Dosing strategies for CD3 / CD20 bispecific antibodies to alleviate cytokine release syndrome

Abstract

Disclosed is an administration regimen for reducing cytokine release syndrome and infusion-related reactions for therapeutic proteins (e.g., T cell activating bispecific antibodies). The method employs an initial partial administration, wherein an additional agent, such as a steroid or cytokine antagonist, is optionally administered with a maximum weekly dosing interruption during the course of the administration regimen.

Description

[0001] Reference to a sequence listing

[0002] This application incorporates by reference a sequence listing filed in computer readable form as File 10496WO01-Sequence, which was created on August 30, 2019 and contains 162,944 bytes. Technical Field

[0003] The present invention belongs to the field of medicine and relates to dosing strategies and administration regimens for therapeutic antibodies (e.g., bispecific antibodies targeting T cells) that reduce the prevalence and severity of cytokine release syndrome or infusion-related reactions in patients undergoing immunotherapy. Background Art

[0004] Cytokine release syndrome (CRS) is a systemic inflammatory response that can be triggered by a variety of factors (including certain drugs). T cell activation cancer immunotherapy carries a particularly high risk of CRS, which is usually due to bispecific antibodies or chimeric antigen receptor (CAR) T cells binding to their antigens and then activating bystander immune cells and non-immune cells (such as endothelial cells). The activation of bystander cells leads to a large amount of release of a series of cytokines. IL-6, IL-10 and interferon (IFN)-γ are core cytokines that are found to be continuously elevated in the serum of patients with CRS. In T cell activation therapy for tumor cells, CRS is triggered by a large amount of IFN-γ released by activated T cells or tumor cells themselves. Secreted IFN-γ induces the activation of other immune cells (most importantly macrophages), which in turn produces excessive other cytokines, such as IL-6, TNF-α and IL-10. In particular, IL-6 can cause many major symptoms of CRS, including vascular leakage and activation of complement and coagulation cascades (including disseminated intravascular coagulation). In addition, IL-6 may cause cardiomyopathy by promoting myocardial dysfunction. Shimabukaro-Vornhagen et al., Journal for Immunotherapy of Cancer, 6:56, pp. 1-14, 2018. In some cases, if symptoms associated with CRS occur less than six hours after the start of the infusion, the symptoms are referred to as infusion-related reactions (IRRs), and if they occur more than six hours after the start of the infusion, they are referred to as CRS.

[0005] Management of the toxicity of cancer immunotherapy is a challenging clinical problem. Reducing CRS or IRR is a sign of administering certain treatment modalities (e.g., CAR T cells and bispecific antibodies targeting T cells). Low-grade CRS is usually treated symptomatically with antihistamines, antipyretics, and fluids. Severe CRS may represent a life-threatening adverse event requiring rapid and active treatment. Reducing tumor burden, limiting the dose of the administered therapy, and premedicating with steroids reduces the incidence of severe CRS as well as using anti-cytokine therapy. In some cases, Tocilizumab (Tocilizumab, anti-IL-6 antibody) has become the standard initial treatment for severe CRS. However, the use of dose restrictions and treatment to minimize cytokine activity may have a deleterious effect on the efficacy of immunotherapy. Therefore, alternative strategies are still needed to mitigate the potential life-threatening effects of CRS without negatively affecting the therapeutic benefits of immunotherapy. Summary of the invention

[0006] In one aspect, the present invention provides a method of administering a therapeutic protein to a subject at a dosing regimen for reducing the side effects of cytokine release syndrome or infusion-related reactions, the method comprising: (i) administering a portion of a primary dose (D1) of the therapeutic protein within week 1 of the dosing regimen, wherein the primary dose comprises no more than 10 mg of the therapeutic protein, a first dose portion (F1D1) comprises 40% to 60% of the total primary dose and is administered to the subject on day 1 of week 1, and a second dose portion (F2D1) comprises the remaining 40% to 60% of the total primary dose and is administered to the subject 12 to 96 hours after administration of the F1D1 said subject; (ii) administering a portion of a secondary dose (D2) of the therapeutic protein during week 2 of the dosing regimen, wherein the secondary dose does not exceed half of the maximum weekly dose of the therapeutic protein, the first dose portion (F1D2) comprises 40% to 60% of the total secondary dose, the second dose portion (F2D2) comprises the remaining 40% to 60% of the total secondary dose, and the F2D2 is administered to the subject 12 to 96 hours after the administration of the F1D2 during week 2 of the dosing regimen; and (iii) administering the maximum weekly dose of the therapeutic protein to the subject as a single dose in a subsequent week of the dosing regimen.

[0007] In some cases, the F2D1 is administered to the subject 24 to 96 hours after the administration of the F1D1. In some cases, the F2D1 is administered to the subject 18 to 72 hours after the administration of the F1D1. In some cases, the F2D2 is administered to the subject 24 to 96 hours after the administration of the F1D2. In some cases, the F2D2 is administered to the subject 18 to 72 hours after the administration of the F1D2. In some cases, the subsequent week is the 3rd week of the dosing regimen. In some cases, the subsequent week is the 4th week of the dosing regimen. In some cases, the subsequent week is the 14th week of the dosing regimen. In some cases, the subsequent week is any week from the 4th to the 36th week of the dosing regimen.

[0008] In some embodiments, the method further comprises: (i) administering a portion of a tertiary dose (D3) of the therapeutic protein during week 3 of the dosing regimen, wherein the tertiary dose is not less than half of the maximum weekly dose of the therapeutic protein and does not exceed the maximum weekly dose of the therapeutic protein, the first dose portion (F1D3) comprises 40% to 60% of the total tertiary dose, the second dose portion (F2D3) comprises the remaining 40% to 60% of the total tertiary dose, and the F2D3 is administered to the subject 12 to 96 hours after administration of the F1D3 during week 3 of the dosing regimen; and (ii) administering the maximum weekly dose of the therapeutic protein to the subject as a single dose in a subsequent week of the dosing regimen.

[0009] In some cases, the F2D3 is administered to the subject 24 to 96 hours after administration of the F1D3. In some cases, the F2D3 is administered to the subject 18 to 72 hours after administration of the F1D3.

[0010] In some cases, the subsequent week is week 4 of the dosing regimen. In some cases, the subsequent week is week 14 of the dosing regimen. In some cases, the subsequent week is any week from week 4 to week 36 of the dosing regimen. In various embodiments, the tertiary dose is administered as a single dose within weeks 4 to 12 of the dosing regimen.

[0011] In one aspect, the present invention provides a method of administering a therapeutic protein to a subject at a dosing regimen for reducing the side effects of cytokine release syndrome or infusion-related reactions, the method comprising: (i) administering a portion of a primary dose (D1) of the therapeutic protein within week 1 of the dosing regimen, wherein the primary dose comprises no more than 10 mg of the therapeutic protein, a first dose portion (F1D1) comprises 40% to 60% of the total primary dose and is administered to the subject on day 1 of week 1, and a second dose portion (F2D1) comprises the remaining 40% to 60% of the total primary dose and is administered to the subject 12 to 14 days after administration of the F1D1. (ii) administering a portion of a secondary dose (D2) of the therapeutic protein during week 2 of the dosing regimen, wherein the secondary dose is equal to the maximum weekly dose of the therapeutic protein, the first dose portion (F1D2) comprises 50% of the total secondary dose, the second dose portion (F2D2) comprises 50% of the total secondary dose, and the F2D2 is administered to the subject 12 to 96 hours after administration of the F1D2 during week 2 of the dosing regimen; and (iii) administering the maximum weekly dose of the therapeutic protein to the subject as a single dose in a subsequent week of the dosing regimen.

[0012] In one aspect, the present invention provides a method for administering a therapeutic protein to a subject in a dosing regimen for reducing the side effects of cytokine release syndrome or infusion-related reactions, the method comprising: (i) administering a portion of a primary dose (D1) of the therapeutic protein in week 1 of the dosing regimen, wherein the primary dose comprises no more than 10 mg of the therapeutic protein, wherein D1 is administered to the subject in multiple dose portions (e.g., F1D1, F2D1, F3D1, F4D1, F5D1) on subsequent days in week 1; (ii) administering a portion of a secondary dose (D2) of the therapeutic protein in week 2 of the dosing regimen, wherein the secondary dose is equal to or less than the maximum weekly dose of the therapeutic protein and is administered to the subject in multiple portions (e.g., F1D2, F2D2, F3D2, F4D2, F5D2) on subsequent days in week 2; and (iii) administering the maximum weekly dose (MD) of the therapeutic protein to the subject in multiple portions of MD or in a single dose in a subsequent week of the dosing regimen.

[0013] In some embodiments, the second dose portion (F2) is administered to the subject 12 to 96 (e.g., 24 to 72) hours after administration of the first dose portion (F1); optionally, the third dose portion (F3) is administered to the subject not less than 24 hours after administration of the second dose portion (F2); optionally, the fourth dose portion (F4) is administered to the subject not less than 24 hours after administration of the third dose portion (F3); and optionally, the fifth dose portion (F5) is administered to the subject not less than 24 hours after administration of the fourth dose portion (F4) during week 1, 2, or 3 of the dosing regimen.

[0014] In some embodiments, the method of the present disclosure further comprises administering one or more "maintenance" doses during the maintenance phase of the dosing regimen, after the weekly phase of the regimen is completed. In some cases, each maintenance dose is administered 2 weeks, 3 weeks, or 4 weeks after the previous dose. In one embodiment, the maintenance dose is the maximum weekly dose of the therapeutic protein administered in a single dose.

[0015] In some cases, the maximum weekly dose (MD) of the therapeutic protein is administered to the subject in a single dose during the weekly phase of the dosing regimen for 1 to 8 weeks, 1 to 12 weeks, or 1 to 16 weeks. In some cases, the maximum weekly dose of the therapeutic protein is administered to the subject in a single dose once every two weeks during the maintenance phase of the dosing regimen, and the maintenance phase is after the completion of the weekly phase of the dosing regimen. In some cases, the maximum weekly dose of the therapeutic protein is administered to the subject in a single dose once every three weeks during the maintenance phase of the dosing regimen, and the maintenance phase is after the completion of the weekly phase of the dosing regimen. In some cases, the maximum weekly dose of the therapeutic protein is administered to the subject in a single dose once every four weeks during the maintenance phase of the dosing regimen, and the maintenance phase is after the completion of the weekly phase of the dosing regimen. In some embodiments, the maintenance phase is a time period of up to 86 weeks. In some embodiments, the maintenance phase is a time period of up to 87 weeks. In some embodiments, the maintenance phase is a time period of up to 88 weeks. In some embodiments, the maintenance phase is greater than 86 weeks, greater than 100 weeks, greater than 150 weeks, greater than 200 weeks, or greater than 250 weeks. In some embodiments, the maintenance phase is at least 24 weeks. In some embodiments, the maintenance phase is 24 weeks.

[0016] In various embodiments, the primary dose is 1 mg. In various embodiments, the secondary dose is 20 mg. In various embodiments, the tertiary dose is 40 mg. In various embodiments, the tertiary dose is 80 mg. In various embodiments, the tertiary dose is 160 mg. In various embodiments, the tertiary dose is 320 mg.

[0017] In various embodiments, the F1D1 comprises 50% of the total primary dose, and the F2D1 comprises 50% of the total primary dose. In various embodiments, the F1D2 comprises 50% of the total secondary dose, and the F2D2 comprises 50% of the total secondary dose. In various embodiments, the F1D3 comprises 50% of the total tertiary dose, and the F2D3 comprises 50% of the total tertiary dose.

[0018] In some cases, the maximum weekly dose (MD) of the therapeutic protein is 5 mg to 320 mg. In various embodiments, the maximum weekly dose of the therapeutic protein is 6-320 mg, 10-320 mg, 5-40 mg, 5-80 mg, 5-160 mg, 12-40 mg, 18-80 mg, 40-80 mg, 80-160 mg, 160-320 mg, 5 mg, 6 mg, 7 mg, 8 mg, 12 mg, 18 mg, 27 mg, 40 mg, 80 mg, 160 mg or 320 mg. In some embodiments, the maximum weekly dose is 80 mg. In some embodiments, the maximum weekly dose is 160 mg. In some embodiments, the maximum weekly dose is 320 mg.

[0019] In some cases, the maintenance dose of the therapeutic protein is 5 mg to 320 mg. In various embodiments, the maintenance dose of the therapeutic protein is 6-320 mg, 10-320 mg, 5-40 mg, 5-80 mg, 5-160 mg, 12-40 mg, 18-80 mg, 40-80 mg, 80-160 mg, 160-320 mg, 5 mg, 6 mg, 7 mg, 8 mg, 12 mg, 18 mg, 27 mg, 40 mg, 80 mg, 160 mg or 320 mg. In some embodiments, the maintenance dose is 80 mg. In some embodiments, the maintenance dose is 160 mg. In some embodiments, the maintenance dose is 320 mg.

[0020] In some cases, each dose or dose portion is administered to the subject over a period of 1 to 6 hours.

[0021] In some embodiments, the subject has been diagnosed with cancer. In some cases, the cancer is a B cell malignancy. In some cases, the B cell malignancy is a CD20+B cell malignancy. In some cases, the cancer is non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom macroglobulinemia, primary mediastinal B cell lymphoma, lymphoblastic lymphoma or Burkitt lymphoma. In some cases, the cancer is selected from pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, large oncocytoma, transitional cell carcinoma, urothelial carcinoma, bladder adenocarcinoma or bladder small cell carcinoma. In some embodiments, the subject has been diagnosed with follicular lymphoma (FL). In some cases, the FL is grade 1-3a. In some embodiments, the subject has been diagnosed with diffuse large B-cell lymphoma (DLBCL). In one embodiment, the subject has been diagnosed with relapsed / refractory DLBCL. In some cases, the subject diagnosed with DLBCL has previously failed CAR-T therapy. In some embodiments, the subject has been diagnosed with mantle cell lymphoma (MCL). In some cases, the subject diagnosed with MCL has failed prior Bruton tyrosine kinase (BTK) inhibitor therapy. In some embodiments, the subject has been diagnosed with marginal zone lymphoma (MZL).

[0022] In some cases, the subject is a human, an adult, or a human child (less than eighteen years of age).

[0023] In various embodiments, the therapeutic protein is an antibody or antigen binding fragment thereof. In some cases, the antibody is a fully human antibody. In some cases, the antibody is a bispecific antibody or antigen binding fragment thereof. In some embodiments, the bispecific antibody or antigen binding fragment comprises a first antigen binding arm that binds to a T cell antigen. In some cases, the T cell antigen is CD3. In some cases, the T cell antigen is CD28. In some embodiments, the bispecific antibody or antigen binding fragment comprises a second antigen binding arm that binds to a tumor cell antigen. In some cases, the tumor cell antigen is selected from the group consisting of: AFP, ALK, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD40, CD70, CDK4, CEA, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein (e.g., GAGE-1, 2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-r af, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, 2, 3, 4, 6, and 12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Thompson-nouvelle antigen antigen, Tn), TRP-1, TRP-2, tyrosinase and uroplakin-3.

[0024] In some embodiments, the tumor cell antigen is CD20. In some instances, the bispecific antibody is an anti-CD20 x anti-CD3 antibody. In some instances, the anti-CD20 x anti-CD3 antibody is REGN1979.

[0025] In some embodiments, the tumor cell antigen is BCMA. In some cases, the bispecific antibody is an anti-BCMA x anti-CD3 antibody.

[0026] In some embodiments, the tumor cell antigen is PSMA. In some cases, the bispecific antibody is an anti-PSMA x anti-CD3 antibody.

[0027] In some embodiments, the tumor cell antigen is MUC 16. In some instances, the bispecific antibody is an anti-MUC16 x anti-CD3 antibody.

[0028] In some embodiments, the tumor cell antigen is STEAP2. In some cases, the bispecific antibody is an anti-STEAP2 x anti-CD3 antibody.

[0029] In various embodiments, after administration of the maximum weekly dose for the duration of the dosing regimen, the therapeutic protein is maintained at a serum concentration at or above about 2000 micrograms per liter (mcg / L). In some cases, after administration of the maximum weekly dose for the duration of the dosing regimen, the therapeutic protein is maintained at a serum concentration at or above about 2600 mcg / L. In some embodiments, after administration of the maximum weekly dose for the duration of the dosing regimen, the therapeutic protein is maintained at a serum concentration at or above about 3700 mcg / L.

