Dosing strategy that mitigates cytokine release syndrome for CD3 / c20 bispecific antibodies

Abstract

To provide a pharmaceutical composition comprising therapeutic proteins (e.g., T cell-activating bispecific antibodies) that mitigate cytokine release syndrome and infusion-related reaction.SOLUTION: Provided is a pharmaceutical composition comprising a bispecific antibody for use in treatment of refractory CD20+B-cell diffuse large B-cell lymphoma that is resistant to CAR-T therapy. Therein the bispecific antibody is a bispecific anti-CD20×anti-CD3 antibody, comprising: a first antigen-binding arm that binds to human CD3 and comprises a heavy chain variable region (HCVR) that comprises a complementarity determining region each comprising a specific amino acid sequence and a light chain variable region (LCVR) that comprises a complementarity determining region each comprising a specific amino acid sequence; and a second antigen-binding arm that binds to human CD20 and comprises a HCVR that comprises a complementarity determining region each comprising a specific amino acid sequence and a LCVR that comprises a complementarity determining region each comprising a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing Reference This application incorporates by reference a sequence listing, which was submitted in computer readable form as File 10496WO01-Sequence, created on Aug. 30, 2019, and contains 162,944 bytes.

[0002] The present invention is in the field of medicine and relates to administration strategies and 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 technology]

[0003] Cytokine release syndrome (CRS) is a systemic inflammatory response that can be triggered by various factors, including certain drugs. T cell-activated cancer immunotherapy is at particular risk for CRS, which is usually due to the on-target effect induced by the binding of bispecific antibody or chimeric antigen receptor (CAR) T cells to antigens and the subsequent activation of bystander immune cells and non-immune cells, such as endothelial cells. Activation of bystander cells results in the massive release of various cytokines. IL-6, IL-10, and interferon (IFN)-γ are among the central cytokines that have been consistently found to be elevated in the serum of CRS patients. In T cell-activated therapy against tumor cells, CRS is triggered by the massive release of IFN-γ by activated T cells or the tumor cells themselves. The secreted IFN-γ induces the activation of other immune cells, most importantly macrophages, which in turn overproduce additional cytokines, such as IL-6, TNF-α, and IL-10. In particular, IL-6 is involved in many of the major symptoms of CRS, including vascular leakage, activation of complement and coagulation cascades that induce disseminated intravascular coagulation, etc. In addition, IL-6 may contribute to cardiomyopathy by promoting myocardial dysfunction. Non-Patent Document 1. In some cases, symptoms related to CRS are called infusion-related reactions (IRR) if they occur within 6 hours after the start of the infusion, and CRS if they occur more than 6 hours after the start of the infusion.

[0004] Managing the toxicity of cancer immunotherapy is a challenging clinical problem. Mitigating CRS or IRR is a hallmark of administering certain therapies, such as CAR T cells and bispecific antibodies targeting T cells. Low-grade CRS is generally treated symptomatically with antihistamines, antipyretics, and fluids. Severe CRS may present with life-threatening adverse events that require prompt and aggressive treatment. Reduction of tumor burden, limiting the dose of administered therapy, and premedication with steroids have reduced the incidence of severe CRS, as has the use of anti-cytokine therapy. Tocilizumab, an anti-IL-6 antibody, has become the standard initial treatment for severe CRS in some circumstances. However, the use of dose restriction and treatments to minimize cytokine activity may negatively impact the efficacy of immunotherapy. Thus, there remains a need for alternative strategies to mitigate the potentially life-threatening effects of CRS without negatively impacting the therapeutic benefits of immunotherapy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Shimabukaro-Vornhagen et al.,Journal for Immunotherapy of Cancer,6:56,pp.1-14,2018 Summary of the Invention

[0006] In one aspect, the invention provides a method of administering a therapeutic protein to a subject in a dosing regimen to reduce adverse effects of cytokine release syndrome or infusion-related reactions, comprising: (i) administering a fraction of a primary dose (D1) of the therapeutic protein during week 1 of the dosing regimen, where the primary dose comprises 10 mg or less of therapeutic protein, a first dose fraction (F1D1) comprising 40%-60% of the total primary dose and is administered to the subject on day 1 of week 1, and a second dose fraction (F2D1) comprising the remaining 40%-60% of the total primary dose and is administered to the subject 12-96 hours after administration of F1D1; and (ii) administering a fraction of a therapeutic protein during week 2 of the dosing regimen, where the primary dose comprises 10 mg or less of therapeutic protein, a first dose fraction (F1D1) comprising 40%-60% of the total primary dose and is administered to the subject 12-96 hours after administration of F1D1. (iii) administering to the subject a fraction of a secondary dose (D2) of the therapeutic protein, wherein the secondary dose is less than or equal to half of the maximum weekly dose of the therapeutic protein, a first dose fraction (F1D2) comprising 40-60% of the total secondary dose and a second dose fraction (F2D2) comprising the remaining 40%-60% of the total secondary dose, F2D2 being administered to the subject 12-96 hours after administration of F1D2 during the second week of the dosing regimen; and (iii) administering to the subject the maximum weekly dose of the therapeutic protein as a single dose during subsequent weeks of the dosing regimen.

[0007] Optionally, F2D1 is administered to the subject 24-96 hours after administration of F1D1. Optionally, F2D1 is administered to the subject 18-72 hours after administration of F1D1. Optionally, F2D2 is administered to the subject 24-96 hours after administration of F1D2. Optionally, F2D2 is administered to the subject 18-72 hours after administration of F1D2. Optionally, the subsequent week is week 3 of the dosing regimen. Optionally, the subsequent week is week 4 of the dosing regimen. Optionally, the subsequent week is week 14 of the dosing regimen. Optionally, the subsequent week is any one of weeks 4 through 36 of the dosing regimen.

