.

Immunogenicity Analysis Set: The anti-drug antibody analysis set (AAS) was defined individually for each study drug and included all treated subjects who received any amount of study drug (safety analysis set) and had at least one non-absent ADA result after the first dose of the respective study drug.

Dose-Limiting Toxicity Analysis Set: The dose-limiting toxicity (DLT) analysis set includes all subjects enrolled in dose escalation, treated with REGN5837 and onitumumab, and evaluable for DLT (defined as subjects who completed the DLT observation period), as well as subjects who discontinued prematurely due to the occurrence of DLT during the DLT observation period. This analysis set will be used to assess DLTs for determining the dose escalation efficacy analysis

Primary Efficacy Analysis: All efficacy endpoints in this study were secondary endpoints.

Secondary Efficacy Analyses: ORR and CR rates according to Lugano classification based on local investigator review will be summarized with two-sided 95% confidence intervals. Individuals for whom best overall response is not evaluable will be considered non-responders. Other secondary efficacy endpoints including DOR, PFS, and OS will be summarized by median and its 95% confidence interval using the Kaplan-Meier method, if applicable. Example 3 : Results

It is expected that administration of REGN5837 in combination with REGN1979 will result in enhanced tumor regression and improved disease control. In addition, it is expected that administration of REGN5837 in combination with REGN1979 will be safe, with no increased incidence of adverse events and/or toxicity compared to monotherapy.

The trial is currently recruiting. Preliminary results indicate that REGN5837 and REGN1979 are safe to administer.

The scope of the present disclosure is not limited to the specific embodiments described herein. In fact, various modifications of the present disclosure other than those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended patent applications.

Figure 1 shows a schematic diagram of the combination therapy study, which includes a 28-day screening period and an onitumumab monotherapy run-in period, followed by combination with REGN5837. Each cycle in the induction period is 21 days, and each cycle in the maintenance period is 28 days. QW: every week. Q2W: every 2 weeks. Q4W: every 4 weeks.

FIG. 2 shows a schematic representation of an example of a combination dosing regimen for dose level 1 (DL1) for an individual.

FIG3 shows a schematic representation of the pre-drug dosing schedule from cycle 1 to cycle 3, from initial onitumumab monotherapy to combination therapy of REGN5837 and onitumumab.

TW202500582A_113108925_SEQL.xml

Claims (72)