[0030] In some embodiments, the therapeutic protein is administered to the subject in combination with a second agent selected from the following: a steroid, an antihistamine, acetaminophen, a nonsteroidal anti-inflammatory drug (NSAID), an IL-6 antagonist or an IL-6R antagonist. In some cases, the steroid is dexamethasone. In some cases, the NSAID is indomethacin. In some cases, the IL-6 antagonist is an anti-IL-6 antibody, or the IL-6R antagonist is an anti-IL-6R antibody. In some embodiments, the anti-IL-6R antibody is sarilumab. In various embodiments, after the maximum weekly dose is administered for the first time for the duration of the dosing regimen, the administration of the second agent is eliminated. In other embodiments, the second agent is administered before the administration of the therapeutic protein (e.g., about one to three hours before the F1D1, the F2D1, the F1D2, the F2D2, the F1D3 and / or the F2D3). In other embodiments, the therapeutic protein is administered by infusion over a period of time (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or more hours).

[0031] In various embodiments, the therapeutic protein is administered to the subject in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises at least one of rituximab, obinutuzumab, cyclophophamide, doxorubicin, vincristine, prednisone, prednisolone, bendamustine, lenalidomide, chlorambucil, ibritumomab In some cases, the second therapeutic agent includes a combination of cyclophosphamide, doxorubicin, vincristine and prednisone. In some cases, the second therapeutic agent includes a combination of ifosfamide, cisplatin and etoposide. In some cases, the second therapeutic agent includes a combination of gemcitabine and oxaliplatin. In some cases, the second therapeutic agent includes a combination of lenalidomide and rituximab. In some cases, the second therapeutic agent is lenalidomide.

[0032] In one aspect, the present invention comprises a method for treating a B cell cancer in a subject, the method comprising: (a) selecting a subject diagnosed with a B cell cancer; and (b) administering a therapeutic protein to the subject according to any of the methods discussed above or herein using a dosing regimen for alleviating the side effects of cytokine release syndrome or infusion-related reactions. In some embodiments, the subject has previously been treated with anti-CD20 antibody therapy. In some embodiments, the subject has previously been treated with CAR-T therapy. In some cases, the B cell cancer is selected from the group consisting of: follicular lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, and marginal zone lymphoma.

[0033] In any of the embodiments discussed above or herein, the incidence of grade 3 CRS and IRR is less than 10%. In some cases, the incidence of grade 3 CRS and IRR is less than 7.5% or less than 7%. In some embodiments, when the incidence of CRS and IRR is less than 10%, less than 9%, less than 8%, less than 7.5%, or less than 7%, the maximum weekly dose is 80 mg or greater. In any of the embodiments, any dose administered as a single dose can be administered within no more than 1 hour.

[0034] In various embodiments, any of the features or components of any embodiment discussed above or herein can be combined, and such combinations are included in the scope of the present disclosure. Any specific value discussed above or herein can be combined with another related value discussed above or herein to list a range of values ​​representing the upper and lower ends of a range, and such ranges are included in the scope of the present disclosure. The use of therapeutic proteins used in any of the methods discussed herein or therapeutic proteins in the manufacture of a drug used in any of the methods discussed herein is included in the scope of the present disclosure.

[0035] Other embodiments will become apparent upon reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The incidence of CRS / IRR during the first five weeks of therapy with different dose levels of REGN1979 is shown. DETAILED DESCRIPTION

[0037] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive, because the scope of the present invention is limited only by the appended claims.

[0038] 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 the invention belongs. When used to refer to a specific listed value, the term "about" means that the value may differ from the listed value by no more than 1%. For example, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0039] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, 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.

[0040] The expression "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR), and the antigen is composed of a homodimer or heterodimer formed by the association of two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-ε includes the amino acid sequence shown in SEQ ID NO: 1; human CD3-δ includes the amino acid sequence shown in SEQ ID NO: 2; human CD3-ζ includes the amino acid sequence shown in SEQ ID NO: 3; and human CD3-γ includes the amino acid sequence shown in SEQ ID NO: 4.

[0041] All references to proteins, polypeptides and protein fragments herein are intended to refer to the human form of the corresponding protein, polypeptide or protein fragment, unless it is explicitly stated that it is from a non-human species. Thus, the expression "CD3" means human CD3, unless specified as being from a non-human species, e.g., "mouse CD3", "monkey CD3", etc.

[0042] "Antigen binding domains that bind to CD3", "antibodies that bind to CD3" or "anti-CD3 antibodies" include antibodies and antigen binding fragments thereof that specifically recognize a single CD3 subunit (e.g., ε, δ, γ or ζ), as well as antibodies and antigen binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., γ / γ, δ / ε and ζ / ζ CD3 dimers). The antibodies and antigen binding fragments of the present invention can bind to soluble CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes natural CD3 protein and recombinant CD3 protein variants such as monomeric and dimeric CD3 constructs that lack a transmembrane domain or are otherwise not associated with a cell membrane.

[0043] The expression "CD20" refers to a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. Because CD20 is expressed by more than 95% of B cell non-Hodgkin's lymphomas (NHL) and other B cell malignancies, but is absent on precursor B cells, dendritic cells and plasma cells, it is considered to be a B cell tumor-associated antigen. The human CD20 protein has the amino acid sequence shown in SEQ ID NO: 5.

[0044] The “antigen binding domain that binds to CD20”, “antibody that binds to CD20” or “anti-CD20 antibody” include antibodies that specifically recognize CD20 and antigen-binding fragments thereof.

[0045] The expression "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is shown in SEQ ID NO:6.

[0046] An “antigen-binding domain that binds to BCMA,” “antibody that binds to BCMA,” or “anti-BCMA antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize BCMA.

[0047] The expression "PSMA" refers to prostate specific membrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is a non-shedding integral membrane glycoprotein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. The amino acid sequence of human PSMA is shown in SEQ ID NO:7.

[0048] An “antigen binding domain that binds to PSMA,” “antibody that binds to PSMA,” or “anti-PSMA antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize PSMA.

[0049] The expression "MUC16" refers to mucin 16. MUC16 is a single-pass transmembrane domain, highly glycosylated, integral membrane glycoprotein that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is shown in SEQ ID NO:8.

[0050] An "antigen binding domain that binds to MUC16," "antibody that binds to MUC16," or "anti-MUC16 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize MUC16.

[0051] The expression "STEAP2" refers to six transmembrane epithelial antigen of the prostate 2. STEAP2 is a six transmembrane intrinsic protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. STEAP2 is a 490 amino acid protein encoded by the STE4P2 gene located at human chromosome region 7q21. The amino acid sequence of human STEAP2 is shown in SEQ ID NO: 9.

[0052] The “antigen-binding domain that binds to STEAP2,” “antibody that binds to STEAP2,” or “anti-STEAP2 antibody” includes antibodies that specifically recognize STEAP2 and antigen-binding fragments thereof.

[0053] The term "therapeutic protein" includes any polypeptide used to prevent, treat or ameliorate any condition, disease or disorder in a subject, including antibodies and antigen-binding fragments thereof and bispecific antibodies and antigen-binding fragments thereof.

[0054] The term "antigen binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0055] The term "antibody" means any antigen binding molecule or molecular complex that includes at least one complementary determining region (CDR) that specifically binds or interacts with a specific antigen (e.g., CD20, BCMA, PSMA, MUC16, STEAP2, or CD3). The term "antibody" includes immunoglobulin molecules that include four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region consists of three domains: C H 1. C H 2 and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region consists of a domain (C L 1) You can set V H Area and V L The V region is further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L It 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 different embodiments of the present invention, the FRs of the antibody (or its antigen-binding portion) may be identical to human germline sequences, or may be natural or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Unless otherwise indicated, the term "antibody" includes "bispecific antibodies".

[0056] The term "antibody" also includes the antigen binding fragment of a complete antibody molecule. The term "antigen binding portion thereof", "antigen binding fragment" of an antibody, etc., includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein, which specifically binds to an antigen to form a complex. The antigen binding fragment of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique (such as proteolytic digestion or recombinant genetic engineering technology, which relates to manipulation and expression of DNA encoding antibody variable domains and optional constant domains). Such DNA is known and / or easy to obtain from, for example, commercial sources, DNA libraries (comprising, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by molecular biological techniques, for example, one or more variable domains and / or constant domains are arranged into a suitable configuration, or codons are introduced, cysteine ​​residues are produced, modified, added or deleted amino acids, etc.

[0057] The expression "bispecific antigen binding molecule" refers to 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 additional CDRs and / or FRs. Bispecific antigen binding molecules include bispecific antibodies.

[0058] 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, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variant IgNAR domains are also encompassed within the expression "antigen-binding fragment".

[0059] The antigen-binding fragment of an antibody generally includes at least one variable domain. The variable domain can be of any size or amino acid composition and will generally include at least one CDR adjacent to or within one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H Domain and V LThe domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L Alternatively, the antigen-binding fragment of the antibody may contain a monomer V H or V L Structural domain.

[0060] 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 within the antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L. In any configuration of variable and constant regions (including any of the exemplary configurations listed above), the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge 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 result in 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 of the antibody of the present invention may include homodimers or heterodimers (or other multimers) of any variable domain and constant domain configuration listed above, which are non-covalently associated with each other and / or with one or more monomer V H or V L The domains are covalently associated (eg, through one or more disulfide bonds).

[0061] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Conventional techniques available in the art can be used to apply any multispecific antibody format (including the exemplary bispecific antibody formats disclosed herein) to the context of antigen-binding fragments of the antibodies of the present invention.

[0062] The antibodies of the present invention can act through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells and thereby cause the lysis of target cells. CDC and ADCC can be measured using assays well known and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337 and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95: 652-656). The constant region of an antibody is important for the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of the antibody can be selected based on whether the antibody needs to mediate cytotoxicity. The antibodies of the present disclosure can comprise a human IgG heavy chain. In various embodiments, the heavy chain can be of IgG1, IgG2, IgG3 or IgG4 isotype.

[0063] In certain embodiments of the present invention, antibody or bispecific antibody is human antibody.Term " human antibody " is intended to include the antibody with variable region and constant region derived from human germline immunoglobulin sequence.Human antibody of the present invention can include amino acid residues (for example, by in vitro random or site-specific mutagenesis or by the mutation introduced by in vivo somatic mutation) that are not encoded by human germline immunoglobulin sequence, for example, in CDR, and specifically in CDR3.However, term " human antibody " is not intended to include the antibody that the CDR sequence derived from the germline of another mammalian species such as mouse has been transplanted on human framework sequence.

[0064] In some embodiments, the antibodies of the present invention may be recombinant human antibodies. The term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies separated from recombinant combined human antibody libraries (described further below), antibodies separated from human immunoglobulin gene transgenic animals (e.g., mice) (see, e.g., Taylor et al., (1992), "Nucl. Acids Res.", 20: 6287-6295) or antibodies prepared, expressed, produced or isolated by any other means, the other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals with transgenic human Ig sequences, undergo in vivo somatic mutagenesis), and thus the V H Area and V L The amino acid sequence of the region is as follows: H Sequence and V L Sequences are related to but may not naturally occur in vivo in the human antibody germline repertoire.

[0065] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule includes a stable four-chain construct of about 150-160 kDa, in which the dimer is held together by an interchain heavy chain disulfide bond. In the second form, the dimer is not connected by an interchain disulfide bond and forms a molecule of about 75-80 kDa, which is composed of covalently coupled light and heavy chains (half antibodies). Even after affinity purification, these forms are extremely difficult to separate.

[0066] The frequency of the second form in various intact IgG isotypes is based on, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to the level typically observed with the human IgG1 hinge. The present invention encompasses substitutions in the hinge, C H Zone 2 or C H Region 3: Antibodies with one or more mutations, which may be desirable, for example, in production to increase the yield of a desired antibody form.

[0067] The antibodies of the present invention can be isolated antibodies. "Isolated antibodies" means antibodies that have been identified and separated and / or recovered from at least one component of its natural environment. For example, antibodies that have been separated or removed from at least one component of an organism or from tissues or cells in which the antibody naturally exists or is naturally produced are "isolated antibodies" for the purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. Isolated antibodies are antibodies that have undergone at least one purification or separation step. According to certain embodiments, the isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0068] As compared to the corresponding germline sequence of the derived antibody, the antibodies disclosed herein include 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. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments derived from any amino acid sequence disclosed herein, wherein one or more amino acid mutations in one or more frameworks and / or CDR regions are mutated to one or more corresponding residues of the germline sequence of the derived antibody, or mutated to one or more corresponding residues of another human germline sequence, or mutated to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, a person of ordinary skill in the art can easily produce many antibodies and antigen-binding fragments including one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LIn other embodiments, all frameworks and / or CDR residues in the domain are mutated back to the residues found in the original germline sequence of the derived antibody. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the residue after the mutation found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the residue after the mutation found in CDR1, CDR2 or CDR3. In other embodiments, one or more mutations in one or more frameworks and / or CDR residues are one or more corresponding residues of different germline sequences (that is, germline sequences different from the germline sequence of the initial derived antibody). In addition, the antibody of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR region, for example, wherein some individual residues are mutated into the corresponding residues of a specific germline sequence, and some other residues different from the original germline sequence can maintain or mutate into the corresponding residues of different germline sequences. Once obtained, antibodies and antigen-binding fragments 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 and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0069] The invention also encompasses antibodies comprising variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention encompasses antibodies having HCVR, LCVR and / or CDR amino acid sequences having, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any HCVR, LCVR and / or CDR amino acid sequences set forth herein, an anti-CD3 antibody disclosed in WO 2014 / 047231 or WO 2017 / 053856, a bispecific anti-CD20×anti-CD3 antibody disclosed in WO 2014 / 047231, an anti-PSMA or anti-PSMA×anti-CD3 antibody disclosed in WO 2017 / 023761, an anti-MUC16 or anti-MUC16×anti-CD3 antibody disclosed in WO 2018 / 067331, an anti-STEAP2 or anti-STEAP2×anti-CD3 antibody disclosed in WO 2018 / 058001, or a US The anti-BCMA or anti-BCMA x anti-CD3 antibodies disclosed in 62 / 700,596 (filed on July 19, 2018), each of which is incorporated herein by reference.

[0070] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule called a paratope. A single antigen can have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may contain a portion of a sugar, phosphoryl, or sulfonyl group on the antigen.

[0071] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof means a nucleotide sequence identity of at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an amino acid sequence that is identical or substantially similar to a polypeptide encoded by the reference nucleic acid molecule.

[0072] When applied to these polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is substituted by another amino acid residue having a side chain (R group) that is similar in chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein. In the case 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. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups having chemically similar side chains include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing 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 glutamic acid; and (7) sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.

[0073] Sequence similarity of polypeptides also referred to as sequence identity is usually measured using sequence analysis software. Protein analysis software uses a measure of the similarity assigned to various substitutions, deletions and other modifications (comprising 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 closely related polypeptides, such as sequence homology or sequence identity between homologous polypeptides from different organism species or between wild-type proteins and their mutant proteins. See, for example, 6.1 version GCG. FASTA using default or recommended parameters can also be used to compare polypeptide sequences, which is a program in 6.1 version GCG. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlap region between the query sequence and the search sequence (Pearson (2000), the same). When comparing the sequence of the present invention with a database containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0074] Dosing strategy and administration schedule

[0075] A dosing strategy is created that provides an administration scheme that reduces the prevalence or severity of cytokine release syndrome (CRS) or infusion-related reactions (IRR) or both by administering therapeutic proteins to patients for various therapies. According to certain embodiments of the present invention, these strategies include multiple doses of therapeutic proteins or antigen binding molecules (e.g., antibodies or bispecific antibodies) that can be administered to subjects over a defined time course to create a scheme. The method according to this aspect of the present invention includes sequentially administering multiple doses of the therapeutic protein of the present invention to the subject. "Sequential administration" means that each dose of the therapeutic protein is administered to the subject at different time points, for example, on different dates separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes the following method: sequentially administering to the patient a separate primary dose of a therapeutic protein, then a separate secondary dose of the therapeutic protein, optionally then a separate tertiary dose of the therapeutic protein, and then a single dose of the maximum weekly dose of the therapeutic protein. The administration scheme of the present invention allows for a higher dose of therapeutic protein that is desirable for enhancing therapeutic efficacy but does not produce adverse effects associated with CRS or IRR. Without intending to be bound by any particular theory, the administration regimens of the invention provide for eliciting an immune response to the administered therapeutic protein during the initial phase of the treatment regimen to minimize the prevalence and severity of CRS and IRR, which then allows for administration of higher doses of the therapeutic protein during subsequent phases of the treatment regimen without producing significant adverse events associated with CRS or IRR.