[0008] In some embodiments, the method further comprises: (i) administering a fraction of a tertiary dose (D3) of a therapeutic protein during week three of the dosing regimen, where the tertiary dose is greater than or equal to half of the maximum weekly dose of the therapeutic protein and less than or equal to the maximum weekly dose of the therapeutic protein, a first dose fraction (F1D3) comprising 40%-60% of the total tertiary dose and a second dose fraction (F2D3) comprising the remaining 40%-60% of the total tertiary dose, where F2D3 is administered to the subject 12-96 hours after administration of F1D3 during week three of the dosing regimen; and (ii) administering the maximum weekly dose of the therapeutic protein as a single dose to the subject during subsequent weeks of the dosing regimen.

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

[0010] Optionally, the subsequent week is week 4 of the dosing regimen. Optionally, the subsequent week is week 14 of the dosing regimen. Optionally, the subsequent week is any one of weeks 4 through 36 of the dosing regimen. In various embodiments, the tertiary dose is administered as a single dose in weeks 4 through 12 of the dosing regimen.

[0011] In one aspect, the invention provides a method of administering a therapeutic protein to a subject in a dosing regimen to reduce adverse effects of cytokine release syndrome or infusion-related reactions, comprising: (i) administering a fraction of a primary dose (D1) of the therapeutic protein in week 1 of the dosing regimen, where the primary dose comprises 10 mg or less of therapeutic protein, a first dose fraction (F1D1) comprising 40%-60% of the total primary dose and is administered to the subject on day 1 of week 1, and a second dose fraction (F2D1) comprising the remaining 40%-60% of the total primary dose and is administered to the subject 12-96 hours after administration of F1D1; and (ii) administering a therapeutic tannin in week 2 of the dosing regimen. (iii) administering to the subject a fraction of a secondary dose (D2) of a protein, wherein the secondary dose is equal to a maximum weekly dose of the therapeutic protein, a first dose fraction (F1D2) constituting 50% of the total secondary dose and a second dose fraction (F2D2) constituting 50% of the total secondary dose, F2D2 being administered 12-96 hours after administration of F1D2 during the second week of the dosing regimen; and (iv) administering to the subject the maximum weekly dose of the therapeutic protein as a single dose during subsequent weeks of the dosing regimen.

[0012] In one aspect, the invention provides a method of administering a therapeutic protein to a subject in a dosing regimen to reduce adverse effects of cytokine release syndrome or infusion-related reactions, comprising: (i) administering a fraction of a primary dose (D1) of the therapeutic protein in week 1 of the dosing regimen, where the primary dose comprises 10 mg or less of therapeutic protein, and D1 is administered to the subject in multiple dose fractions (e.g., F1D1, F2D1, F3D1, F4D1, F5D1) on subsequent days within the first week; and (i) administering a fraction of a primary dose (D1) of the therapeutic protein in week 1 of the dosing regimen, where the primary dose comprises 10 mg or less of therapeutic protein, and D1 is administered to the subject in multiple dose fractions (e.g., F1D1, F2D1, F3D1, F4D1, F5D1) on subsequent days within the first week. The methods include (i) administering a fraction of a secondary dose (D2) of a therapeutic protein in the second week of the dosing regimen, where the secondary dose is less than or equal to the maximum weekly dose of the therapeutic protein and is administered to the subject in multiple dose fractions (e.g., F1D2, F2D2, F3D2, F4D2, F5D2) on subsequent days within the second week; and (iii) administering a maximum weekly dose (MD) of a therapeutic protein to the subject in subsequent weeks of the dosing regimen, either as multiple fractions of the MD or as a single dose.

[0013] In some embodiments, the second dose fraction (F2) is administered to the subject 12-96 (e.g., 24-72) hours after administration of the first dose fraction (F1), optionally a third dose fraction (F3) is administered to the subject 24 hours or more after administration of the second dose fraction (F2), optionally a fourth dose fraction (F4) is administered to the subject 24 hours or more after administration of the third dose fraction (F3), and optionally a fifth dose fraction (F5) is administered to the subject 24 hours or more after administration of the fourth dose fraction (F4) during the first, second or third week of the dosing regimen.

[0014] In some embodiments, the disclosed methods further comprise administering one or more "maintenance" doses during the maintenance phase of the dosing regimen following completion of the weekly phase of the regimen. In some cases, each maintenance dose is administered 2, 3, or 4 weeks after the immediately preceding dose. In one embodiment, the maintenance dose is the maximum weekly dose of the therapeutic protein administered as a single dose.

[0015] In some cases, during the weekly phase of the dosing regimen, the maximum weekly dose (MD) of the therapeutic protein is administered to the subject as a single dose for 1-8 weeks, 1-12 weeks, or 1-16 weeks. In some cases, following completion of the weekly phase of the dosing regimen, the maximum weekly dose of the therapeutic protein is administered to the subject as a single dose once every 2 weeks during the maintenance phase of the dosing regimen. In some cases, following completion of the weekly phase of the dosing regimen, the maximum weekly dose of the therapeutic protein is administered to the subject as a single dose once every 3 weeks during the maintenance phase of the dosing regimen. In some cases, following completion of the weekly phase of the dosing regimen, the maximum weekly dose of the therapeutic protein is administered to the subject as a single dose once every 4 weeks during the maintenance phase of the dosing regimen. In some embodiments, the maintenance phase is up to 86 weeks in duration. In some embodiments, the maintenance phase is up to 87 weeks in duration. In some embodiments, the maintenance phase is up to 88 weeks in duration. 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, F1D1 constitutes 50% of the total primary dose and F2D1 constitutes 50% of the total primary dose. In various embodiments, F1D2 constitutes 50% of the total secondary dose and F2D2 constitutes 50% of the total secondary dose. In various embodiments, F1D3 constitutes 50% of the total tertiary dose and F2D3 constitutes 50% of the total tertiary dose.