A method for treating a B cell proliferative disorder or a cell malignancy expressing CD20 in an individual, the method comprising administering to the individual a therapeutically effective amount of a combination of a bispecific CD22×CD28 antibody or an antigen-binding fragment thereof and a bispecific CD3×CD20 antibody or an antigen-binding fragment thereof, wherein the bispecific CD22×CD28 antibody or an antigen-binding fragment thereof comprises a first antigen-binding domain that binds to cluster of differentiation factor 28 (CD28) and a second antigen-binding domain that binds to cluster of differentiation factor 22 (CD22), and the bispecific CD3×CD20 antibody or an antigen-binding fragment thereof comprises a first antigen-binding domain that binds to cluster of differentiation factor 3 (CD3) and a second antigen-binding domain that binds to cluster of differentiation factor 20 (CD20), The B cell proliferative disorder or CD20-expressing cell malignancy in the individual is thereby treated. The method of claim 1, wherein the B cell proliferative disorder is B cell lymphoma. The method of claim 2, wherein the lymphoma is B-cell non-Hodgkin lymphoma (B-NHL). The method of claim 3, wherein the non-Hodgkin lymphoma is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), high-grade B-cell lymphoma, Burkitt's lymphoma, primary longitudinal large B-cell lymphoma, and follicular lymphoma. The method of any one of claims 1 to 4, further comprising selecting an individual, wherein the individual has aggressive B-NHL. The method of any of claims 1 to 5, wherein the individual meets at least one of the following criteria, or is selected based on at least one of the following criteria: a.   Has CD20+ aggressive B-NHL; b.   Has disease progression after at least 2 lines of systemic therapy containing a CD20 inhibitor and an alkylating agent; c.   Has measurable disease on cross-sectional imaging; d.   Has adequate bone marrow and liver function; and/or e.  Patients with any of the following cancer types: DLBCL, primary lateral (thymic) large B-cell lymphoma, T-cell/tissue-rich large B-cell lymphoma, grade 3b follicular lymphoma, and high-grade B-cell lymphoma (HGBL) with or without MYC, BCL2, or BCL6 translocations. The method of any one of claims 1 to 6, wherein the individual has been treated with a prior therapy and has relapsed, or the disorder has progressed during or after the prior therapy. The method of any one of claims 1 to 7, wherein the individual has received CAR-T therapy. The method of any one of claims 1 to 8, wherein the individual has measurable CD20+ aggressive B-NHL that has progressed after ≥2 lines of systemic therapy containing at least a CD20 inhibitor and an alkylating agent. The method of claim 9, wherein the CD20 inhibitor is an anti-CD20 antibody. The method of claim 9 or 10, wherein the individual has been treated with CAR-T cell therapy. The method of any one of claims 9 to 11, wherein the individual has not been previously treated with a CD3 antibody, a CD3 bispecific antibody, or a CD3×CD20 bispecific antibody. The method of any one of claims 1 to 12, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg to about 400 mg. The method of any one of claims 1 to 13, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered in an amount of about 0.01 mg, 0.03 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg or 400 mg. The method of any one of claims 1 to 14, wherein the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in an amount of about 0.1 mg to about 400 mg. The method of any one of claims 1 to 15, wherein the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in an amount of about 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg. The method of any one of claims 1 to 16, wherein the method comprises administering one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof in combination with one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof. The method of claim 17, wherein each of the one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is about 0.01 mg to about 400 mg. The method of claim 18, wherein each of the one or more doses is about 0.01 mg, 0.03 mg, 0.05 mg, 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg. The method of any one of claims 17 to 19, wherein each of the one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is about 0.1 mg to about 400 mg. The method of any of claims 17 to 20, wherein each of the one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is about 0.1 mg, 0.2 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 200 mg, 240 mg, 280 mg, 300 mg, 320 mg, 350 mg, or 400 mg. The method of any one of claims 17 to 21, wherein the one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered 1 day to 8 weeks after the previous dose. A method as in any one of claims 17 to 22, wherein each of the one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks or once every six weeks. The method of any one of claims 17 to 23, wherein one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered once a week. The method of any one of claims 17 to 24, wherein one or more doses of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered once every two weeks. The method of any one of claims 17 to 25, wherein one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered once a week. The method of any one of claims 17 to 26, wherein one or more doses of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered once every two weeks. The method of any one of claims 17 to 27, wherein a dose of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered in a single administration or is divided and administered on two days no more than 3 days apart. The method of any one of claims 1 to 28, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered intravenously. The method of any one of claims 1 to 29, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered subcutaneously. The method of any one of claims 1 to 30, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered on the same day. The method of any one of claims 1 to 31, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and/or the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered on different days. The method of claim 32, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered before or after the bispecific CD3×CD20 antibody or antigen-binding fragment thereof. The method of claim 33, wherein the bispecific CD22×CD28 antibody or antigen-binding fragment thereof is administered one day before the bispecific CD3×CD20 antibody or antigen-binding fragment thereof. A method as claimed in any one of claims 1 to 34, comprising the following steps: (i) administering to the individual subcutaneously or intravenously a dose of 0.1 mg to 160 mg of the bispecific CD3×CD20 antibody or its antigen-binding fragment weekly for a single treatment period, wherein the single treatment period is at least 2 weeks; and (ii) administering to the individual subcutaneously or intravenously a dose of 0.01 mg to 400 mg of the bispecific CD22×CD28 antibody or its antigen-binding fragment weekly and administering to the individual intravenously or subcutaneously a dose of 80 mg to 160 mg per week. mg of the bispecific CD3×CD20 or its antigen-binding fragment to continuously induce the combination therapy period. The method of claim 35, wherein the single treatment period is at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks. The method of claim 36 or 37, wherein during the single treatment period the dose of the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is split and administered on two different days no more than 3 days apart, or is administered in a single administration. The method of any one of claims 35 to 37, wherein the single treatment period in step (i) comprises administering an initial dose of the bispecific CD3×CD20 antibody and increasing the dose to a full dose by the end of the single treatment period. The method of claim 38, wherein the complete dose of the bispecific CD3×CD20 in step (i) is 80 or 160 mg. The method of any one of claims 35 to 39, wherein the induction combination therapy period in step (ii) comprises: (a) administering an initial dose of the bispecific CD22×CD28 antibody, wherein the initial dose comprises 0.03 mg to 2 mg; (b) administering an intermediate dose of the bispecific CD22×CD28 antibody, wherein the intermediate dose comprises 0.1 mg to 20 mg; and (c) administering a full dose of