[0076] An exemplary administration regimen comprises: (i) administering a portion of a primary dose of the therapeutic protein within week 1 (W1) of the dosing regimen, wherein the primary dose comprises no more than 1 mg of the therapeutic protein, a first dose portion (F1D1) comprises 50% of the primary dose and is administered to the subject on day 1 of week 1, and a second dose portion (F2D1) comprises 50% of the total primary dose and is administered to the subject within 96 hours after administration of the F1D1; (ii) administering a portion of a secondary dose of the therapeutic protein within week 2 (W2) of the dosing regimen, wherein the secondary dose does not exceed half of the maximum weekly dose of the therapeutic protein, a first dose portion (F1D2) comprises 50% of the secondary dose, a second dose portion (F2D2) comprises 50% of the secondary dose, and the F1D2 and the F2D2 are administered to the subject within 96 hours of each other during week 2 of the dosing regimen; and (iii) administering the maximum weekly dose of the therapeutic protein to the subject as a single dose within a subsequent week (Ws) of the dosing regimen.

[0077] Another exemplary administration regimen comprises: (i) administering a portion of a primary dose of the therapeutic protein during week 1 (W1) of the dosing regimen, wherein the primary dose comprises no more than 1 mg of the therapeutic protein, a first dose portion (F1D1) comprises 50% of the primary dose and is administered to the subject on day 1 of week 1, and a second dose portion (F2D1) comprises 50% of the total primary dose and is administered to the subject within 96 hours after administration of F1D1; (ii) administering a portion of a secondary dose of the therapeutic protein during week 2 (W2) of the dosing regimen, wherein the secondary dose does not exceed half of the maximum weekly dose of the therapeutic protein, a first dose portion (F1D2) comprises 50% of the secondary dose, and a second dose portion (F2D2) comprises 50% of the secondary dose , and the F1D2 and the F2D2 are administered to the subject within 96 hours of each other during week 2 of the dosing regimen; (iii) administering a portion of a tertiary dose of the therapeutic protein in week 3 (W3) of the dosing regimen, wherein the tertiary dose is not less than half of the maximum weekly dose of the therapeutic protein and does not exceed the maximum weekly dose of the therapeutic protein, the first dose portion (F1D3) includes 50% of the tertiary dose, the second dose portion (F2D3) includes 50% of the tertiary dose, and the F1D3 and the F2D3 are administered to the subject within 96 hours of each other during week 3 of the dosing regimen; and (iv) administering the maximum weekly dose of the therapeutic protein to the subject as a single dose in the subsequent week (Ws) of the dosing regimen.

[0078] In various embodiments, the primary dose of the therapeutic protein can range from 0.1 mg to 10 mg or more. In some cases, the primary dose of the therapeutic protein is 0.5 mg to 10 mg, 1-10 mg, 2-5 mg, or 5-10 mg. In some cases, the primary dose of the therapeutic protein is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg. In various embodiments, the primary dose of the therapeutic protein includes a range (e.g., 1-5 mg) including any of the values ​​listed above as the upper or lower end of the range.

[0079] In various embodiments, the maximum weekly dose of the therapeutic protein is 5 mg to 320 mg. In some embodiments, the maximum weekly dose of the therapeutic protein is greater than 5 mg up to 320 mg. In some embodiments, the maximum weekly dose of the therapeutic protein is 6-320 mg, 10-320 mg, 5-40 mg, 5-80 mg, 5-160 mg, 12-40 mg, 18-80 mg, 40-80 mg, 80-160 mg, 160-320 mg, 5 mg, 6 mg, 7 mg, 8 mg, 12 mg, 18 mg, 27 mg, 40 mg, 80 mg, 160 mg or 320 mg. In some cases, the maximum weekly dose of the therapeutic protein is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 11 0, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315 or 320 mg. In various embodiments, the maximum weekly dose of the therapeutic protein includes a range (e.g., 200-300 mg) comprising any of the values ​​listed above as the upper or lower end of the range.

[0080] In various embodiments, the secondary dose comprises 50% of the maximum weekly dose of the therapeutic protein. In some cases, the secondary dose comprises 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% of the maximum weekly dose of the therapeutic protein. In some cases, the secondary dose of the therapeutic protein comprises a range of percentages of the maximum weekly dose that includes any of the values ​​listed above as the upper or lower end of the range (e.g., 35-50%).

[0081] In various embodiments, the tertiary dose comprises 50% of the maximum weekly dose of the therapeutic protein. In some cases, the tertiary dose comprises 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90% of the maximum weekly dose of the therapeutic protein. In some cases, the tertiary dose of the therapeutic protein comprises a range of percentages of the maximum weekly dose (e.g., 50-75%) that includes any of the values ​​listed above as the upper or lower end of the range.

[0082] In various embodiments, the first dose portion and the second dose portion of the primary, secondary and / or tertiary dose each include 50% of the dose. In some cases, the first dose portion and the second dose portion of the primary, secondary and / or tertiary dose include different percentages of the total dose (totaling 100%). For example, the first dose portion may include 45% of the dose and the second dose portion may include 55% of the dose. Alternatively, the first dose portion may include 55% of the dose and the second dose portion may include 45% of the dose. In various embodiments, the first and second dose portions may include 10% / 90%, 15% / 85%, 20% / 80%, 25% / 75%, 30% / 70%, 35% / 65%, 40% / 60%, 46% / 54%, 47% / 53%, 48% / 52%, 49% / 51% of the primary, secondary or tertiary dose, respectively, or vice versa.

[0083] In various embodiments, the primary, secondary and / or tertiary doses (D1, D2 and / or D3) can be divided into two or more parts. Various options for dividing the dose into two parts are discussed above. However, in some cases, the dose is divided into 3, 4 or 5 parts. For example, the primary dose can be divided into 5 parts, each of which includes 20% of the total primary dose, and each dose portion (F1D1, F2D1, F3D1, F4D1 and F5D1) can be administered to the subject for five consecutive days during the first week of the administration regimen. In other cases, the percentage of the total dose (e.g., the primary dose) may vary between each dose portion. For example, if the primary dose is divided into 3 parts, the first dose portion (F1D1) may contain 30% of the total primary dose, the second dose portion (F2D1) may contain 30% of the total primary dose, and the third dose portion (F3D1) may contain the remaining 40% of the total primary dose. Other combinations of percentages and quantities of partial doses equal to 100% of the total dose are clearly contemplated herein.

[0084] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 6 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 3 mg, the tertiary dose comprises 12 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 6 mg, and the maximum weekly dose comprises 12 mg of the therapeutic protein.

[0085] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 9 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 4.5 mg, the tertiary dose comprises 18 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 9 mg, and the maximum weekly dose comprises 18 mg of the therapeutic protein.

[0086] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 13.5 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 6.75 mg, the tertiary dose comprises 27 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 13.5 mg, and the maximum weekly dose comprises 27 mg of the therapeutic protein.

[0087] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 40 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 20 mg, and the maximum weekly dose comprises 40 mg of the therapeutic protein.

[0088] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 60 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 30 mg, and the maximum weekly dose comprises 80 mg of the therapeutic protein.

[0089] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 80 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 40 mg, and the maximum weekly dose comprises 160 mg of the therapeutic protein.

[0090] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 120 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 60 mg, and the maximum weekly dose comprises 240 mg of the therapeutic protein.

[0091] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 160 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 80 mg, and the maximum weekly dose comprises 320 mg of the therapeutic protein.

[0092] In an exemplary embodiment of the dosing regimen, the primary dose (D1) comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D2) of the primary dose comprises 500 mcg, the secondary dose (D2) comprises 3 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) of the secondary dose comprises 1.5 mg, and the maximum weekly dose comprises 3 mg of the therapeutic protein.

[0093] In an exemplary embodiment of the dosing regimen, the primary dose (D1) comprises 3 mg, each of the first dose portion (F1D1) and the second dose portion (F2D2) of the primary dose comprises 1.5 mg, the secondary dose (D2) comprises 9 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) of the secondary dose comprises 4.5 mg, and the maximum weekly dose comprises 9 mg of the therapeutic protein.

[0094] In an exemplary embodiment of the dosing regimen, the primary dose (D1) comprises 5 mg, each of the first dose portion (F1D1) and the second dose portion (F2D2) of the primary dose comprises 2.5 mg, the secondary dose (D2) comprises 15 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) of the secondary dose comprises 7.5 mg, and the maximum weekly dose comprises 15 mg of the therapeutic protein.

[0095] In an exemplary embodiment of the dosing regimen, the primary dose (D1) comprises 10 mg, each of the first dose portion (F1D1) and the second dose portion (F2D2) of the primary dose comprises 5 mg, the secondary dose (D2) comprises 30 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) of the secondary dose comprises 15 mg, and the maximum weekly dose comprises 30 mg of the therapeutic protein.

[0096] In various embodiments, the therapeutic protein is administered at a dose that maintains a serum concentration of at least about 2000 mcg / L after administration of the maximum weekly dose for the duration of the dosing regimen. In some cases, the therapeutic protein is administered at a dose that maintains a serum concentration of at least about 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, or 3500 mcg / L after administration of the maximum weekly dose for the duration of the dosing regimen.

[0097] In various embodiments, the therapeutic protein is administered at a dose that maintains an average serum concentration of at least about 2600 mcg / L after administration of the maximum weekly dose for the duration of the dosing regimen. In some cases, the therapeutic protein is administered at a dose that maintains an average serum concentration of at least about 2000, 2100, 2200, 2300, 2400, 2500, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 mcg / L after administration of the maximum weekly dose for the duration of the dosing regimen.

[0098] In various embodiments, the subsequent week (Ws) of the dosing regimen is week 3 (W3), week 4 (W4), week 5 (W5), week 6 (W6), week 7 (W7), week 8 (W8), week 9 (W9), week 10 (W10), week 11 (W11), week 12 (W12), week 13 (W13), week 14 (W14), week 15 (W15), week 16 (W16), week 17 (W17), week 18 (W19). 8), Week 19 (W19), Week 20 (W20), Week 21 (W21), Week 22 (W22), Week 23 (W23), Week 24 (W24), Week 25 (W25), Week 26 (W26), Week 27 (W27), Week 28 (W29), Week 30 (W30), Week 31 (31), Week 32 (32), Week 33 (33), Week 34 (W34), Week 35 (W35) or Week 36 (W36).

[0099] In various embodiments, the second portion of the dose in any given week of the dosing regimen is administered within 24, 36, 48, 60, 72, 84, or 96 hours after administration of the first portion of the dose.

[0100] In various embodiments, the maximum weekly dose of the therapeutic protein is administered to the subject in a single dose during the weekly phase of the dosing regimen for 1 to 8 weeks or for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks or more weeks. In some cases, the maximum weekly dose of the therapeutic protein is administered to the subject in a single dose within a range of weeks (e.g., 1-12 weeks) that includes any of the values ​​listed above as the upper or lower end of the range.

[0101] In various embodiments, the maximum weekly dose of the therapeutic protein is administered as a single dose (maintenance dose) once every two weeks for up to 24 weeks or for up to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 8, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or more weeks of administration to the subject, and the maintenance phase may be after the weekly phase of the dosing regimen (i.e., weekly administration of the maximum weekly dose or separate administration of the primary, secondary, and (optionally) tertiary doses) is completed. In some cases, the maximum weekly dose of the therapeutic protein is administered to the subject as a single dose (maintenance dose) once every two weeks, once every three weeks, or once every four weeks within a range of weeks (e.g., 24-86 weeks) that includes any of the values ​​listed above as the upper or lower end of the range.

[0102] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 80 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 40 mg, and the maximum weekly dose comprises 160 mg of the therapeutic protein, wherein the tertiary dose is administered weekly (QW) as a single dose (i.e., 80 mg) during weeks 4 to 12 of the dosing regimen, and the maximum weekly dose is administered once every two weeks (Q2W) as a single dose (i.e., 160 mg) from week 14 onwards of the dosing regimen.

[0103] In some instances, the dosing regimens identified above are for use in a method of treating an aggressive lymphoma (eg, mantle cell lymphoma or marginal zone lymphoma).

[0104] In an exemplary embodiment of the dosing regimen, the primary dose comprises 1 mg, each of the first dose portion (F1D1) and the second dose portion (F2D1) comprises 500 mcg, the secondary dose comprises 20 mg, each of the first dose portion (F1D2) and the second dose portion (F2D2) comprises 10 mg, the tertiary dose comprises 160 mg, each of the first dose portion (F1D3) and the second dose portion (F2D3) comprises 80 mg, and the maximum weekly dose comprises 320 mg of the therapeutic protein, wherein the tertiary dose is administered weekly (QW) as a single dose (i.e., 160 mg) during weeks 4 to 12 of the dosing regimen, and the maximum weekly dose is administered once every two weeks (Q2W) as a single dose (i.e., 320 mg) from week 14 onwards of the dosing regimen.

[0105] In some cases, the dosage regimen identified above is used in the method for treating aggressive lymphoma (e.g., mantle cell lymphoma or marginal zone lymphoma). In some cases, the dosage regimen identified above is used in the method for treating follicular lymphoma (e.g., 1-3a grade). In some cases, the dosage regimen identified above is used for treating diffuse large B cell lymphoma (including recurrent or refractory DLBCL in patients with failure of previous CAR-T therapy, for example).

[0106] In various embodiments, each dose or partial dose of the therapeutic protein is administered to the subject (e.g., by infusion) within a time period of 1-4 hours, 1-5 hours, or 1-6 hours. In some cases, the dose or partial dose is administered within a time period of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours or more. In some cases, each dose or partial dose of the therapeutic protein is administered to the subject within a time range (e.g., 1-8 hours) that includes any value listed above as the upper or lower end of the range. In various embodiments, each maximum weekly dose is administered with a single infusion.

[0107] In some embodiments of the administration scheme discussed herein, the second agent is used as a pretreatment or in combination with a therapeutic protein. In some cases, before the primary partial dose and the secondary partial dose are administered, and optionally before the tertiary partial dose, a steroid (such as dexamethasone) is administered to the patient as a pretreatment. In certain embodiments, the dexamethasone is administered to the patient about one to three hours before the first dose portion (F1D1). In certain embodiments, the dexamethasone dose is administered by intravenous infusion. In some cases, during the administration of primary, secondary and optional tertiary doses, a cytokine antagonist (such as an anti-IL-6 antibody or an anti-IL-6R antibody) is administered in combination with a therapeutic protein. In some cases, before the primary partial dose and optionally the secondary partial dose, and optionally before the tertiary partial dose, an anti-CD20 monospecific antibody (e.g., rituximab) is administered to the patient as a pretreatment. In embodiments, pretreatment or combined administration of the second agent is interrupted with a single dose administration of the maximum weekly dose beginning, for example, at week 3, 4, 5, or 6 of the dosing regimen so as not to artificially suppress cytokine activity and thereby hinder the therapeutic effect of the therapeutic protein.

[0108] The terms "primary dose", "secondary dose" and "tertiary dose" refer to the time sequence of the antigen binding molecules of the present invention. Therefore, "primary dose" is the dose applied at the beginning of the treatment regimen; "secondary dose" is the dose applied after the primary dose (all dose portions comprising the primary dose); and "tertiary dose" is the dose applied after the secondary dose (all dose portions comprising the secondary dose). The primary, secondary and tertiary doses (or their dose portions) may contain a certain amount of therapeutic proteins discussed herein. In some cases, "primary dose" and "secondary dose" and optional "tertiary dose" may be referred to as "loading doses", and the subsequent maximum weekly dose may be referred to as "maintenance doses". In some cases, "primary dose" may be referred to as "initial dose". In some cases, "secondary dose" may be referred to as "intermediate dose". In some cases, "tertiary dose" may be referred to as "ascending dose".

[0109] The phrase "previous dose" refers to the dose of antigen binding molecule administered to a patient in a sequence of multiple administrations, prior to the next dose in the sequence, without an intervening dose.

[0110] Antigen Binding Molecules

[0111] Antibodies used in connection with the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a target polypeptide, or may contain antigen binding domains that are specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The antibodies and bispecific antibodies of the present invention may be linked or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities (e.g., another antibody or antibody fragment) to produce a bispecific or multispecific antibody fragment having a second or additional binding specificity.