[0018] In some cases, the maximum weekly dose (MD) of the therapeutic protein is between 5 mg and 320 mg. In various embodiments, the maximum weekly dose of the therapeutic protein is between 6 and 320 mg, 10 and 320 mg, 5 and 40 mg, 5 and 80 mg, 5 and 160 mg, 12 and 40 mg, 18 and 80 mg, 40 and 80 mg, 80 and 160 mg, 160 and 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. In some cases, the maintenance dose of the therapeutic protein is between 5 mg and 320 mg. In various embodiments, the maintenance dose of the therapeutic protein is between 6 mg and 320 mg, 10 mg and 320 mg, 5 mg and 40 mg, 5 mg and 80 mg, 5 mg and 160 mg, 12 mg and 40 mg, 18 mg and 80 mg, 40 mg and 80 mg, 80 mg and 160 mg, 160 mg and 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.

[0019] In some cases, each dose or dose fraction is administered to the subject over a period of 1 to 6 hours.

[0020] In some embodiments, the subject is 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'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 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, melanomaglioma, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, oncocytoma, transitional cell carcinoma, urothelial carcinoma, bladder adenocarcinoma, or bladder small cell carcinoma. In some embodiments, the subject is diagnosed with follicular lymphoma (FL). In some embodiments, the FL is grade 1-3a. In some embodiments, the subject is diagnosed with diffuse large B-cell lymphoma (DLBCL). In one embodiment, the subject is diagnosed with relapsed / refractory DLBCL. In some embodiments, the subject diagnosed with DLBCL has failed a previous CAR-T therapy. In some embodiments, the subject is diagnosed with mantle cell lymphoma (MCL). In some cases, the subject diagnosed with MCL has failed a previous Bruton's tyrosine kinase (BTK) inhibitor therapy. In some embodiments, the subject is diagnosed with marginal zone lymphoma (MZL).

[0021] In some cases, the subject is a human, a human adult, or a human child (under 18 years of age).

[0022] In various embodiments, the therapeutic protein is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a fully human antibody. In some embodiments, the antibody is a bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the bispecific antibody or antigen-binding fragment thereof. comprises a first antigen-binding arm that binds to a T cell antigen. Optionally, the T cell antigen is CD3. Optionally, 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-raf, HLA / k-ras, HLA / MAGE-A3, h Selected from the group consisting of TERT, 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 (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0023] In some embodiments, the tumor cell antigen is CD20. Optionally, the bispecific antibody is an anti-CD20 x anti-CD3 antibody. Optionally, the anti-CD20 x anti-CD3 antibody is REGN1979.

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

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

[0026] In some embodiments, the tumor cell antigen is MUC16. Optionally, the bispecific antibody is an anti-MUC16 x anti-CD3 antibody.

[0027] In some embodiments, the tumor cell antigen is STEAP2. Optionally, the bispecific antibody is an anti-STEAP2 x anti-CD3 antibody.

[0028] In various embodiments, the therapeutic protein is maintained at a serum concentration of about 2000 micrograms per liter (mcg / L) or greater after administration of the maximum weekly dose for the duration of the dosing regimen. In some cases, the therapeutic protein is maintained at a serum concentration of about 2600 mcg / L or greater after administration of the maximum weekly dose for the duration of the dosing regimen. In some embodiments, the therapeutic protein is maintained at a serum concentration of about 3700 mcg / L or greater after administration of the maximum weekly dose for the duration of the dosing regimen.

[0029] In some embodiments, the therapeutic protein is administered to the subject in combination with a second agent selected from a steroid, an antihistamine, acetaminophen, a nonsteroidal anti-inflammatory drug (NSAID), an IL-6 antagonist, or an IL-6R antagonist. In some embodiments, the steroid is dexamethasone. In some embodiments, the NSAID is indomethacin. In some embodiments, 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, administration of the second agent is omitted after the first administration of the maximum weekly dose during the dosing regimen. In other embodiments, the second agent is administered prior to administration of the therapeutic protein (e.g., about 1-3 hours prior to F1D1, F2D1, F1D2, F2D2, F1D3 and / or F2D3). In still other embodiments, the therapeutic protein is administered by infusion over a period of time, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours, or more.

[0030] 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, cyclophosphamide, doxorubicin, vincristine, prednisone, prednisolone, bendamustine, lenalidomide, chlorambucil, ibritumomab tiuxetan, idelalisib, copanlisib, duvelisib, etoposide, methylprednisolone, cytarabine, cisplatin, mesna, ifosfamide, mitoxantrone, and procarbazine.In some cases, the second therapeutic agent comprises a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone.In some cases, the second therapeutic agent comprises a combination of ifosfamide, cisplatin, and etoposide. In some cases, the second therapeutic agent comprises a combination of gemcitabine and oxaliplatin. In some cases, the second therapeutic agent comprises a combination of lenalidomide and rituximab. In some cases, the second therapeutic agent is lenalidomide.