the bispecific CD22×CD28, wherein the full dose comprises 0.3 mg to 160 mg. The method of any one of claims 35 to 40, wherein during step (ii), the bispecific CD3×CD20 antibody or antigen-binding fragment thereof, such as the bispecific CD22×CD28 antibody or antigen-binding fragment thereof, is administered on different days. The method of claim 41, wherein the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is administered one day after the bispecific CD22×CD28 antibody or antigen-binding fragment thereof. The method of any one of claims 35 to 40, wherein during step (ii), the bispecific CD3×CD20 antibody or antigen-binding fragment thereof and the bispecific CD22×CD28 antibody or antigen-binding fragment thereof are administered on the same day. The method of any one of claims 35 to 43, wherein in step (ii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered in combination for at least 9 weeks. The method of claim 44, wherein in step (ii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered in combination for at least 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks or 15 weeks. The method of any one of claims 35 to 44, further comprising: (iii) after step (ii), administering the combination of the bispecific CD22×CD28 antibody or its antigen-binding fragment and 160 mg or 320 mg of the bispecific CD3×CD20 antibody or its antigen-binding fragment every two weeks or more for the duration of the combination therapy. The method of claim 46, wherein in step (iii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 antibody or antigen-binding fragment thereof are administered on the same day. The method of claim 46 or 47, wherein in step (iii), the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 or antigen-binding fragment thereof are administered every two weeks or every four weeks. The method of any one of claims 1 to 48, further comprising administering to the individual one or more additional agents to treat or prevent one or more symptoms of the adverse event. The method of any one of claims 1 to 49, wherein the bispecific antibodies are administered to the subject in combination with a second agent, wherein the second agent is selected from the group consisting of: dexamethasone, diphenhydramine, acetaminophen, steroids, antihistamines, nonsteroidal anti-inflammatory drugs (NSAIDs), IL-6 antagonists, and IL-6R antagonists. The method of any one of claims 1 to 50, wherein the individual has stable disease, a partial response, or a complete response at least one week after administration of a dose of about 0.01 mg to about 400 mg of the combination of the bispecific CD22×CD28 antibody or antigen-binding fragment thereof and the bispecific CD3×CD20 antibody or antigen-binding fragment thereof. A method as claimed in any one of claims 1 to 51, wherein the bispecific CD3×CD20 antibody or its antigen-binding fragment comprises: i) a CD3 binding arm comprising: a heavy chain complementary determining region (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 5; and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 6; and ii) a CD20 binding arm comprising: a heavy chain complementary determining region (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 4; and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: The three light chain complementation determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable region (LCVR) of the amino acid sequence of 6. A method as claimed in any one of claims 1 to 52, wherein the bispecific CD3×CD20 antibody or antigen-binding fragment thereof comprises i) a CD3 binding arm comprising three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 10; HCDR2 comprises the amino acid sequence of SEQ ID NO: 11; HCDR3 comprises the amino acid sequence of SEQ ID NO: 12; LCDR1 comprises the amino acid sequence of SEQ ID NO: 13; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15; and ii) a CD20 binding arm comprising three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8; HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; LCDR1 comprises the amino acid sequence of SEQ ID NO: 13; LCDR2 comprises the amino acid sequence of SEQ ID NO: 14; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 15. The method of claim 53, wherein the HCVR of the CD3 binding arm comprises the amino acid sequence of SEQ ID NO: 5, the HCVR of the CD20 binding arm comprises the amino acid sequence of SEQ ID NO: 4, and the common LCVR comprises the amino acid sequence of SEQ ID NO: 6. A method as described in any one of claims 52 to 54, wherein the bispecific CD3×CD20 antibody or its antigen-binding fragment comprises: a heavy chain of a CD3 binding arm comprising the amino acid sequence of SEQ ID NO: 2; a heavy chain of a CD20 binding arm comprising the amino acid sequence of SEQ ID NO: 1; and a common light chain comprising the amino acid sequence of SEQ ID NO: 3. The method of any one of claims 1 to 55, wherein the bispecific CD3×CD20 antibody or antigen-binding fragment thereof is odronextamab. A method as described in any one of claims 1 to 56, wherein the first antigen binding domain that binds to CD28 comprises: three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) contained in a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 20; and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 21. The method of claim 57, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 25, HCDR2 comprises the amino acid sequence of SEQ ID NO: 26, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 27. The method of claim 58, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 30. A method as described in any one of claims 57 to 59, wherein the first antigen binding domain that binds CD28 comprises: a HCVR comprising the amino acid sequence of SEQ ID NO: 20; and a LCVR comprising the amino acid sequence of SEQ ID NO: 21. A method as described in any one of claims 1 to 60, wherein the second antigen binding domain that binds to CD22 comprises: three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) contained in a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 19; and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 21. The method of claim 61, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 22, HCDR2 comprises the amino acid sequence of SEQ ID NO: 23, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 24. The method of claim 62, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 28, LCDR2 comprises the amino acid sequence of SEQ ID NO: 29, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 30. A method as in any one of claims 61 to 63, wherein the second antigen binding domain that binds CD22 comprises: a HCVR comprising the amino acid sequence of SEQ ID NO: 19; and a LCVR comprising the amino acid sequence of SEQ ID NO: 21. A method as claimed in any one of claims 1 to 64, wherein: (a) the first antigen-binding domain that binds to human CD28 comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21; and (b) the second antigen-binding domain that binds to CD22 comprises: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 19; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21. The method of any one of claims 57 to 65, wherein the bispecific CD22×CD28 antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 17. The method of any one of claims 57 to 66, wherein the bispecific CD22×CD28 antibody comprises a second chain comprising the amino acid sequence of SEQ ID NO: 16. The method of any one of claims 57 to 67, wherein the bispecific CD22×CD28 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 18. A method as in any one of claims 57 to 68, wherein the bispecific CD22×CD28 antibody comprises: a first heavy chain comprising the amino acid sequence of SEQ ID NO: 17; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 16; and a common light chain comprising the amino acid sequence of SEQ ID NO: 18. A method as described in any one of claims 57 to 69, wherein the first antigen-binding domain comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 17; and a light chain comprising the amino acid sequence of SEQ ID NO: 18. A method as in any one of claims 57 to 70, wherein the second antigen-binding domain comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 16; and a light chain comprising the amino acid sequence of SEQ ID NO: 18. The method of any one of claims 1 to 71, wherein the bispecific CD22×CD28 antibody is REGN5837 or an antigen-binding fragment thereof.