[0112] The present invention comprises antibodies having the HCVR, LCVR and / or CDR amino acid sequences of the antibodies described herein, an anti-CD3 antibody disclosed in WO2014 / 047231 or WO 2017 / 053856, a bispecific anti-CD20×anti-CD3 antibody disclosed in WO 2014 / 047231, an anti-PSMA or anti-PSMA×anti-CD3 antibody disclosed in WO 2017 / 023761, an anti-MUC16 or anti-MUC16×anti-CD3 antibody disclosed in WO2018 / 067331, an anti-STEAP2 or anti-STEAP2×anti-CD3 antibody disclosed in WO 2018 / 058001, or an anti-BCMA or anti-BCMA×anti-CD3 antibody disclosed in US 62 / 700,596 (filed on July 19, 2018), each of which is incorporated herein by reference.

[0113] The expressions "anti-CD3 antibody", "anti-CD20 antibody", "anti-PSMA antibody", "anti-MUC16 antibody", "anti-STEAP2 antibody", "anti-BCMA antibody", etc., used herein are intended to include both monospecific antibodies as well as bispecific antibodies comprising the corresponding antigen binding arms (e.g., CD3). Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds human CD3 and the other arm of the immunoglobulin is specific for, for example, human CD20, human PSMA, human MUC16, human STEAP2, or human BCMA.

[0114] In certain embodiments, the CD3 binding arm is bound to human CD3 and induces human T cell activation. In certain embodiments, the CD3 binding arm is weakly bound to human CD3 and induces human T cell activation. In other embodiments, the CD3 binding arm is weakly bound to human CD3 and induces tumor-associated antigen-expressing cell killing in the context of bispecific or multispecific antibodies. In other embodiments, the CD3 binding arm is bound to or weakly associated with human and cynomolgus macaque (monkey) CD3, but the binding interaction cannot be detected by in vitro assays known in the art. In certain embodiments, the bispecific antibody or antigen-binding fragment used for the present invention includes an antigen binding arm that is bound to induce T cell activation with CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1 or TIM2.

[0115] In certain exemplary embodiments of the present invention, bispecific antigen binding molecules are bispecific antibodies.Each antigen binding domain of a bispecific antibody includes 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) including a first antigen binding domain and a second antigen binding domain, the CDR of the first antigen binding domain can be represented by the prefix "A1", and the CDR of the second antigen binding domain can be represented by the prefix "A2".Therefore, the CDR of the first antigen binding domain can be referred to as A1-HCDR1, A1-HCDR2 and A1-HCDR3 in this article; and the CDR of the second antigen binding domain can be referred to as A2-HCDR1, A2-HCDR2 and A2-HCDR3 in this article.

[0116] In one embodiment, the therapeutic protein is a bispecific anti-CD20×anti-CD3 antibody referred to as REGN1979. REGN1979 includes: an anti-CD20 binding arm including a heavy chain including the amino acid sequence of SEQ ID NO: 10; an anti-CD3 binding arm including a heavy chain including the amino acid sequence of SEQ ID NO: 11 and a common light chain including the amino acid sequence of SEQ ID NO: 12 (corresponding to both the anti-CD20 binding arm and the anti-CD3 binding arm). In some cases, the mature form of the antibody may not contain the C-terminal lysine residues of SEQ ID NOs: 10 and 11. Therefore, in some cases, the anti-CD20 binding arm of REGN1979 includes a heavy chain including residues 1-452 of SEQ ID NO: 10, and the anti-CD3 binding arm of REGN1979 includes a heavy chain including residues 1-448 of SEQ ID NO: 11. REGN1979 also includes the HCVR, LCVR and CDR sequences shown in the attached sequence listing. The anti-CD20 HCVR corresponds to SEQ ID NO: 13, the anti-CD3 HCVR corresponds to SEQ ID NO: 14, and the common LCVR corresponds to SEQ ID NO: 15. The anti-CD20 HCDR1-HCDR2-HCDR3 domains correspond to SEQ ID NOs: 16-17-18, respectively. The anti-CD3 HCDR1-HCDR2-HCDR3 domains correspond to SEQ ID NOs: 19-20-21, respectively. The common LCDR1-LCDR2-LCDR3 domains correspond to SEQ ID NOs: 22-23-24, respectively.

[0117] The bispecific antigen binding molecules discussed above or herein can be bispecific antibodies. In some cases, the bispecific antibody includes a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is an isotype IgG1. In some cases, the human IgG heavy chain constant region is an isotype IgG4. In various embodiments, the bispecific antibody includes a chimeric hinge that reduces Fcγ receptor binding relative to a wild-type hinge of the same isotype. In some cases, the bispecific antigen binding molecules of the present invention include Brinkmann et al., MABS, 9 (2): 182-212, any of the forms discussed in 2017, which are incorporated herein by reference.

[0118] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form a bispecific antigen binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The association of a multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen binding molecule. "Multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid with the ability to associate with a second multimerization domain having the same or similar structure or composition. For example, the multimerization domain can be an immunoglobulin C H A non-limiting example of a multimeric component is the Fc portion of an immunoglobulin (including the C H 2-C H 3 domains), e.g., an IgG selected from the group consisting of isotypes IgG1, IgG2, IgG3, and IgG4, and an Fc domain of any isotype within each isotype group.

[0119] The bispecific antigen binding molecules of the present invention generally include two multimerization domains, for example, two Fc domains, each of which is independently a part of a separate antibody heavy chain. The first multimerization domain and the second multimerization domain can be the same IgG isotype as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first multimerization domain and the second multimerization domain can be different IgG isotypes such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0120] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a peptide containing short cysteine. Other multimerization domains include peptides or polypeptides including or consisting of a leucine zipper, a spiral loop motif, or a coiled coil motif.

[0121] Any bispecific antibody form or technology can be used to prepare the bispecific antigen binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked to one or more other molecular entities such as another antibody or antibody fragment having a second antigen binding specificity (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to produce a bispecific antigen binding molecule. Specific exemplary bispecific forms that can be used in the context of the present invention include but are not limited to the following: for example, based on scFv or double antibody bispecific forms, IgG-scFv fusion, dual variable domains (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs into holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, DuobodV, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab 2 Bispecific formats (see, e.g., Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the aforementioned formats).

[0122] In the context of the bispecific antigen binding molecules of the present invention, a multimerization domain, e.g., an Fc domain, may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) as compared to a naturally occurring Fc domain. For example, the present invention comprises bispecific antigen binding molecules comprising one or more modifications in the Fc domain that result in a modified Fc domain with a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule comprises a C H Zone 2 or C H3 region, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); modifications at positions 250 and 428 (e.g., L or F); modifications at positions 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., 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).

[0123] The present invention also includes a bispecific antigen binding molecule comprising a first C H 3 domain and the second Ig C H 3 domains, of which the first C H 3 domain and the second Ig C H The three 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 as compared to a bispecific antibody lacking the amino acid difference. H 3 domain binding protein A, and the second IgC H The 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (by IMGT exon numbering; H435R by EU numbering). H 3 may further include a Y96F modification (by IMGT; Y436F by EU). See, for example, U.S. Pat. No. 8,586,713. HFurther modifications found within 3 include: in the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU); in the case of IgG2 antibodies, N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU).

[0124] In certain embodiments, the Fc domain may be chimeric, with a combination of Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include a Fc sequence derived from human IgG1 C H Region 2, human IgG2 C H Region 2 or human IgG4 C H C in Zone 2 H 2 sequence and a portion or all of a C sequence derived from human IgG1, human IgG2 or human IgG4 H 3 sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may include a combination of an "upper hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region and a "lower hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any antigen-binding molecule shown herein include, 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 listed herein includes, 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 can be included in any antigen binding molecule of the invention are described in U.S. Publication No. 2014 / 0243504, published on August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector function.

[0125] Binding properties of antigen-binding molecules

[0126] In the context of binding of an antibody, immunoglobulin, antibody binding fragment or Fc-containing protein to, for example, a predetermined antigen (e.g., a cell surface protein) or a fragment thereof, the term "binding" generally refers to the interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.

[0127] For example, when measured by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using an antigen as a ligand and an antibody, Ig, antibody binding fragment or Fc-containing protein as the analyte (or anti-ligand), the binding affinity is typically about 10 -7 M or lower K D Value, such as about 10 -8 M or lower, such as about 10 -9 Cell-based binding strategies such as fluorescence activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlate strongly with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods 1997, 201(2):223-31; Geuijen, CA et al., J Immunol Methods 2005, 302(1-2):68-77).

[0128] Therefore, the antibody or antigen-binding protein of the present invention binds to a predetermined antigen or cell surface molecule (receptor) having a K D The affinity corresponding to the K value is at least ten times lower than the affinity of the antigen or cell surface molecule binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, an affinity corresponding to the K value equal to or less than ten times that of the non-specific antigen is at least ten times lower than the affinity of the antigen or cell surface molecule binding to the non-specific antigen (e.g., BSA, casein). D The affinity of the antibody corresponding to the value can be considered as undetectable binding, but such an antibody can be paired with a second antigen-binding arm to generate a bispecific antibody of the invention.

[0129] The term "K D "(M)" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction or the dissociation equilibrium constant of the binding of an antibody or antibody binding fragment to an antigen. K D There is an inverse relationship between K and binding affinity, so K D The smaller the value, the higher the affinity, ie the stronger it is. Therefore, the term "higher affinity" or "stronger affinity" relates to a higher ability to form an interaction and therefore to a smaller K D value, and conversely, the term "lower affinity" or "weaker affinity" relates to a lower ability to form an interaction, and thus K DIn some cases, a particular molecule (e.g., an antibody) has a higher binding affinity (or K) for a partner molecule (e.g., antigen X) with which it interacts than the binding affinity of the molecule (e.g., antibody) for another interacting partner molecule (e.g., antigen Y). D ) can be expressed as D value (lower or weaker affinity) divided by the smaller K D (higher or stronger affinity), for example, a binding ratio determined by a 5-fold or 10-fold greater binding affinity, as appropriate.

[0130] The term “k d "(seconds-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction or the dissociation rate constant of an antibody or antibody binding fragment. This value is also called k off value.

[0131] The term “k a "(M-1×sec-1 or 1 / M) refers to the association rate constant for a specific antibody-antigen interaction or the association rate constant of an antibody or antibody binding fragment.

[0132] The term "K A ” (M-1 or 1 / M) refers to the association equilibrium constant of a specific antibody-antigen interaction or the association equilibrium constant of an antibody or antibody binding fragment. By dividing k a Divide by k d The association equilibrium constant was obtained.

[0133] The term "EC50" or "EC 50 ” refers to the half-maximal effective concentration, which comprises the concentration of the antibody that induces a response intermediate between the baseline and maximum after a specified exposure time. EC 50 Substantially represents the concentration of the antibody at which 50% of its maximal effect is observed. In certain embodiments, EC 50 The value is equal to the concentration of an antibody of the invention that has half-maximal binding to cells expressing, for example, CD3 or a tumor-associated antigen (e.g., CD20) as determined by, for example, a FACS binding assay. Thus, decreased or weaker binding is observed, with an increased EC 50 Or half of the maximum effective concentration.

[0134] In one embodiment, decreased binding can be defined as an increased EC 50 The concentration of antibody that is able to bind half the maximal number of target cells.

[0135] In another embodiment, EC 50The values ​​represent the concentration of the antibody of the invention that induces half-maximal depletion of target cells by T cell cytotoxic activity. Thus, increased cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed with a decreased EC 50 Or the effective concentration value of half the maximum value.

[0136] Sequence variants

[0137] As compared with the corresponding germline sequences derived from a single antigen binding domain, the antibodies and bispecific antigen binding molecules of the present invention can include 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. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences that can be obtained from, for example, public antibody sequence databases. The antigen binding molecules of the present invention may include antigen binding domains derived from any exemplary amino acid sequence disclosed herein, wherein one or more amino acid mutations in one or more frameworks and / or CDR regions are mutated to one or more corresponding residues of the germline sequence of the derived antibody, or mutated to one or more corresponding residues of another human germline sequence, or mutated to conservative amino acid substitutions of one or more corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, a person of ordinary skill in the art can easily produce many antibodies and antigen binding fragments including one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll mutations of framework and / or CDR residues in the domain are back to the residues found in the original germline sequence of the initial derived antigen-binding domains. In other embodiments, only some residues are mutated back to the original germline sequence, for example, only the residue after the mutation found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the residue after the mutation found in CDR1, CDR2 or CDR3. In other embodiments, one or more mutations of one or more CDR residues in the framework are one or more corresponding residues of different germline sequences (that is, germline sequences different from the germline sequences of the initial derived antigen-binding domains). In addition, the antigen-binding domains of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR districts, for example, wherein some individual residues are mutated to the corresponding residues of specific germline sequences, and some other residues different from the original germline sequence can maintain or mutate to the corresponding residues of different germline sequences. 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 antigen binding domains obtained in this general manner are encompassed by the present invention.

[0138] pH-dependent binding

[0139] The present invention includes antibodies and bispecific antigen binding molecules with pH-dependent binding characteristics. For example, as compared with neutral pH, the antibodies of the present invention can show reduced binding with, for example, tumor antigens (such as CD20) at acidic pH. Alternatively, as compared with neutral pH, the antibodies of the present invention can show enhanced binding with, for example, tumor antigens (such as CD20) at acidic pH. The expression "acidic pH" includes a pH value less than about 6.2, for example, 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 less. The expression "neutral pH" means that the pH is about 7.0 to about 7.4. The expression "neutral pH" encompasses pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

[0140] In some cases, "reduced binding at acidic pH as compared to neutral pH" means that the K of the antibody bound to the antigen at acidic pH is D The K value of an antibody binding to its antigen at neutral pH DFor example, if an antibody or antigen-binding fragment thereof exhibits an acidic / neutral K of about 3.0 or greater, D For purposes of the present invention, an antibody or antigen-binding fragment thereof may be considered to exhibit "reduced binding to, for example, CD20 at acidic pH as compared to neutral pH" if the ratio of acidic / neutral K to neutral pH is greater than or equal to 1.5. D The ratio 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.

[0141] Antibodies with pH-dependent binding characteristics can be obtained by, for example, screening for a population of antibodies that have reduced (or enhanced) binding to a specific antigen at an acidic pH as compared to a neutral pH. In addition, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by replacing one or more amino acids of an antigen-binding domain (e.g., within a CDR) with histidine residues, an antibody with reduced binding to an antigen at an acidic pH relative to a neutral pH can be obtained.

[0142] Antibodies including Fc variants

[0143] According to certain embodiments of the present invention, antibodies and bispecific antigen-binding molecules are provided, which include an Fc domain, wherein the Fc domain includes one or more mutations that enhance or reduce the binding of the antibody to the FcRn receptor at acidic pH, such as compared to neutral pH. For example, the present invention includes a C in the Fc domain. H Zone 2 or C HAn antibody comprising mutations in region 3, wherein one or more mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome at a pH 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., modification at position 250 (e.g., E or Q); modification at positions 250 and 428 (e.g., L or F); modification at positions 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 modification 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 modification at positions 250 and / or 428; or modification 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).

[0144] For example, the present invention comprises antibodies and bispecific antigen binding molecules, the molecules comprising an Fc domain, the Fc domain comprising one or more mutation pairs or mutation groups 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 within the scope of the present invention.

[0145] Preparation of antigen binding domains and construction of bispecific molecules

[0146] Antigen binding domains specific to specific antigens can be prepared by any antibody production technology known in the art. Once obtained, two different antigen binding domains specific to two different antigens (e.g., CD3 and CD20) can be appropriately arranged relative to each other to produce the bispecific antigen binding molecules of the present invention using conventional methods. (Discussion of exemplary bispecific antibody forms that can be used to construct bispecific antigen binding molecules of the present invention is provided elsewhere herein). In certain embodiments, one or more individual components (e.g., heavy chain and light chain) of the multispecific antigen binding molecules of the present invention are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used TM One or more heavy chains and / or light chains of the bispecific antigen-binding molecules of the present invention are prepared using VELOCIMMUNE TM Using the PCR technology (or any other human antibody generation technology), high affinity chimeric antibodies are initially isolated for a specific antigen (e.g., CD3 or CD20) having a human variable region and a mouse constant region. The antibodies are characterized and selected to obtain the desired properties including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0147] Genetically engineered animals can be used to prepare human bispecific antigen binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, wherein the mouse only expresses one or two human light chain variable domains encoded by human immunoglobulin sequences, and the human immunoglobulin sequences are operably connected to the mouse κ constant gene at the endogenous mouse κ locus. This genetically modified mouse can be used to produce fully human bispecific antigen binding molecules including two different heavy chains, and the heavy chain is associated with the same light chain including the variable domain derived from one of the two different human light chain variable region gene segments. (See, for example, US 2011 / 0195454). Fully human refers to an antibody or its antigen-binding fragment or immunoglobulin domain comprising an amino acid sequence encoded by DNA, and the DNA is derived from a human sequence within the entire length of each polypeptide of an antibody or its antigen-binding fragment or immunoglobulin domain. In some cases, fully human sequences are derived from endogenous proteins of people. In other cases, fully human proteins or protein sequences include chimeric sequences, wherein each component sequence is derived from a human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are typically designed to minimize the generation of immunogenic epitopes at the junction of the component sequences, for example compared to any wild-type human immunoglobulin region or domain.