[0031] In one aspect, the present invention includes a method of treating a B cell cancer in a subject, 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 described above or discussed herein, utilizing a dosing regimen to reduce the adverse effects of cytokine release syndrome or infusion-related reactions. In some embodiments, the subject has previously been treated with an anti-CD20 antibody therapy. In some embodiments, the subject has previously been treated with a 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.

[0032] 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 more. In any of the embodiments, any dose administered as a single dose may be administered within 1 hour.

[0033] In various embodiments, any of the features or components of any of the embodiments described above or discussed herein can be combined, and such combinations are encompassed within the scope of the present disclosure. Any particular value described above or discussed herein can be combined with another related value described above or discussed herein to recite a range in which those values ​​represent the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure. Therapeutic proteins for use in any of the methods discussed herein, or the use of therapeutic proteins in the manufacture of a medicament for use in any of the methods discussed herein, are also encompassed within the scope of the present disclosure.

[0034] Other embodiments will become apparent from review of the following detailed description. [Brief description of the drawings]

[0035] [Figure 1] 1 shows the incidence of CRS / IRR during the first 5 weeks of treatment with REGN1979 at various dose levels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Before describing the present invention, it is to be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value can vary by 1% or less from the recited value.For example, as used in the present invention, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0038] 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 herein are incorporated by reference in their entirety.

[0039] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multi-molecule T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acid sequence set forth in SEQ ID NO:1, human CD3-delta comprises the amino acid sequence set forth in SEQ ID NO:2, human CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO:3, and CD3-gamma comprises the amino acid sequence set forth in SEQ ID NO:4.

[0040] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless specifically identified as being from a non-human species. Thus, the term "CD3" refers to human CD3, unless specifically identified as being from a non-human species, e.g., "mouse CD3," "monkey CD3," etc.

[0041] An "antigen-binding domain that binds to CD3," an "antibody that binds to CD3," or an "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3 and / or cell surface expressed CD3. Soluble CD3 includes native CD3 protein, as well as recombinant CD3 protein variants that lack a transmembrane domain or are not associated with a cell membrane, such as monomeric and dimeric CD3 constructs.

[0042] The term "CD20" refers to a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. CD20 is considered a B cell tumor-associated antigen because it is expressed in 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. The human CD20 protein is represented by SEQ ID NO: 5.

[0043] An "antigen-binding domain that binds to CD20," "antibody that binds to CD20," or "anti-CD20 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize CD20.

[0044] As used herein, the term "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.

[0045] An "antigen binding domain that binds BCMA", "antibody that binds BCMA" or "anti-BCMA antibody" includes antibodies and antigen-binding fragments thereof that specifically recognise BCMA.

[0046] The term "PSMA" refers to the enzyme folate hydrolase 1 (PSMA). PSMA refers to prostate-specific membrane antigen (FOLH1), also known as prostate specific membrane antigen (PSMA). PSMA is an integral, non-shed 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.

[0047] 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.

[0048] The term "MUC16" refers to mucin 16. MUC16 is a single 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.

[0049] An "antigen-binding domain that binds MUC16," "antibody that binds MUC16," or "anti-MUC16 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize MUC16.

[0050] The term "STEAP2" refers to six-transmembrane epithelial antigen 2 of the prostate. STEAP2 is an integral six-transmembrane 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 STEAP2 gene located in human chromosomal region 7q21. The amino acid sequence of human STEAP2 is shown in SEQ ID NO:9.

[0051] An "antigen-binding domain that binds to STEAP2", "antibody that binds to STEAP2" or "anti-STEAP2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize STEAP2.

[0052] The term "therapeutic protein" includes any polypeptide, including antibodies and antigen-binding fragments thereof, as well as bispecific antibodies and antigen-binding fragments thereof, that are used to prevent, treat or ameliorate any condition, disease or disorder in a subject.

[0053] The term "antigen-binding molecule" includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies.

[0054] The term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD20, BCMA, PSMA, MUC16, STEAP2, or CD3). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules that consist of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or VVR). H The heavy chain constant region is made up of three domains: H 1. C H 2, and C H Each light chain comprises a light chain variable region (herein referred to as LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H Area and V L The regions can be 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 Each of the CDRs consists of three CDRs and four FRs arranged from amino terminus to carboxy 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 antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs. The term "antibody" includes "bispecific antibodies" unless otherwise specified.

[0055] The term "antibody" also includes antigen-binding fragments of complete antibody molecules. An "antigen-binding portion" of an antibody, an "antigen-binding fragment" of an antibody, and similar terms include naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from complete antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion techniques or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding the antibody variable domains and, optionally, the antibody constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated, for example, chemically or by using molecular biology techniques, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0056] The term "bispecific antigen-binding molecule" refers to a protein, polypeptide or molecular complex that comprises at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. Bispecific antigen-binding molecules include bispecific antibodies.

[0057] 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 mimicking the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody, or constrained FR3-CDR3-FR4 peptides. 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 other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment".

[0058] An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. H Domain is V L In the antigen-binding fragment linked to the V domain, H Domains and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer and the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H or V L It may include a domain.

[0059] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H1, (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 the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of the antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Or V LThe variable domain configurations and constant domain configurations may include homodimers or heterodimers (or other multimers) of any of the above listed variable domain configurations and constant domain configurations in non-covalent association with the domains (e.g., via disulfide bond(s)).