[0148] Bioequivalence

[0149] The present invention encompasses antigen binding molecules having an amino acid sequence that is different from the amino acid sequence of the exemplary molecules disclosed herein but retains the ability to bind to one or more of the same antigens. Such variant molecules may include one or more additions, deletions or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity substantially the same as that of the described antibodies or bispecific antigen binding molecules.

[0150] The present invention includes antigen binding molecules that are bioequivalent to any exemplary antigen binding molecules listed herein. If for example two antigen binding proteins or antibodies are that absorption rate and absorption extent do not show drug equivalents or drug substitutes of significant differences when using (single dose or multiple doses) with identical molar doses under similar experimental conditions, then it is considered to be bioequivalent. If some antigen binding proteins are equivalent in absorption extent but their absorption rate is not equivalent, then it is considered to be equivalent or drug substitute, and can be considered to be bioequivalent, because this intentional and the difference in absorption rate reflected on the label is not necessary for obtaining effective body drug concentration when for example long-term use, and is considered to be medically insignificant for the specific drug product studied.

[0151] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in safety, purity, and potency.

[0152] In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased effectiveness, as compared to continued treatment without such switching.

[0153] In one implementation, two antigen binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the disorder or condition of use (provided such mechanisms are known).

[0154] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example: (a) in vivo tests performed in humans or other mammals, where the concentration of the antibody or its metabolites is measured over time in blood, plasma, serum or other biological fluids; (b) in vitro tests that are correlated with and reasonably predictive of bioavailability data in humans; (c) in vivo tests performed in humans or other mammals, where appropriate acute pharmacological effects of the antibody (or its target) are measured over time; and (d) in well-controlled clinical trials to establish the safety, efficacy or bioavailability or bioequivalence of the antigen-binding protein.

[0155] Bioequivalent variants of the exemplary antibodies and bispecific antigen-binding molecules listed 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 necessary for biological activity. For example, cysteine ​​residues that are not necessary for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bonds during renaturation. In other cases, bioequivalent antigen-binding proteins can include variants of the exemplary antibodies and bispecific antigen-binding molecules listed herein that include amino acid changes that alter the glycosylation characteristics of the molecule, such as mutations that eliminate or remove glycosylation.

[0156] Therapeutic formulations and delivery

[0157] The present invention provides pharmaceutical compositions comprising antigen binding molecules of the present invention. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients and other agents that provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption creams, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) "Journal of Pharmaceutical Science and Technology ( ...

[0158] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the invention, e.g., 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 composition can be administered by any convenient route, e.g., by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other biologically active agents. Administration can be systemic or local. In one embodiment, the therapeutic protein of the invention is administered by intravenous infusion.

[0159] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices are readily applicable to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once the entire 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. Instead, the disposable pen delivery device is preloaded with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0160] Many reusable pen and auto-injector delivery devices are useful for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN70 / 30 TM PEN (Eli Lilly and Co., Indianapolis, Indiana), NOVOPEN TM I, II and III (NovoNordisk, Copenhagen, Denmark), NOVOPENJUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TMPEN (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR TM Pen (Sanofi), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM PEN (Abbott Labs, Abbott Technology Park, IL), to name a few.

[0161] 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; Seffon, 1987, CRC: Biomedical Engineering Review (CRC Crit. Ref. Biomed.) Eng., 14: 201). In another embodiment, a polymer material can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, so only a small portion of the systemic dose is required (see, for example, Goodson, 1984, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in a review by Langer, 1990, Science, 249: 1527-1533.

[0162] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, instillations, etc. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by, for example, dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or a conventional oily medium for injection. As aqueous media for injection, for example, there are physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizers such as alcohol (for example, ethanol), polyols (for example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)], etc. As oily media, for example, sesame oil, soybean oil, etc. can be used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injection prepared in this way is preferably filled in a suitable ampoule.

[0163] Therapeutic uses of antigen binding molecules

[0164] The present invention includes methods comprising administering a therapeutic composition comprising a therapeutic protein to a subject in need thereof. The therapeutic composition may include any antibody or bispecific antigen binding molecule disclosed herein and a pharmaceutically acceptable carrier or diluent. The expression "subject in need thereof" means a human or non-human animal (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned below) that exhibits one or more symptoms or signs of cancer.

[0165] The antibodies and bispecific antigen binding molecules of the present invention (and therapeutic compositions comprising the same) are particularly useful for treating any disease or condition in which stimulation, activation and / or targeting of an immune response would be beneficial. Specifically, the antibodies or bispecific antigen binding molecules of the present invention can be used to treat, prevent and / or improve any disease or condition associated with or mediated by the expression or activity of CD20, PSMA, MUC16, STEAP2 or BCMA or the proliferation of CD20+, PSMA+, MUC16+, STEAP2+ or BCMA+ cells. The mechanism of action for achieving the therapeutic methods of the present invention includes, for example, killing cells expressing such antigens in the presence of effector cells by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0166] The antigen binding molecules of the invention can be used to treat, for example, primary and / or metastatic tumors arising in the brain and meninges, head and neck, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood-forming cells and bone marrow, liver and urinary tract, kidneys, bladder and / or special sensory organs (such as the eye). In certain embodiments, the antibodies and bispecific antigen binding molecules of the invention are used to treat one or more of the following cancers, but are not limited to the following cancers: pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, breast cancer (e.g., ductal or intraductal breast cancer), squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, (kidney) large oncocytoma, (kidney) transitional cell carcinoma, urothelial carcinoma, (bladder) adenocarcinoma or (bladder) small cell carcinoma. According to certain embodiments of the invention, the antibodies or bispecific antibodies can be used to treat patients with refractory or difficult to treat cancers (e.g., castration-resistant prostate cancer).

[0167] In some embodiments, the antigen binding molecule is a bispecific anti-CD3×anti-CD20 antibody that can be used to treat cancers expressing CD20 (including non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom's macroglobulinemia, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma or Burkitt's lymphoma). In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is mantle cell lymphoma. In some embodiments, the cancer is marginal zone lymphoma. In some embodiments, the cancer is follicular lymphoma and the maximum weekly dose of the bispecific antibody is 80 mg. In some embodiments, the cancer is DLBCL and the maximum weekly dose of the bispecific antibody is 80 mg. In some embodiments, the cancer is DLBCL and the maximum weekly dose of the bispecific antibody is 160 mg. In some embodiments, the cancer is DLBCL and the maximum weekly dose of the bispecific antibody is 320 mg. In any of these embodiments or other embodiments discussed herein, the cancer patient may have been pretreated with an anti-CD20 monospecific antibody therapy.

[0168] Non-Hodgkin lymphoma (NHL) is the most common hematological malignancy. In a group of heterogeneous NHL, 85-90% are of B cell origin and include follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and several other B-NHL. Anti-CD20 antibodies in combination with chemotherapy are standard care for the treatment of B-NHL; however, despite initial responses, many patients relapse, and the duration of response is usually gradually shortened in subsequent lines of therapy and the prognosis is poor. Therefore, in some embodiments, the antigen binding molecule is a bispecific anti-CD3×anti-CD20 that binds to CD3+T cells and CD20+B cells targeting CD20+ tumor cells through T cell-mediated cytotoxicity. In some cases, the anti-CD3×CD20 bispecific antibody is used to treat B cell cancers (e.g., NHL) in subjects who have previously failed therapy with anti-CD20 monospecific antibodies.

[0169] For patients who do not fully respond to CAR-T therapy, the prognosis is generally poor and there are no standard of care treatment options. Therefore, in some cases, the anti-CD3×CD20 bispecific antibodies of the present invention are used to treat B cell cancers (e.g., NHL, such as DLBCL) in subjects who have failed previous CAR-T therapy or have not responded to previous CAR-T therapy (e.g., anti-CD19 CAR-T therapy).

[0170] In some embodiments, the antigen binding molecule is a bispecific anti-CD3 x anti-PSMA antibody useful for treating PSMA-expressing cancers including prostate cancer, kidney cancer, bladder cancer, colorectal cancer, and gastric cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0171] In some embodiments, the antigen binding molecule is a bispecific anti-CD3×anti-MUC16 antibody that can be used to treat cancers expressing MUC16 (including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic bile duct carcinoma-mass type, cervical adenocarcinoma, gastrointestinal adenocarcinoma). In some embodiments, the cancer is ovarian cancer.

[0172] In some embodiments, the antigen binding molecule is a bispecific anti-CD3×anti-STEAP2 antibody that can be used to treat cancers expressing STEAP2 (including prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, uterine cancer, ovarian cancer). In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0173] In some embodiments, the antigen binding molecule is a bispecific anti-CD3 x anti-BCMA antibody that can be used to treat BCMA-expressing cancers including multiple myeloma or other B cell or plasma cell cancers (such as Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and diffuse large B-cell lymphoma), non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin's lymphoma. In some embodiments, the cancer is multiple myeloma.

[0174] Combination therapy

[0175] The present invention provides a method including administering a pharmaceutical composition, the pharmaceutical composition including any exemplary antibody described herein and a combination of bispecific antigen binding molecules and one or more other therapeutic agents. The exemplary other therapeutic agents that can be combined with the antigen binding molecules of the present invention or used in combination with it include, for example, anti-tumor agents (e.g., chemotherapeutic agents, including melphalan (melphalan), vincristine (Oncovin (Oncovin)), cyclophosphamide (Cytoxan (Cytoxan)), etoposide (VP-16), doxorubicin (adriamycin (Adriamycin)), liposomal doxorubicin (Doxil (Doxil)), obendamustine (obendamustine, bendamustine (Treanda)) or any other chemotherapeutic agents known to be effective for treating plasma cell tumors of subjects). In certain embodiments, the second therapeutic agent is a scheme including radiotherapy or hematopoietic stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulator. In certain embodiments, the second therapeutic agent can be a proteasome inhibitor, including bortezomib (Velcade), carfilzomib / Kyprolis, ixazomib / Ninlaro. In certain embodiments, the second therapeutic agent can be a histone deacetylase inhibitor, such as panobinostat / Farydak. In certain embodiments, the second therapeutic agent can be a monoclonal antibody, an antibody drug conjugate, a bispecific antibody conjugated to an anti-tumor agent, an immune checkpoint inhibitor, or a combination thereof. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising bispecific antigen binding molecules as disclosed herein) can also be administered as part of a treatment regimen comprising one or more therapeutic combinations selected from the following: a monoclonal antibody different from the monoclonal antibodies described herein, which can interact with different antigens on the surface of plasma cells; a bispecific antibody, one arm of which binds to an antigen on the surface of a tumor cell and the other arm binds to an antigen on a T cell; an antibody drug conjugate; a bispecific antibody conjugated to an anti-tumor agent; a checkpoint inhibitor, such as a checkpoint inhibitor targeting PD-1 or CTLA-4; or a combination thereof. In certain embodiments, the checkpoint inhibitor can be selected from a PD-1 inhibitor, such as pembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab.In certain embodiments, the checkpoint inhibitor may be selected from a PD-L1 inhibitor, such as atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). In certain embodiments, the checkpoint inhibitor may be selected from a CTLA-4 inhibitor, such as ipilimumab (Yervoy). Other combinations that may be used in combination with the antibodies of the present invention are described above.

[0176] The present invention also comprises a therapeutic combination comprising any antigen binding molecule mentioned herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9 or uroplastin, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, 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 a double antibody, a three-antibody, a four-antibody, a mini-antibody and a minimum recognition unit). The antigen binding molecules of the present invention can also be administered in combination with antiviral agents, antibiotics, analgesics and / or NSAIDs and / or co-formulated. The antigen binding molecules of the invention may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0177] One or more additional therapeutically active ingredients may be administered prior to, concurrently with, or after administration of the antigen binding molecules of the invention; (for purposes of this disclosure, such administration regimens are considered administration of the antigen binding molecules "in combination" with the additional therapeutically active components).

[0178] The invention encompasses pharmaceutical compositions in which the antigen binding molecules of the invention are co-formulated with one or more additional therapeutically active ingredients as described elsewhere herein.

[0179] Examples

[0180] The following examples are presented to provide a complete disclosure and description of how to prepare and use the methods and compositions of the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy regarding the numerals used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, and pressure is or is close to atmospheric pressure.

[0181] Example 1: Clinical evaluation and dose escalation of bispecific antibodies

[0182] The clinical study described below is an open-label, multicenter Phase 1 study designed to investigate the safety and tolerability of REGN1979, an anti-CD20 × anti-CD3 bispecific monoclonal antibody, in patients with CD20+ B-cell malignancies previously treated with CD20-directed antibody therapy.

[0183] Purpose: The primary objectives of this study are to evaluate the safety, tolerability, and dose-limiting toxicities (DLTs) of REGN1979 administered intravenously (IV) and to investigate the antitumor activity of REGN1979 in an expansion cohort that includes diffuse large B-cell lymphoma (DLBCL) following failure of chimeric antigen receptor T-cell (CAR-T) therapy, aggressive lymphomas (different from DLBCL following failure of CAR-T therapy), follicular lymphoma (FL) grades 1-3a, and chronic lymphocytic leukemia (CLL). The secondary objectives of the study are: (1) characterize the pharmacokinetic (PK) profile of REGN1979; (2) evaluate the immunogenicity of REGN1979; (3) investigate the preliminary antitumor activity of REGN1979 in patients with CD20+ B-cell malignancies (non-Hodgkin lymphoma [NHL] or chronic lymphocytic leukemia [CLL] previously treated with anti-CD20 antibody therapy); and (4) investigate the preliminary antitumor activity of REGN1979 in the dose escalation portion of this study. Patients with CLL were evaluated for minimal residual disease (MRD). The exploratory objectives of this study were to evaluate biomarkers that may be associated with the mechanism of action, observed toxicities, and potential antitumor activity, including but not limited to: (1) cytokine profiling and assessment of inflammatory markers (e.g., C-reactive protein [CRP]); (2) peripheral blood B cell and T cell subset and immunophenotyping; and (3) changes in gene expression in peripheral blood.

[0184] Study Design: Patients are assigned to dose level (DL) groups consisting of an initial starting dose, followed by an increase in ascending dose for the second and subsequent dose administrations. Patients are enrolled based on indications (NHL or CLL). At each DL, there are 2 groups (one group per indication), with 3 to 6 patients per NHL group and 1 to 6 patients per CLL group. Patients with small lymphocytic lymphoma (SLL) are enrolled in the CLL group and are evaluated for NHL.

[0185] Patients who initially showed clinical benefit and subsequently relapsed or progressed or had a suboptimal response to treatment could have been retreated at the time of relapse or progression with the highest DL of REGN 1979 deemed tolerable.

[0186] Patients underwent a screening procedure to determine eligibility within 28 days prior to the first dose of REGN 1979. Patients were enrolled sequentially based on indication (NHL or CLL) in the order of eligibility confirmed by the sponsor until each cohort was filled according to protocol criteria.

[0187] At each DL, there are separate independent dose escalation cohorts for NHL and CLL. Each DL consists of an initial dose and a second and subsequent dose that is higher than the starting dose, provided that the initial dose can be tolerated.

[0188] For patients with NHL, dose escalation was performed according to a traditional 3+3 dose escalation design, with three to six patients planned per cohort based on observed toxicities.

[0189] For patients with CLL, dose escalation was performed according to an accelerated 3+3 schedule with modified titration components. One to six patients were planned per cohort, depending on observed toxicities.

[0190] After completion of the dose escalation phase, and after the recommended dose is determined for further study, in patients with NHL, three expansion cohorts will be opened for the following: 1) patients with DLBCL after failure of CAR-T therapy (20 patients), 2) aggressive lymphoma (different from DLBCL after failure of CAR-T therapy) (40 patients; 20 patients were each included in aggressive lymphoma cohort 1 [160 mg ascending dose]) and cohort 2 [80 mg ascending dose]) and 3) patients with relapsed / refractory follicular lymphoma grades 1-3a (60 patients; 30 patients were each included in cohort 1 [80 mg ascending dose] and cohort 2 [160 mg ascending dose]). The weekly ascending dose of REGN1979 in the expansion cohorts in these expansion cohorts (except aggressive lymphoma cohort 2 and follicular lymphoma cohort 1) was set to 160 mg per week, followed by Q2W maintenance treatment with 320 mg REGN1979. In the aggressive lymphoma cohort 2 and follicular lymphoma cohort 1, the weekly escalation dose of REGN1979 was 80 mg, and the Q2W maintenance dose of REGN1979 was 160 mg. For patients assigned to the 80 mg escalation dose, after the initial dose escalation, the patients received 80 mg weekly during the 4-week induction period, then received an additional 8 weekly doses, and were treated with 160 mg Q2W until progression, and the patient chose to interrupt treatment after a durable response lasting at least 9 months after the first CR. For patients assigned to the 160 mg escalation dose, after the initial dose escalation, the patients received 160 mg weekly during the 4-week induction period, then received an additional 8 weekly doses, and were treated with 320 mg Q2W until progression, and the patient chose to interrupt treatment after a durable response lasting at least 9 months after the first CR.

[0191] The utility of a rituximab lead-in dose prior to the first administration of REGN1979 was studied in patients with NHL to determine whether this intervention could reduce the incidence and severity of infusion-related reactions (IRRs) and cytokine release syndrome (CRS). In the rituximab lead-in cohort, REGN1979 was administered using ascending doses of 160 mg during weekly treatment and 320 mg during Q2W maintenance therapy. Subsequently, an additional 24 patients will be treated with the optimal dose regimen and dose; together with the 6 patients treated at the optimal dose in the rituximab lead-in group, a total of 30 patients were reviewed for safety and tolerability.

[0192] In the first DL of the dose escalation portion, the first 3 patients within the same indication required a 48-hour waiting period between initial study drug administrations. Regardless of indication, subsequent patients in the first DL were not treated on the same day. In subsequent cohorts, the first infusions of the first 3 patients were administered at least 24 hours apart if no unexpected toxicity was observed in the previous cohort or in the cohort.

[0193] After patients in each cohort are enrolled, treated, and complete the DLT observation period, a determination is made to open a subsequent DL cohort for enrollment (or expand a current open DL cohort) once safety data have been reviewed by the Sponsor and Investigator(s).

[0194] The DLT observation period was defined as the first 28 days of treatment, which in this study corresponded to the induction period. During the induction period, patients were treated with REGN1979 4 times per week.

[0195] To be able to be evaluated for DLT, individual patients must have received at least the first 2 administrations of REGN1979 (Week 1 Day 1 ["initial or primary dose"] and Week 2 Day 1 ["secondary and subsequent doses"]) or experienced a DLT. Patients enrolled at DL11 and above must have received at least the first 3 administrations of REGN1979 (initial dose in Week 1, intermediate or secondary dose in Week 2, and higher tertiary or ascending doses in Week 3) or experienced a DLT. Additionally, patients must have been evaluated at least 28 days after the first administration and at least 21 days after the second administration.

[0196] Table 1 below shows dose escalation and cohorts for NHL and CLL patients.

[0197] Table 1. Dose escalation and cohorts

[0198]

[0199] In this study, each dose level includes an initial REGN1979 dose followed by an ascending dose; for the DL11 and above groups, a secondary dose has been added before reaching the ascending dose (see Table 1). The first administration of the initial (primary) dose, the intermediate (secondary) dose (if applicable), and the ascending (tertiary) dose is each optionally separated by at least 2 days. All patients in the DL7 and higher maximum dose groups receive separate dosing (e.g., partial dosing) of the initial (primary) dose and the intermediate (secondary) dose. Patients in the DL11 and higher maximum dose groups receive separate dosing (e.g., partial dosing) of the initial (primary) dose, the intermediate (secondary) dose, and the first ascending (tertiary) dose, followed by the ascending dose (maximum weekly dose).

[0200] Study duration:The treatment period is 9 months. Patients will be treated with up to 24 doses of REGN1979 - 4 weekly doses during the 4-week induction phase, then an additional 8 weekly doses, and 12 or more doses twice a month (Q2W) during the maintenance phase until progression, with the option to interrupt treatment after 9 months when patients achieve a complete response. Patients can be followed up for up to 15 months after the end of treatment for efficacy and safety.

[0201] Study Group: Assuming inclusion until DL17 and full inclusion in all expansion groups, up to 370 patients are planned to be arranged in approximately 15 locations across the United States and Germany. During the dose escalation phase, for two indications (NHL and CLL), up to 204 patients will be included in the dose escalation group until DL17. Up to 100 patients (including 90 NHL patients (DLBCL patients after 20 CAR-T therapy failures, 40 CAR-T therapy failures after aggressive lymphoma patients [different from DLBCL after CAR-T therapy failure], 30 FL 1-3a grade patients) and 10 CLL patients) will be included in the disease-specific expansion group. Up to 42 patients will be included in the rituximab introduction group to determine the best dose regimen. Once the best rituximab introduction dose regimen is determined, another 24 patients will be included in the rituximab introduction expansion and will be combined with 6 patients from the above-mentioned rituximab introduction group treated with the best dose regimen and dose. Therefore, a total of 30 patients will be evaluated at the optimal rituximab induction dose schedule and dose.

[0202] Patients must have documented CD20+ B-cell malignancies with active disease that has not responded to previous therapy, for whom there are no standard of care options and for whom treatment with anti-CD20 antibodies may be appropriate. Patients with NHL must have been previously treated with CD20-directed antibody therapy.

[0203] Inclusion criteria: Patients must meet the following criteria to be eligible for study inclusion:

[0204] 1. Patients with documented CD20+ B-cell malignancies whose active disease has not responded to previous therapy, for whom standard of care options do not exist and for whom treatment with anti-CD20 antibodies may be appropriate:

[0205] B-NHL confirmed by the 2007 NCI Working Group criteria (Cheson 2007, Appendix 2); and

[0206] Patients with CLL-small lymphocytic lymphoma (SLL) confirmed by the 2008 International Workshop on Chronic Lymphocytic Leukemia (IWCLL) Working Group criteria (Hallek 2008, Appendix 3) will be included in the CLL group and evaluated for NHL.

[0207] NOTE - Patients who have a CD20-negative lymph node (NHL) biopsy just prior to enrollment as per standard of care are still eligible for the study provided that the patient has previously been documented to have CD20+ disease and has been previously treated with rituximab or other CD20-directed antibody therapy within approximately 6 months. Individual circumstances may be discussed with the Medical Monitor.

[0208] 2. Patients with NHL must have been previously treated with anti-CD20 antibody therapy. Patients with CLL do not need to have previously received treatment with anti-CD20 antibody therapy, provided that the patient has failed both a BTK inhibitor or a PI3K inhibitor and the treating physician believes that it is appropriate for the patient to enter the Phase 1 trial. To be included in the FL 1-3a grade expansion cohort, patients must have previously received at least 2 prior lines of systemic therapy, including an anti-CD20 antibody and an alkylating agent. To be included in the disease-specific expansion cohort that includes patients with DLBCL after failure of CAR-T therapy, patients must have recovered from the toxicity of lymphodepleting therapy and CAR-T infusion. It is not necessary to have previous CAR-T therapy as the most recent line of therapy prior to study inclusion.

[0209] 3. If CT scan was not available, all patients (B-cell NHL and CLL) must have at least one bidimensionally measurable lesion (≥1.5 cm) documented by CT or MRI scan.

[0210] 4. Patients with CLL must have a white blood cell count (WBC) ≤ 200 × 109 / L

[0211] 5. Age ≥ 18 years old

[0212] 6. Eastern Cooperative Oncology Group (ECOG) performance status ≤ 1

[0213] 7. Life expectancy is at least 6 months

[0214] 8. Adequate bone marrow function as documented by: a. Platelet count ≥ 75 × 109 / L; b. Hb level ≥ 9 g / dL; c. ANC ≥ 1 × 109 / L

[0215] NOTE - Patients may be considered for inclusion if, in the investigator's opinion, the reason their cell counts are below the thresholds listed above is due to bone marrow infiltration from underlying disease. In such cases, the investigator must discuss eligibility with the sponsor and obtain written approval for inclusion.

[0216] 9. Adequate organ function as documented by:

[0217] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 × ULN

[0218] Total bilirubin ≤1.5×ULN

[0219] NOTE - Patients with Gilbert's syndrome do not have to meet this requirement provided their total bilirubin does not change from their baseline.

[0220] Creatinine clearance calculated by Cockcroft-Gault ≥ 50 mL / min

[0221] NOTE - Patients may be considered for inclusion if the abnormal laboratory results are, in the investigator's opinion, due to an underlying disease. In such cases, the investigator must discuss eligibility with the sponsor and obtain written approval for inclusion.

[0222] NOTE - Patients with borderline creatinine clearance by Cockcroft-Gault may be considered for inclusion if the measured creatinine clearance (based on 24-hour urine or other reliable method) is ≥50 mL / min.

[0223] 10. Willing to undergo mandatory tumor biopsy pretreatment if, in the investigator's opinion, the patient's accessible lesions can be biopsied without significant risk to the patient.

[0224] 11. Willing and able to comply with clinic visits and study-related procedures

[0225] 12. Provide signed informed consent.

[0226] Exclusion criteria: Patients who meet any of the following criteria will be excluded from this study:

[0227] 1. Primary central nervous system (CNS) lymphoma or known or suspected non-primary CNS NHL involving the CNS

[0228] 2. History of CNS pathology or current related CNS pathology, such as

[0229] Epilepsy, seizure, mild paralysis, aphasia, stroke, severe brain injury, cerebellar disease, organic brain syndrome, psychosis; or

[0230] Evidence of inflammatory lesions and / or vasculitis on brain MRI

[0231] 3. Prior to the first administration of study drug, standard anti-tumor chemotherapy (non-biological) was administered within 5 times the half-life or 28 days, whichever is shorter.

[0232] 4. Standard radiotherapy within 14 days of the first administration of study drug.

[0233] NOTE - Ablative radiation therapy for symptomatic lymph nodes / lesions is permitted provided that one or more irradiated lesions or lymph nodes are not included as target lesions for tumor evaluation

[0234] 5. Allogeneic stem cell transplantation

[0235] 6. Treatment with rituximab, alemtuzumab, or other investigational or commercial biologics within 12 weeks prior to the first administration of study drug.

[0236] NOTE - For patients with aggressive lymphomas who require immediate treatment, the washout period may be shortened to 28 days. This will require discussion with the sponsor and written approval.

[0237] 7. Immunosuppressive therapy (except biologics) within 28 days of first administration of study drug.

[0238] 8. Treatment with an investigational non-biological agent within 28 days of the first administration of study drug.

[0239] 9. History of allergic reactions attributed to compounds with chemical or biological components similar to the study drug.

[0240] 10. A history of hypersensitivity to any compound in the tetracycline antibiotic group.

[0241] 11. The patient is undergoing treatment for concurrent active malignant tumors.

[0242] 12. Known active bacterial, viral, fungal, mycobacterial or other infection or any major infection episode requiring hospitalization or treatment with IV anti-infective drugs within 4 weeks of first administration.

[0243] 13. Evidence of significant concurrent illness or medical conditions that may interfere with the conduct of the study or place the patient 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) and / or significant pulmonary disease (e.g., obstructive pulmonary disease and history of symptomatic bronchospasm).

[0244] Caution - Patients with a medical history of cardiac disease should be evaluated by ECHO or multiple depth acquisition scanning (MUGA) prior to the first administration of REGN1979 to ensure adequate cardiac reserve and function.

[0245] 14. Ongoing systemic steroid therapy, up to 10 mg / day of prednisone or equivalent in addition to steroids used for other (non-neoplastic and non-immunosuppressive) indications.

[0246] 15. Infection with human immunodeficiency virus (HIV) or chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Patients with hepatitis B (HepBsAg+) whose infection is under control (serum hepatitis B virus DNA is below the limit of detection and receiving antiviral therapy for hepatitis B) are allowed after consultation with the physician managing the infection.

[0247] 16. Known hypersensitivity to both allopurinol and rasburicase.

[0248] 17. Pregnant or breastfeeding women.

[0249] 18. Women of childbearing potential* who are unwilling to use highly effective contraception before the first study drug treatment, during the study, and for at least 6 months after the last dose. Highly effective contraception includes stable use of combined (estrogen and progestin) hormonal contraception (oral, vaginal, transdermal) or progestin-only hormonal contraception (oral, injectable, implantable) and suppression of ovulation for the first 2 or more menstrual cycles prior to screening; intrauterine device; intrauterine hormone-releasing system; bilateral tubal ligation; vasectomized partner; and / or sexual abstinence.

[0250] *Postmenopausal women must have had amenorrhea for at least 12 months to not be considered of childbearing potential. Women with a documented hysterectomy or tubal ligation are exempt from pregnancy testing and contraception.

[0251] Sexual abstinence is considered a highly effective method only if it is defined as abstention from heterosexual intercourse during the entire risk period associated with study treatment. The reliability of sexual abstinence needs to be assessed with respect to the duration of the clinical trial and the patient's preferences and usual lifestyle.

[0252] Periodic abstinence (calendar, ovulatory, postovulatory methods), withdrawal (coitus interruptus), spermicides alone, and the lactational amenorrhea method (LAM) are unacceptable methods of contraception. Female and male condoms should not be used together.

[0253] 19. Live vaccine administration within 28 days of first administration of study drug

[0254] 20. Members of the clinical site research team and / or his / her immediate family members, unless approved in advance by the Sponsor.

[0255] treat: REGN1979 is provided in liquid form in sterile single-use vials. Each vial contains REGN1979 at a concentration of 2 mg / mL. Detailed preparation and administration instructions are provided for the field in the pharmacy manual. Diluent is provided for REGN1979 study drug preparation.

[0256] Patients received REGN1979 weekly during a 4-week induction period, followed by 8 additional weekly doses and Q2W dosing until progression, reaching the dose according to their assigned cohort.

[0257] In the rituximab introduction cohort and expansion only, a single dose of rituximab (375 mg / m 2 ). REGN1979 started on week 1 day 1, and the treatment period of REGN1979 was 9 months. Patients were treated with up to 24 doses of REGN1979: 4 weekly doses during the 4-week induction period, then an additional 8 weekly doses, and 12 or more doses were administered Q2W during the maintenance period until progression. In the rituximab introduction group, REGN1979 was administered using an ascending dose of 160 mg during weekly treatment and an ascending dose of 320 mg during Q2W maintenance treatment. Dose groups with ascending doses of REGN1979 lower than 160 mg weekly treatment and lower than 320 mg REGN1979 Q2W maintenance treatment can also be evaluated. Subsequently, an additional 24 patients were evaluated with the optimal dose regimen and dose; together with the 6 patients treated with the optimal dose regimen and dose in the rituximab introduction group, a total of 30 patients were reviewed for safety and tolerability.

[0258] Endpoints

[0259] main: The primary endpoints are safety (specifically, adverse events [AEs] and DLTs) to determine the maximum tolerated dose (MTD) and / or optimal biologic dose (OBD) as the recommended phase 2 dose (RP2D) of REGN1979; and efficacy as measured by objective response rate (ORR) in an expansion cohort of patients with DLBCL following failure of CAR-T therapy, expansion cohorts 1 and 2 of aggressive lymphomas (different from DLBCL following failure of CAR-T therapy), an expansion cohort of FL grade 1-3a, and an expansion cohort of CLL.

[0260] secondary: Secondary endpoints are:

[0261] Pharmacokinetics: Concentration of REGN1979

[0262] Immunogenicity: Anti-REGN1979 Antibodies

[0263] Antitumor activity:

[0264] -Objective response rate (ORR)

[0265] Tumor response was assessed according to the revised Response Criteria for Malignant Lymphoma of the NCI International Working Group (NCI-WG).