[0060] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in conjunction with the antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0061] The antibodies of the present invention may function 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 non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby resulting in the lysis of the target cells. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. 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 the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity. The antibody of the present disclosure can include a human IgG heavy chain. In various embodiments, the heavy chain can be of the IgG1, IgG2, IgG3 or IgG4 isotype.

[0062] In certain embodiments of the present invention, the antibody or bispecific antibody is a human antibody. The term "human antibody" is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation), for example in the CDRs, particularly CDR3. However, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, for example, a mouse, have been grafted onto human framework sequences.

[0063] The antibodies of the present invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibodies" is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means (described below), such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library (described below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including splicing of 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 are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), and thus the V and V regions of the recombinant antibodies are not subject to any mutations. H Area and V L The amino acid sequence of the region is H Array and V LThese are sequences that are derived from and related to the human antibody germline repertoire but may not naturally occur in vivo within the human antibody germline repertoire.

[0064] Human antibodies can exist in two forms related to hinge heterogeneity: in the first form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming molecules of approximately 75-80 kDa consisting of covalently linked light and heavy chains (half antibodies). These forms have proven extremely difficult to separate, even after affinity purification.

[0065] The frequency of occurrence of the second form in various intact IgG isotypes is due to, 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 a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for the identification of a second form that is highly specific to the hinge region, C H 2 area, or C H Antibodies with one or more mutations in three regions are included, which may be desirable, for example, to improve the yield of a desired antibody form in production.

[0066] The antibody of the present invention may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for the purposes of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0067] The antibodies discussed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibodies were derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residues are found only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or the mutated residues are found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. 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 antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the scope of the present invention.

[0068] The invention also includes 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 includes antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or less, 8 or less, 6 or less, 4 or less, any conservative amino acid substitutions relative to the HCVR, LCVR, and / or CDR amino acid sequences described herein, the anti-CD3 antibodies disclosed in WO2014 / 047231 or WO2017 / 053856, the bispecific anti-CD20 x anti-CD3 antibodies disclosed in WO2014 / 047231, the bispecific anti-CD20 x anti-CD3 antibodies disclosed in WO2017 / No. 023761, anti-MUC16 or anti-MUC16 x anti-CD3 antibodies disclosed in WO2018 / 067331, anti-STEAP2 or anti-STEAP2 x anti-CD3 antibodies disclosed in WO2018 / 058001, or anti-BCMA or anti-BCMA x anti-CD3 antibodies disclosed in US 62 / 700,596 (filed July 19, 2018), each of which is incorporated herein by reference.

[0069] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are produced by adjacent amino acids within a polypeptide chain. In certain circumstances, an epitope may comprise a saccharide, phosphoryl, or sulfonyl moiety on an antigen.

[0070] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), the nucleotide sequence is identical in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases as measured by any known sequence identity algorithm, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0071] When applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using a predefined gap weight. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other in conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic hydroxyl side chains: serine and threonine, (3) amide-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: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0072] Sequence similarity of polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provide alignment and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) (see above)). The sequences of the present invention can be compared to a database containing a large number of sequences from different organisms. Another preferred algorithm for comparing with a database is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0073] Dosing strategies and regimens By administering therapeutic proteins to patients for various treatments, dosing strategies have been created that provide dosing regimens that reduce the prevalence or severity, or both, of cytokine release syndrome (CRS) or infusion-related reactions (IRR). According to certain embodiments of the invention, these strategies include multiple administrations of therapeutic proteins or antigen-binding molecules (e.g., antibodies or bispecific antibodies) that can be administered to a subject over a defined time course to create a dosing regimen. The method according to this aspect of the invention includes sequentially administering multiple doses of a therapeutic protein of the invention to a subject. As used herein, "sequentially administering" means that each dose of a therapeutic protein is administered to a subject at different time points, e.g., different days separated by a predefined interval (e.g., hours, days, weeks, or months). The invention includes methods that include sequentially administering to a patient a split primary dose of a therapeutic protein, followed by a split secondary dose of a therapeutic protein, optionally followed by a split tertiary dose of a therapeutic protein, followed by a single dose of the maximum weekly dose of a therapeutic protein. The present dosing regimen allows for higher doses of therapeutic protein that are desirable for enhancing therapeutic efficacy, but without the adverse effects associated with CRS or IRR. Without intending to be bound by a particular theory, the present dosing regimen provides priming of the immune response to administration of the therapeutic protein to minimize the incidence and severity of CRS and IRR during the early stages of the treatment regimen, thereby allowing for higher doses of therapeutic protein during the subsequent periods of the treatment regimen without significant adverse events associated with CRS or IRR.

[0074] An exemplary dosing regimen includes (i) administering a primary dose fraction of a therapeutic protein during week 1 (W1) of the dosing regimen, the primary dose comprising 1 mg or less of therapeutic protein, a first dose fraction (F1D1) constituting 50% of the primary dose and administered to the subject on day 1 of week 1, and a second dose fraction (F2D1) constituting 50% of the total primary dose and administered to the subject within 96 hours after administration of F1D1; and (ii) administering a secondary dose fraction of a therapeutic protein during week 2 (W2) of the dosing regimen. (iii) administering dose fractions, wherein the secondary dose is less than or equal to half of the maximum weekly dose of the therapeutic protein, a first dose fraction (F1D2) constituting 50% of the secondary dose and a second dose fraction (F2D2) constituting 50% of the secondary dose, wherein F1D2 and F2D2 are administered to the subject within 96 hours of each other during the second week of the dosing regimen; and (iii) in subsequent weeks (Ws) of the dosing regimen, administering the maximum weekly dose of the therapeutic protein to the subject as a single dose.