[0266] Tumor response assessment according to the International Chronic Lymphocytic Leukemia Workshop guidelines for the diagnosis and treatment of CLL

[0267] For patients included in the NHL expansion cohort, tumor response was assessed according to the Lugano Classification

[0268] -Progression-free survival (PFS) and overall survival (OS)

[0269] - Minimal residual disease (MRD) in patients with CLL

[0270] Exploratory endpoints include:

[0271] Pharmacodynamic (PD) measures, including:

[0272] - B cell and T cell subsets and phenotypes

[0273] - Circulating cytokine levels

[0274] -CRP

[0275] - Changes in gene expression in peripheral blood

[0276] Procedures and Assessment

[0277] Baseline Procedure: Brain MRI, electrocardiogram (ECG), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) testing, and blood coagulation.

[0278] Safety procedures: medical history, physical examination, symptom assessment, performance status evaluation, clinical laboratory tests, vital signs, AEs, and concomitant medications.

[0279] Efficacy program: Tumor assessment, including CT or MRI scans, 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) scans, bone marrow puncture and biopsy, lymph node and / or tumor biopsy, and peripheral blood samples (for CLL patients only). Blood samples for PK and anti-drug antibody (ADA) evaluations are collected. Biomarker samples are collected to monitor changes in cytokine production, serum levels of pro-inflammatory cytokines, and changes in lymphocyte subsets and activation states. In addition, these samples allow for tumor or somatic genetic analysis of variants that affect the clinical course of the underlying disease or modulate the side effects of treatment.

[0280] Statistics Program: The study design is based on a traditional 3+3 design with 3 to 6 patients per DL for patients with NHL and a modified 3+3 design with 1 to 6 patients per DL for patients with CLL. The exact number of patients included will depend on the number of patients (NHL and CLL) with protocol-defined DLTs observed and treatment-related toxicities in which acute effects (except for relevant laboratory abnormalities) resolved within 72 hours to ≤ Grade 1 or Grade 2 or higher from baseline (for CLL during the accelerated titration component) and the need to expand the currently defined DL or open additional cohorts with lower DLs.

[0281] Patient inclusion is ongoing and plans to arrange up to 370 patients. During the dose escalation phase, for two indications (NHL and CLL), up to 204 patients will be included in the dose escalation group until DL17. Up to 100 patients (including 90 NHL patients (DLBCL patients after 20 CAR-T therapy failures, 40 CAR-T therapy failures, aggressive lymphoma patients [different from DLBCL after CAR-T therapy failures] and 30 FL 1-3a grade patients) and 10 CLL patients) will be included in the disease-specific expansion group. Up to 42 patients will be included in the rituximab introduction group to determine the best dose regimen. Another 24 patients will be included in the rituximab introduction expansion and will be combined with 6 patients from the above-mentioned rituximab introduction group treated with the best dose regimen and dose. Therefore, a total of 30 patients will be evaluated with the best rituximab introduction dose regimen and dose.

[0282] Data were summarized using descriptive statistics only. In general, data were summarized by DL and indication (NHL or CLL). Within the NHL indication, data were also summarized by subgroups and doses for DLBCL after CAR-T failure, aggressive lymphoma (different from DLBCL after CAR-T failure) cohorts 1 and 2, and FL grade 1-3a. Within the NHL indication, data were also summarized by subgroups of indolent and aggressive NHL. Demographic and baseline characteristics were summarized descriptively by group.

[0283] Safety summaries and analyses were performed on the Safety Analysis Set (SAF). The primary analysis of safety was based on treatment-emergent AEs (TEAEs). This analysis comprised the basis for drawing conclusions about the safety of REGN1979. All AEs reported in this study were reported using the currently available version of the Medical Dictionary for Regulatory Activities. Coding. The lowest level of terms will be used for coding. Verbatim text, preferred terms (PT) and major system organ classes (SOC) are listed. The analysis of efficacy and baseline variables is performed on the efficacy analysis set (FAS). Efficacy analysis will be performed separately on the expansion groups including DLBCL after CAR-T therapy failure, aggressive lymphoma (different from DLBCL after CAR-T therapy failure) groups 1 and 2, FL 1-3a grades and CLL after all patients in the corresponding groups have completed 24 weeks of visits before or have interrupted the study.

[0284] result:The results of patients administered with a maximum weekly dose of 5 to 320 mg indicate that the incidence of CRS is low. It was observed that the serum concentration of REGN1979 in patients receiving a maximum weekly dose of as low as 12 mg was close to or exceeded the serum concentration level that has been proven to be effective in the Raji tumor xenograft mouse model (data not shown). Patients receiving a maximum weekly dose of as low as 40 mg maintained a serum concentration (data not shown) that exceeded the minimum concentration level (2000 μg / L) that has been proven to be effective in the Raii tumor xenograft mouse model. In addition, many partial responses and complete responses were also observed in patients treated at these levels, as shown in Tables 2-9 below. Cumulative REGN1979 safety and PK experiences until the DLT evaluation period of group 13N (27,000mcg REGN1979) proved that the management algorithm for CRS or IRR responses (i.e., stepwise dose escalation, separate administration within the first few weeks of REGN1979 administration, and premedication with steroids) has been proven to effectively prevent severe CRS or IRR, despite the gradual increase in administration in subsequent dose groups. Separate dosing provides benefits for patient safety (existing data) during weeks 1 to 4, with fewer overall incidences of severe CRS / IRR observed. In particular, the dosing strategy discussed herein provides a safer strategy for increasing the dose to levels greater than 80 mg, even 160 mg or higher, with less severe events occurring in weeks 3 and 4 when the higher doses reach and exceed the desired serum concentrations discussed above. Figure 1 The incidence of CRS / IRR was demonstrated in patients receiving up to the maximum dose of 320 mg. To date, no patient discontinued due to CRS / IRR adverse events.

[0285] Table 2. Responses observed in follicular lymphoma grades 1-3a

[0286]

[0287] No patients were dosed with 80 mg of REGN1979

[0288] After data cutoff (Table 2), two additional evaluable patients showed a complete response (CR), one patient at 40 mg and the second patient at 320 mg.

[0289] Table 3. Responses observed in diffuse large B-cell lymphoma

[0290]

[0291] Of the three 80 mg patients who achieved CR, two failed CART cell therapy. All complete responses mentioned in Table 3 are complete metabolic responses.

[0292] All CRs at the 80 mg, 160 mg, and 320 mg doses during study treatment were ongoing CRs, demonstrating durability of response.

[0293] Table 4. Responses observed in DLBCL after failure of CAR-T therapy

[0294]

[0295] Table 5. Responses observed in mantle cell lymphoma

[0296]

[0297] Table 6. Responses observed in marginal zone lymphoma

[0298]

[0299] Table 7. Dose Level Response in Patients with MCL

[0300]

[0301]

[0302] Table 8. Dose Level Response in Patients with MZL

[0303]

[0304] Table 9. Dose Level Responses in Patients with Other NHL

[0305]

[0306] In general, activity was observed broadly in heavily pretreated relapsed / refractory B-NHL patients treated with REGN1979 (including some patients who had progressed after prior CAR T-cell therapy), including:

[0307] (i) FL 1-3a: 12 / 13 (92.3%) ORR and 8 / 13 CR (61.5%) at a dose ≥5 mg;

[0308] (ii) DLBCL: 4 / 6 (66.7%) ORR (all CR) at doses of 80-160 mg, including two patients who achieved CR after failure of CD19-guided CAR T-cell therapy;

[0309] (iii) MCL: 3 / 3 responses, including one CR, at a dose ≥5 mg;

[0310] (iv) MZL: 3 / 5 responses, including two CRs, at a dose ≥5 mg;

[0311] (v) doses up to 320 mg per week have been demonstrated to be well tolerated in patients with B-NHL, with no DLTs observed in patients with B-NHL;

[0312] (vi) Most adverse events were mild to moderate in severity;

[0313] (vii) infections were reported in 49.4% of patients (14.8% grade 3-4 infections, including two deaths (2.5%));

[0314] (viii) No patient discontinued treatment due to CRS or neurological adverse events. Of the initial 96 patients, only seven experienced Grade 3 CRS; and

[0315] (ix) Dexamethasone did not inhibit the cytotoxicity of REGN1979, moderately affected the upregulation of T cell activation, and inhibited cytokine release.

[0316] Example 2: Clinical evaluation of bispecific antibodies

[0317] The clinical study described below is an open-label, multicenter Phase 2 study designed to evaluate the anti-tumor activity and safety of REGN1979, an anti-CD20 × anti-CD3 bispecific antibody, in patients with relapsed or refractory follicular lymphoma.

[0318] Purpose:The primary objective of this study was to evaluate the antitumor activity of single-agent REGN1979 in patients with follicular lymphoma (FL) who had relapsed or were refractory to at least 2 prior lines of systemic therapy, including an anti-CD20 antibody and an alkylating agent, as measured by objective response rate (ORR) according to the Lugano classification for responses in malignant lymphomas by independent central review (Cheson, 2014). The secondary objectives of this study were to: (1) evaluate the antitumor activity of single-agent REGN1979 in patients with relapsed or refractory FL as measured by: (a) ORR according to the Lugano classification (Cheson, 2014) as assessed by local investigator review, (b) complete response (CR) rate according to the Lugano classification as assessed by independent central review and local investigator review, (c) progression-free survival (PFS) according to the Lugano classification as assessed by independent central review and local investigator review, (d) overall survival (OS), (e) The objectives of the present invention are to evaluate the duration of response (DOR) according to the Lugano classification, (f) disease control rate (DCR) according to the Lugano classification as assessed by independent central review and local investigator evaluation, and (g) duration of disease control (DDC) according to the Lugano classification as assessed by independent central review and local investigator evaluation; (2) evaluate the safety and tolerability of REGN1979; (3) evaluate the pharmacokinetics (PK) of REGN1979; (4) evaluate the immunogenicity of REGN1979; and (5) evaluate the effect of REGN1979 on quality of life, as measured by the validated tools European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and EuroQoL 5 Dimensions 3 Levels (EQ-5D-3L).

[0319] Study Design: The study consisted of a screening period of up to 28 days, a total treatment period of up to 98 weeks, which included 12 weekly (QW) doses followed by every 2 weeks (Q2W) dosing for up to 86 weeks, and a 96-week post-treatment follow-up period.

[0320] REGN1979 was administered intravenously (IV) as a single dose with an initial (primary) dose of 1 mg, followed by an intermediate (secondary) dose of 20 mg, and subsequently a nominal (tertiary and maximum weekly) dose of 80 mg over a 12 QW treatment regimen, followed by 80 mg REGN1979 Q2W.

[0321] Enrollment followed an open single-arm design.

[0322] The Screening Period begins with the signing of the Informed Consent Form (ICF) and ends when the patient has been confirmed eligible for the study and treatment has begun or when the patient is determined to be ineligible and has been designated as a Screening Failure.

[0323] The treatment period began with the first administration of REGN1979 and consisted of 12 QW infusions of REGN1979 followed by Q2W dosing for 86 weeks, with a total treatment period of 98 weeks of study drug administration unless the patient discontinued study treatment due to disease progression, initiation of subsequent lymphoma therapy, adverse events (AEs), or any other reason.

[0324] The post-treatment follow-up period will be 96 weeks after the last dose of study treatment. All patients will be followed for survival every 12 weeks until death, loss to follow-up, patient withdrawal of consent for follow-up, or study termination by the sponsor (whichever comes first). For patients who discontinue study treatment for any reason other than disease progression, initiation of subsequent lymphoma therapy, or death, disease response will be assessed every 12 weeks during the post-treatment follow-up period until disease progression, death, initiation of subsequent lymphoma therapy, or patient withdrawal of consent for follow-up (whichever comes first).

[0325] Study duration: The study duration for each patient (excluding the screening period) will be approximately 194 weeks, unless the patient has disease progression or starts subsequent therapy, or until death, loss to follow-up, withdrawal of patient consent for follow-up, or termination of the study by the sponsor. The end of the study is defined as the last visit to the last patient.

[0326] Study Group: Up to 481 patients will be enrolled at up to 100 sites. The study population will consist of patients aged 18 years and older with previously treated FL grades 1 to 3a who have relapsed or are refractory to at least 2 prior lines of systemic therapy (including anti-CD20 antibodies and alkylating agents). Central histopathological confirmation of the FL diagnosis will be required prior to enrollment. Patients with FL grade 3b are not eligible. Refractory disease is defined as lack of response to standard regimens or progression within 6 months of the last treatment.

[0327] Inclusion criteria: Each patient must meet the following criteria to be eligible for inclusion in the study:

[0328] 1. Aged 18 or above

[0329] 2. Central histopathological confirmation of the diagnosis of FL grade 1 to 3a must be obtained before study inclusion. Patients with FL grade 3b are not eligible. Follicular lymphoma subtyping is based on the World Health Organization (WHO) classification (Swerdlow, 2017).

[0330] 3. The disease must have relapsed or must be refractory to ≥2 prior lines of systemic therapy, including anti-CD20 antibodies and alkylating agents. At study inclusion, patients should require FL-directed therapy at the investigator's discretion.

[0331] 4. Measurable disease on cross-sectional imaging (defined as at least 1 bidimensionally measurable nodular lesion ≥1.5 cm in the greatest transverse diameter (GTD) regardless of short-axis diameter) documented by diagnostic imaging (computed tomography [CT] or magnetic resonance imaging [MRI]).

[0332] 5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0333] 6. Adequate bone marrow function as documented by: (a) platelet count ≥ 50 × 10 9 / L. To meet the platelet eligibility criteria, patients could have not received platelet transfusion within 7 days before the first dose of REGN1979; (b) hemoglobin ≥9.0 g / dL; (c) absolute neutrophil count (ANC) ≥1.0×10 9 / L. To meet ANC eligibility criteria, patients could not have received granulocyte colony-stimulating factor within 2 days before the first dose of REGN1979.

[0334] 7. Adequate liver function: (a) total bilirubin ≤1.5× upper limit of normal (ULN) (≤3×ULN if due to hepatic lymphoma infiltration); (b) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤2.5×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—patients with AST>2.5×ULN and / or ALT>2.5×ULN and total bilirubin>1.5×ULN will be excluded regardless of the presence or absence of hepatic lymphoma infiltration, and patients with known Gilbert's syndrome are not required to meet this total bilirubin requirement, provided that the value is unchanged from baseline.

[0335] 8. Serum creatinine ≤1.5×ULN, or creatinine clearance calculated by the Cockcroft-Gault formula ≥50 mL / min; Note - Patients 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 / min.

[0336] 9. Willing to undergo a tumor biopsy at baseline. If the Investigator has determined that a baseline tumor biopsy cannot be obtained safely, the Sponsor may exceptionally approve the need for a biopsy only after discussion with and approval from the Medical Monitor.

[0337] 10. Be able to understand the purpose and risks of the research and provide signed and dated informed consent and authorization for the use of protected health information (in accordance with national and local subject privacy regulations).

[0338] 11. Willing and able to comply with clinic visits and study-related procedures.

[0339] 12. Provide the informed consent signed by the research patient or legal representative.

[0340] 13. Ability to understand and complete research-related questionnaires.

[0341] Exclusion criteria: Patients who meet any of the following criteria will be excluded from this study:

[0342] 1. Primary central nervous system (CNS) lymphoma or known non-primary CNS NHL involvement (in addition to mandatory head CT or MRI, suspected CNS lymphoma should also be evaluated by lumbar puncture).

[0343] 2. Treatment with any systemic anti-lymphoma therapy within 5 half-lives or 28 days, whichever is shorter, prior to the first administration of study drug.

[0344] 3. Patients with a history of allogeneic stem cell transplantation.

[0345] 4. Previous treatment with any chimeric antigen receptor T-cell (CAR-T) therapy.

[0346] 5. Ongoing systemic steroid treatment with more than 10 mg of prednisone or anti-inflammatory equivalent per day within 72 hours of study drug initiation.

[0347] 6. History of neurodegenerative conditions or CNS movement disorders. History of uncontrolled epilepsy (defined as any epilepsy within 12 months prior to study entry).

[0348] 7. Vaccination with a replication-competent vector within 28 days prior to the first study drug administration.

[0349] 8. Another malignancy other than FL in the past 5 years, except non-melanoma skin cancer or cervical carcinoma in situ that has undergone potentially curative therapy or any other tumor that is considered to have been effectively treated with clear local control and curative intent.

[0350] 9. Evidence of significant concurrent illness or medical conditions that may interfere with the conduct of the study or place the patient 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) and / or significant pulmonary disease (e.g., obstructive pulmonary disease and history of symptomatic bronchospasm).

[0351] 10. Cardiac ejection fraction <40% as determined by echocardiography or multiple depth acquisition (MUGA) scan.

[0352] 11. Any infection requiring hospitalization or treatment with IV anti-infectives within 2 weeks of the first administration of study drug.

[0353] 12. Uncontrolled infection with human immunodeficiency virus (HIV), hepatitis B, or hepatitis C; or other uncontrolled infection, except for the following: (a) patients with HIV whose infection is controlled (undetectable viral load spontaneously or on a stable antiviral regimen and CD4 count exceeds 350 cells / μL); (b) patients with hepatitis B (HepBsAg+) whose infection is controlled (serum hepatitis B virus DNA polymerase chain reaction [PCR] is below the limit of detection and receiving antiviral therapy for hepatitis B); (c) patients who are hepatitis C virus antibody-positive (HCVAb+) whose infection is controlled (undetectable HCV RNA by PCR either spontaneously or in response to a previous successful course of anti-HCV therapy).

[0354] 13. History of severe allergic reactions attributed to compounds with chemical or biological components similar to those of the study drug or excipients. For this purpose, severe allergic reactions are defined as anaphylaxis requiring hospitalization and / or treatment with epinephrine.

[0355] 14. Known hypersensitivity to both allopurinol and rasburicase.

[0356] 15. Members of the clinical site research team or his / her immediate family, unless approved in advance by the Sponsor.

[0357] 16. Women with a positive serum beta-hCG pregnancy test at the screening visit. If positive, pregnancy must be excluded by ultrasound for the patient to qualify.

[0358] 17. Patients who are institutionalized pursuant to an order issued by a judicial or administrative authority.

[0359] 18. Pregnant or breastfeeding women.

[0360] 19. Women of childbearing potential*, or males who are unwilling to use highly effective contraception before the initial dose / start of the first treatment, during the study, and for at least 6 months after the last dose. Highly effective contraception includes: (a) stable use of combined (estrogen and progestin) hormonal contraception (oral, vaginal, transdermal) or progestin-only hormonal contraception (oral, injectable, implantable) and suppression of ovulation for 2 or more menstrual cycles prior to screening; (b) intrauterine device (IUD); intrauterine hormone-releasing system (IUS); (c) bilateral tubal ligation; (d) vasectomized partner; (e) and / or sexual abstinence.

[0361] Postmenopausal women must have had no menstruation for at least 12 months to not be considered of childbearing potential. Women with a documented hysterectomy or tubal ligation do not need pregnancy testing or contraception.

[0362] Sexual abstinence is considered a highly effective method only if it is defined as abstention from heterosexual intercourse during the entire risk period associated with the study drug. The reliability of sexual abstinence needs to be assessed with respect to the duration of the clinical trial and the subjects' preferences and usual lifestyle.

[0363] Periodic abstinence (calendar, ovulatory, postovulatory methods), withdrawal (coitus interruptus), spermicides alone, and the lactational amenorrhea method (LAM) are unacceptable methods of contraception. Female and male condoms should not be used together.

[0364] treat: REGN1979 will be administered by IV infusion at an initial dose of 1 mg during Week 1, an intermediate dose of 20 mg during Week 2, and a nominal dose of 80 mg or 160 mg during subsequent administrations. For the initial dose, intermediate dose, and first nominal dose (primary, secondary, and tertiary doses, respectively), treatment will be divided into 2 separate infusions, each lasting 4 hours on each of 2 days (e.g., Week 1, Day 1 and Week 1, Day 2), preferably consecutive but no more than 3 days apart. Subsequent treatments (maximum weekly dose; e.g., 320 mg) may be administered as a single infusion or as 2 separate infusions and may be administered over 1 to 4 hours depending on tolerability. Study treatment consists of 12 QW administrations followed by Q2W dosing for 86 weeks, for a total of 98 weeks of study drug administration.

[0365] Endpoints:The primary endpoint of this study was ORR from the first dose until 194 weeks after the first dose in patients whose FL had relapsed or was refractory to at least 2 prior lines of systemic therapy (including anti-CD20 antibodies and alkylating agents), as measured by the Lugano classification for responses in malignant lymphomas (Cheson, 2014) and according to independent central review. Secondary endpoints were: (1) ORR from the first dose until 194 weeks after the first dose according to the Lugano classification as assessed by local investigator review; (2) CR rate from the first dose until 194 weeks after the first dose according to the Lugano classification as assessed by independent central review and local investigator review; (3) PFS from the first dose until 194 weeks after the first dose according to the Lugano classification as assessed by independent central review and local investigator review; (4) OS from the first dose until 194 weeks after the first dose; (5) OS from the first dose until 194 weeks after the first dose as assessed by independent central review and local investigator review. The following items were used to determine the clinical response to the regimen: (1) DOR according to the Lugano classification from the first dose until 194 weeks after the first dose as assessed by independent central review and local investigator evaluation; (2) DCR according to the Lugano classification from the first dose until 194 weeks after the first dose as assessed by independent central review and local investigator evaluation; (3) DDC according to the Lugano classification from the first dose until 194 weeks after the first dose as assessed by independent central review and local investigator evaluation; (4) incidence and severity of treatment-emergent adverse events (TEAEs) from the first dose until 194 weeks after the first dose; and (5) change in patient-reported outcome scores from the first dose until 194 weeks after the first dose as measured by the validated tools EORTC QLQ-C30 and EQ-5D-3L.

[0366] Procedure and Assessment: For all patients, computed tomography (CT) or magnetic resonance imaging (MRI) and 18The disease will be evaluated radiologically by F-fluorodeoxyglucose-positron emission tomography (FDG-PET) procedure. Tumor response according to the Lugano classification criteria will be determined by independent central radiology review. Bone marrow puncture, bone marrow biopsy, lymph node and / or tumor biopsy will be performed, and the samples will be evaluated histologically and can be used for other studies, including immunohistochemistry. Safety will be evaluated by evaluation of vital signs, physical examination, Eastern Cooperative Oncology Group (ECOG) performance status, electrocardiogram (ECG), AE incidence and concomitant medication reports. Laboratory evaluation includes differences in complete blood counts, blood chemistry values, serum immunoglobulin G (IgG), serum pregnancy test (if relevant), ferritin and C-reactive protein (CRP). Blood samples for PK and anti-drug antibody (ADA) evaluation will be collected. Peripheral blood samples will be collected to evaluate changes in biomarkers (e.g., cytokine production, serum levels of proinflammatory cytokines, and changes in lymphocyte subsets and activation states). Additionally, these samples will allow for tumor or somatic genetic analysis of variants that may influence the clinical course of the disease or modulate side effects of treatment.Quality of life assessments will be performed using the self-administered EORTC QLQ-C30 and EQ-5D-3L questionnaires.

[0367] Statistics Program: This study is designed to evaluate the efficacy and safety of REGN1979 for patients whose FL has relapsed or is refractory to at least 2 previous lines of systemic therapy. Analysis of the primary efficacy endpoint will be performed after all patients have completed the 28-week study treatment period and have been evaluated for tumor response or have withdrawn from the study. The primary endpoint of the ORR, adjusted for sample size, uses a single-stage exact binomial design. The two-sided 95% confidence interval for the observed ORR is calculated based on a sample size of 100. For 100 patients, if the observed ORR is at least 60%, 66%, 70%, and 75%, the lower limit of the 95% CI will exclude an ORR of 49%, 55%, 60%, and 65%, respectively; that is, the ORR is significantly different from 49%, 55%, 60%, and 65% as shown in Table 10 below.

[0368] Table 10. Two-sided 95% exact confidence intervals for observed ORR given a sample size of 100 patients

[0369]

[0370]

[0371] For a sample size of 100 patients, if the true treatment effect of REGN1979 is 64%, 70%, 75%, or 80%, the probability of observing the lower limit of the 95% CI is 82%, 83%, 89%, or 91% to exclude 49%, 55%, 60%, or 65%, respectively. The sample size will be further increased by 10% to account for patients who drop out of the study prematurely. Therefore, the total sample size will be 112 patients.

[0372] Statistical methods: Demographics and baseline characteristics will be summarized descriptively. The primary efficacy endpoint is the ORR according to the Lugano classification based on independent central review. ORR will be summarized with a bilateral 95% confidence interval. Patients whose best overall response cannot be evaluated will be considered non-responders. According to the Lugano classification, the secondary efficacy endpoints of the ORR as determined by investigator review, the CR rate and DCR by local investigator evaluation and independent central review according to the Lugano classification will be summarized with bilateral 95% confidence intervals. Other secondary efficacy endpoints (including DOR, DDC, PFS and OS) will be summarized by median and its 95% confidence interval using the Kaplan-Meier method according to the Lugano classification. Disease control rate will be summarized with bilateral confidence intervals. Quality of life measured by the validation tools EORTC QLQ-C30 and EQ-5D-3L will be summarized by descriptive statistics. Safety observations and measurements including drug exposure, AEs, laboratory data, vital signs and ECOG performance status will be summarized and presented in tables and lists.

[0373] Interim analysis: The interim analysis will be conducted after the first 50 patients have completed tumor assessments at 28 weeks or have withdrawn from the study earlier. ORRs and associated 95% confidence intervals will be summarized. Because the primary objective of this interim analysis is to make a point estimate of the ORR and characterize the precision of the point estimate, there are no hypothesis tests associated with this interim analysis. Therefore, type I error adjustment is not applicable for this planned interim analysis. Two-sided 95% confidence intervals will also be presented for other efficacy endpoints.

[0374] Additional efficacy studies of REGN1979 may be included in this or additional studies, including (a) patients with follicular lymphoma (Grade 1-3a) who have received third-line or higher (3L+) therapy, (b) patients with follicular lymphoma (Grade 1-3a) who have received second-line or higher (2L+) therapy and are eligible for full-dose chemotherapy, (c) patients with follicular lymphoma (Grade 1-3a) who have received 2L+ therapy and are not eligible for full-dose chemotherapy, (d) patients with follicular lymphoma (Grade 1-3a) who have not been previously treated and are eligible for full-dose chemoimmunotherapy, (e) patients with follicular lymphoma who have not been previously treated and are not eligible for full-dose chemoimmunotherapy. (f) patients with follicular lymphoma (grade 1-3a) who are eligible for full-dose chemoimmunotherapy with first-line (1L) therapy to standard of care, (g) patients with follicular lymphoma (grade 1-3a) who are not eligible for full-dose chemoimmunotherapy with 1L therapy to standard of care, (h) patients with follicular lymphoma (grade 1-3a) who are eligible for full-dose chemoimmunotherapy with 2L+ therapy to standard of care, (i) patients with follicular lymphoma (grade 1-3a) who are not eligible for full-dose chemoimmunotherapy with 2L+ therapy to standard of care, and / or (j) patients with follicular lymphoma in combination with standard of care.

[0375] Additional efficacy studies of REGN1979 may be included in this study or in additional studies, including (a) patients with diffuse large B-cell lymphoma (DLBCL) who are starting de novo or switching to 3L+ therapy; (b) patients with DLBCL after failure of CAR-T therapy; (c) patients with DLBCL who are eligible for autologous HSCT (hematopoietic stem cell transplantation) on 2L+ therapy; (d) patients with DLBCL who are not eligible for HSCT on 2L+ therapy; (e) patients with DLBCL who are previously untreated and have poor molecular prognostic factors (non-germinal center B, double hits, or triple hits) and are eligible for full-dose chemoimmunotherapy; (f) patients with DLBCL who are previously untreated and have poor molecular prognostic factors (non-germinal center B , double hit or triple hit) and are not suitable for full-dose chemoimmunotherapy; (g) patients with DLBCL who are CAR-T-naive; (h) patients with DLBCL who have a maximum weekly dose of 320 mg; (i) patients with DLBCL in combination with standard of care; (j) patients with DLBCL who have full-dose chemoimmunotherapy with 1L therapy to standard of care; (k) patients with DLBCL who are not suitable for full-dose chemoimmunotherapy with 1L therapy to standard of care; (1) patients with DLBCL who are eligible for autologous HSCT with 2L+ therapy to standard of care and / or (m) patients with DLBCL who are not eligible for autologous HSCT with 2L+ therapy to standard of care.

[0376] Additional efficacy studies of REGN1979 may be included in this study or in additional studies, including (a) patients with mantle cell lymphoma (MCL) after failure of a BTK inhibitor on 2L+ therapy, (b) patients with marginal zone lymphoma (MZL) on 2L+ therapy, and / or (c) patients with lymphoblastic lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma or other B-NHL subtypes on 2L+ therapy.

[0377] Additional efficacy studies of REGN1979 may be included in this study or in additional studies, including (a) patients with CD20+ B-cell malignancies who have received a single dose of rituximab one day before the first dose of REGN1979. In this rituximab lead-in cohort and expansion only, a single dose of rituximab (375 mg / m2) will be administered one day before the first dose of REGN1979 [i.e., on study day (-1)]. 2). REGN1979 will start on the first day of week 1, and the treatment period of REGN1979 will be 9 months. Patients will be treated with up to 24 doses of REGN1979: 4 weekly doses during the 4-week induction period, then an additional 8 weekly doses, and 12 doses are administered Q2W during the 6-month maintenance period. In the first part of this rituximab introduction group, REGN1979 will be administered using an ascending dose of 80 mg. Once the optimal dose regimen is identified, an additional dose group with 6 patients with an ascending dose of 320 mg REGN1979 will be evaluated with the optimal dose regimen. The dose group with an ascending dose of REGN1979 between 80 mg and 320 mg can also be evaluated. Subsequently, an additional 24 patients will be evaluated with the optimal dose regimen and the optimal dose; together with the 6 patients treated with the optimal dose in the rituximab introduction group, a total of 30 patients will be reviewed for safety and tolerability.

[0378] In any combination study with standard of care, the combination could contain REGN1979 plus CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), ICE (ifosfamide, carboplatin, and etoposide), Gem-Ox (gemcitabine and oxaliplatin), lenalidomide, or lenalidomide plus rituximab.

[0379] The present invention is not limited to a certain scope by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. Use of a bispecific antibody in the manufacture of a medicament for treating refractory CD20+ B-cell diffuse large B-cell lymphoma that has failed previous CAR-T therapy, wherein the bispecific antibody is a bispecific anti-CD20 x anti-CD3 antibody comprising: a first antigen binding arm that binds to human CD3 and comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 consisting of the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and the LCVR comprises complementarity determining regions LCDR1, LCDR2 and LCDR3 consisting of the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively; and A second antigen binding arm, which binds to human CD20 and comprises a HCVR and a LCVR, wherein the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 consisting of the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, and the LCVR comprises complementarity determining regions LCDR1, LCDR2 and LCDR3 consisting of the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively.

2. The use according to claim 1, wherein the HCVR and LCVR of the first antigen-binding arm that binds to human CD3 consist of the amino acid sequences of SEQ ID NOs: 14 and 15, respectively, and the HCVR and LCVR of the second antigen-binding arm that binds to human CD20 consist of the amino acid sequences of SEQ ID NOs: 13 and 15, respectively.

3. The use according to claim 2, wherein the bispecific anti-CD20 x anti-CD3 antibody comprises a first heavy chain consisting of the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence comprising residues 1-448 of SEQ ID NO: 11, a second heavy chain consisting of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence comprising residues 1-452 of SEQ ID NO: 10, and a common light chain consisting of the amino acid sequence of SEQ ID NO:

12.

4. The use according to any one of claims 1 to 3, wherein the medicament is administered to the subject in combination with a steroid, an antihistamine, acetaminophen, a nonsteroidal anti-inflammatory drug (NSAID), an IL-6 antagonist or an IL-6R antagonist.

5. The use according to any one of claims 1 to 3, wherein the medicament is administered to a subject in combination with a second therapeutic agent.

6. The use according to claim 5, wherein the second therapeutic agent (a) comprising at least one of the following: rituximab, obinutuzumab, cyclophophamide, doxorubicin, vincristine, prednisone, prednisolone, bendamustine, lenalidomide, chlorambucil, ibritumomab tiuxetan, idelalisib, copanlisib, duvelisib, etoposide, methylprednisolone, cytarabine, cisplatin, mesna, ifosfamide, mitoxantrone and procarbazine; (b) a combination comprising cyclophosphamide, doxorubicin, vincristine, and prednisone; (c) a combination comprising ifosfamide, cisplatin and etoposide; (d) a combination comprising gemcitabine and oxaliplatin; (e) a combination comprising lenalidomide and rituximab; or (d) is lenalidomide.

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