[0075] Another exemplary dosing regimen includes (i) administering a primary dose fraction of a therapeutic protein during week 1 (W1) of the dosing regimen, the primary dose comprising 1 mg or less of therapeutic protein, a first dose fraction (F1D1) constituting 50% of the primary dose and administered to the subject on day 1 of week 1, and a second dose fraction (F2D1) constituting 50% of the total primary dose and administered to the subject within 96 hours after administration of F1D1; and (ii) administering a primary dose fraction of a therapeutic protein during week 2 (W2) of the dosing regimen. (iii) administering a secondary dose fraction of a therapeutic protein in week 3 (W3) of the dosing regimen, wherein the secondary dose is less than or equal to half of the maximum weekly dose of the therapeutic protein, a first dose fraction (F1D2) comprising 50% of the secondary dose and a second dose fraction (F2D2) comprising 50% of the secondary dose, F1D2 and F2D2 being administered to the subject within 96 hours of each other during week 2 of the dosing regimen; and (iv) administering a tertiary dose fraction of a therapeutic protein in week 3 (W3) of the dosing regimen. (iv) administering to the subject the maximum weekly dose of the therapeutic protein as a single dose during subsequent weeks (Ws) of the dosing regimen, wherein the tertiary dose is greater than or equal to half of the maximum weekly dose of the therapeutic protein, and less than the maximum weekly dose of the therapeutic protein, wherein a first dose fraction (F1D3) constitutes 50% of the tertiary dose and a second dose fraction (F2D3) constitutes 50% of the tertiary dose, and wherein F1D3 and F2D3 are administered to the subject within 96 hours of each other during week 3 of the dosing regimen.

[0076] In various embodiments, the primary dose of a therapeutic protein can range from 0.1 mg to 10 mg or more. In some cases, the primary dose of a therapeutic protein is 0.5 mg to 10 mg, 1 to 10 mg, 2 to 5 mg, or 5 to 10 mg. In some cases, the primary dose of a 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 a therapeutic protein includes a range including any of the values ​​listed above as the upper or lower limit of the range (e.g., 1 to 5 mg).

[0077] In various embodiments, the maximum weekly dose of the therapeutic protein is between 5 mg and 320 mg. In some embodiments, the maximum weekly dose of the therapeutic protein is greater than 5 mg and up to 320 mg. In some cases, the maximum weekly dose of the therapeutic protein is between 6 and 320 mg, 10 and 320 mg, 5 and 40 mg, 5 and 80 mg, 5 and 160 mg, 12 and 40 mg, 18 and 80 mg, 40 and 80 mg, 80 and 160 mg, 160 and 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 a 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, 110, 112, 114, 116, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 5, 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 therapeutic protein includes a range including any of the values ​​listed above as the upper or lower limit of the range (e.g., 200-300 mg).

[0078] 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%, 10% of the maximum weekly dose of the therapeutic protein. In some cases, the secondary dose of the therapeutic protein comprises a percentage range of the maximum weekly dose (e.g., 35-50%), including any of the values ​​listed above as the upper or lower limits of the range.

[0079] 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 percentage range of the maximum weekly dose (e.g., 50-75%), including any of the values ​​set forth above as the upper or lower limits of the range. .

[0080] In various embodiments, the first and second dose fractions of the primary, secondary, and / or tertiary doses each comprise 50% of the dose. In some cases, the first and second dose fractions of the primary, secondary, and / or tertiary doses comprise different percentages of the total dose (totaling 100%). For example, the first dose fraction may comprise 45% of the dose and the second dose fraction may comprise 55% of the dose. Alternatively, the first dose fraction may comprise 55% of the dose and the second dose fraction may comprise 45% of the dose. In various embodiments, the first and second dose fractions may comprise 10% / 90%, 15% / 85%, 20% / 80%, 25% / 75%, 30% / 70%, 35% / 65%, 40% / 60%, 46% / 54%, 47% / 53%, 48% / 52%, 49% / 51%, or vice versa, of the total primary, secondary, or tertiary dose, respectively.

[0081] In various embodiments, the primary, secondary and / or tertiary dose (D1, D2 and / or D3) can be divided into two or more fractions. Various options for dividing a dose into two fractions have been discussed above. However, in some cases, the dose is divided into three, four or five fractions. For example, the primary dose can be divided into five fractions, each of which constitutes 20% of the total primary dose, and each dose fraction (F1D1, F2D1, F3D1, F4D1 and F5D1) can be administered to the subject for five consecutive days during the first week of the dosing regimen. In other cases, the percentage of the total dose (e.g., the primary dose) can vary between each dose fraction. For example, if the primary dose is divided into three fractions, the first dose fraction (F1D1) may include 30% of the total primary dose, the second dose fraction (F2D1) may include 30% of the total primary dose, and the third dose fraction (F3D1) may include the remaining 40% of the total primary dose. Other combinations of percentages and numbers of fractional doses that equal 100% of the total dose are expressly contemplated herein.

[0082] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 6 mg in the secondary dose, 3 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 12 mg in the tertiary dose, 6 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 12 mg in the maximum weekly dose.

[0083] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 9 mg in the secondary dose, 4.5 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 18 mg in the tertiary dose, 9 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 18 mg in the maximum weekly dose.

[0084] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 13.5 mg in the secondary dose, 6.75 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 27 mg in the tertiary dose, 13.5 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 27 mg in the maximum weekly dose.

[0085] In one exemplary embodiment of the dosing regimen, the therapeutic protein is administered in a primary dose of 1 mg, in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), at 500 mg. cg, 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 40 mg in the tertiary dose, 20 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 40 mg in the maximum weekly dose.

[0086] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 60 mg in the tertiary dose, 30 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 80 mg in the maximum weekly dose.

[0087] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 80 mg in the tertiary dose, 40 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 160 mg in the maximum weekly dose.

[0088] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 120 mg in the tertiary dose, 60 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 240 mg in the maximum weekly dose.

[0089] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 160 mg in the tertiary dose, 80 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), and 320 mg in the maximum weekly dose.

[0090] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose (D1), 500 mcg in each of the first dose fraction (F1D1) and second dose fraction (F2D2) of the primary dose, 3 mg in the secondary dose (D2), 1.5 mg each in the first dose fraction (F1D2) and second dose fraction (F2D2) of the secondary dose, and 3 mg at the maximum weekly dose.

[0091] In one exemplary embodiment of the dosing regimen, the therapeutic protein is present at 3 mg in the primary dose (D1), 1.5 mg in each of the first dose fraction (F1D1) and second dose fraction (F2D2) of the primary dose, 9 mg in the secondary dose (D2), 4.5 mg in each of the first dose fraction (F1D2) and second dose fraction (F2D2) of the secondary dose, and 9 mg in the maximum weekly dose.

[0092] In one exemplary embodiment of the dosing regimen, the therapeutic protein is present at 5 mg in the primary dose (D1), 2.5 mg in each of the first dose fraction (F1D1) and second dose fraction (F2D2) of the primary dose, 15 mg in the secondary dose (D2), 7.5 mg in each of the first dose fraction (F1D2) and second dose fraction (F2D2) of the secondary dose, and 15 mg at the maximum weekly dose.

[0093] In one exemplary embodiment of the dosing regimen, the therapeutic protein is present at 10 mg in the primary dose (D1), 5 mg in each of the first dose fraction (F1D1) and second dose fraction (F2D2) of the primary dose, 30 mg in the secondary dose (D2), 15 mg in each of the first dose fraction (F1D2) and second dose fraction (F2D2) of the secondary dose, and 30 mg in the maximum weekly dose.

[0094] 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.

[0095] 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 during 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 during the dosing regimen.

[0096] In various embodiments, subsequent weeks (Ws) of the dosing regimen are 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 (W18), 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).

[0097] In various embodiments, the second divided 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 divided dose.

[0098] In various embodiments, the maximum weekly dose of a therapeutic protein is administered to a subject as a single dose for 1-8 weeks, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more weeks during the weekly phase of the dosing regimen. In some cases, the maximum weekly dose of a therapeutic protein is administered to a subject as a single dose over a range of weeks (e.g., 1-12 weeks) including any of the values ​​listed above as the upper or lower limits of the range.

[0099] In various embodiments, the maximum weekly dose of a therapeutic protein is administered once every 2 weeks, once every 3 weeks, once every 4 weeks, or once every 5 weeks (maintenance phase) for up to 24 weeks, or 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 0, 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, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180 , 190, 200, 210, 220, 230, 240, 250, 260 or more weeks, which may be after completion of the weekly phase of the dosing regimen (i.e., either weekly administration of the maximum weekly dose or split administration of the first, second and (optionally) third doses). 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 over a range of weeks (e.g., 24-86 weeks), including any of the values ​​listed above as the upper or lower limits of the range.

[0100] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 80 mg in the tertiary dose, and 40 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), with a maximum weekly dose of 160 mg, with the tertiary dose being administered as a single dose once weekly (QW) (i.e., 80 mg) between weeks 4 and 12 of the dosing regimen, and the maximum weekly dose being administered as a single dose once every two weeks (Q2W) (i.e., 160 mg) from week 14 of the dosing regimen onwards.

[0101] In some cases, the above identified dosing regimens are for use in methods for treating aggressive lymphoma (eg, mantle cell lymphoma or marginal zone lymphoma).

[0102] In one exemplary embodiment of the dosing regimen, the therapeutic protein comprises 1 mg in the primary dose, 500 mcg in each of the first dose fraction (F1D1) and the second dose fraction (F2D1), 20 mg in the secondary dose, 10 mg in each of the first dose fraction (F1D2) and the second dose fraction (F2D2), 160 mg in the tertiary dose, and 80 mg in each of the first dose fraction (F1D3) and the second dose fraction (F2D3), with a maximum weekly dose of 320 mg, with the tertiary dose being administered as a single dose once weekly (QW) (i.e., 160 mg) between weeks 4 and 12 of the dosing regimen, and the maximum weekly dose being administered as a single dose once every two weeks (Q2W) (i.e., 320 mg) from week 14 onwards of the dosing regimen.

[0103] In some cases, the dosing regimen identified above is for use in a method for treating an aggressive lymphoma (e.g., mantle cell lymphoma or marginal zone lymphoma). In some cases, the dosing regimen identified above is for use in a method for treating a follicular lymphoma (e.g., grades 1-3a). In some cases, the dosing regimen identified above is for use in a method for treating a diffuse large B-cell lymphoma (including, for example, relapsed or refractory DLBCL in patients who have previously failed CAR-T therapy).

[0104] In various embodiments, each dose or sub-dose of a therapeutic protein is administered to a subject (e.g., via infusion) over a period of 1-4, 1-5, or 1-6 hours. In some cases, a dose or sub-dose is administered over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more hours. In some cases, each dose or sub-dose of a therapeutic protein is administered to a subject over a range of time (e.g., 1-8 hours) including any of the values ​​listed above as the upper or lower limits of the range. In various embodiments, each maximum weekly dose is administered as a single infusion.

[0105] In some embodiments of the dosing regimens discussed herein, a second agent is administered as a pretreatment or in combination with the therapeutic protein. In some cases, a steroid such as dexamethasone is administered to the patient as a pretreatment prior to administration of the first and second fractional doses, and optionally prior to the third fractional dose. In some embodiments, dexamethasone is administered to the patient about 1-3 hours prior to the first dose fraction (F1D1). In an embodiment, the dexamethasone dose is administered by intravenous infusion. Optionally, a cytokine antagonist, such as an anti-IL-6 antibody or an anti-IL-6R antibody, is administered in combination with the therapeutic protein during administration of the first, second, and optionally third doses. Optionally, an anti-CD20 monospecific antibody (e.g., rituximab) is administered to the patient as a pretreatment before administration of the first and optionally second fractional doses, and optionally before the third fractional dose. These pretreatments or co-administrations of the second agent are, in one embodiment, discontinued by a single dose of the maximum weekly dose, for example, starting in the third, fourth, fifth, or sixth week of the dosing schedule, so as not to artificially attenuate cytokine activity and thereby prevent the therapeutic effect of the therapeutic protein.

[0106] The terms "primary dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the antigen-binding molecules of the present invention. Thus, a "primary dose" is a dose administered at the beginning of a treatment regimen, a "secondary dose" is a dose administered after the primary dose (including all dose fractions of the primary dose), and a "tertiary dose" is a dose administered after the secondary dose (including all dose fractions of the secondary dose). The primary, secondary, and tertiary doses (or their dose fractions) may contain amounts of therapeutic protein as discussed herein. In some cases, the "primary dose" and the "secondary dose", as well as the optional "tertiary dose", may be referred to as "loading doses", and the maximum weekly dose thereafter may be referred to as "maintenance doses". In some cases, the "primary dose" may be referred to as "initial dose". In some cases, the "secondary dose" may be referred to as "intermediate dose". In some cases, the "tertiary dose" may be referred to as "step-up dose".

[0107] The phrase "the immediately preceding dose" refers to a dose of an antigen-binding molecule that is administered to a patient prior to administration of the next dose in the sequence, without any intervening doses, in a multiple dose series.

[0108] antigen binding molecule Antibodies for use in connection with the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide, or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, 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 invention can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second or additional binding specificity.

[0109] The invention includes antibodies having the HCVR, LCVR and / or CDR amino acid sequences of the antibodies described herein, the anti-CD3 antibodies disclosed in WO2014 / 047231 or WO2017 / 053856, the bispecific anti-CD20 x anti-CD3 antibodies disclosed in WO2014 / 047231, the anti-PSMA or anti-PSMA x anti-CD3 antibodies disclosed in WO2017 / 023761, the anti-MUC16 or anti-MUC16 x anti-CD3 antibodies disclosed in WO2018 / 067331, the anti-STEAP2 or anti-STEAP2 x anti-CD3 antibodies disclosed in WO2018 / 058001, or the anti-BCMA or anti-BCMA x anti-CD3 antibodies disclosed in US 62 / 700,596 (filed July 19, 2018), each of which is incorporated herein by reference.

[0110] The use of expressions such as "anti-CD3 antibody," "anti-CD20 antibody," "anti-PSMA antibody," anti-MUC16 antibody, "anti-STEAP2 antibody," "anti-BCMA antibody," and the like herein refers to a single The present invention is intended to include both specific antibodies as well as bispecific antibodies that contain respective antigen-binding arms (e.g., CD3). Thus, the present invention includes bispecific antibodies in which one immunoglobulin arm binds to human CD3 and the other immunoglobulin arm is specific for, for example, human CD20, human PSMA, human MUC16, human STEAP2, or human BCMA.

[0111] In certain embodiments, the CD3 binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3 binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3 binding arm binds weakly to human CD3 and induces tumor-associated antigen expressing cell killing in the context of the bispecific or multispecific antibody. In other embodiments, the CD3 binding arm binds or associates weakly with human and cynomolgus (monkey) CD3, but the binding interaction is undetectable by in vitro assays known in the art. In certain embodiments, the bispecific antibody or antigen-binding fragment for use in the present invention comprises an antigen-binding arm that binds to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2, which induces T cell activation.

[0112] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "A1", and the CDRs of the second antigen-binding domain may be designated with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0113] In one embodiment, the therapeutic protein is a bispecific anti-CD20 x anti-CD3 antibody known as REGN1979. REGN1979 comprises an anti-CD20 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, an anti-CD3 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, and a common light chain (corresponding to both the anti-CD20 and anti-CD3 binding arms) comprising the amino acid sequence of SEQ ID NO: 12. In some cases, the mature form of the antibody may not include the C-terminal lysine residues of SEQ ID NOs: 10 and 11. Thus, in some cases, the anti-CD20 binding arm of REGN1979 comprises a heavy chain comprising residues 1-452 of SEQ ID NO: 10, and the anti-CD3 binding arm of REGN1979 comprises a heavy chain comprising residues 1-448 of SEQ ID NO: 11. REGN1979 also comprises HCVR, LCVR, and CDR sequences as set forth 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 HCDR1-HCDR2-HCDR3 domains of anti-CD20 correspond to SEQ ID NOs: 16-17-18, respectively. The HCDR1-HCDR2-HCDR3 domains of anti-CD3 correspond to SEQ ID NOs: 19-20-21, respectively. The common LCDR1-LCDR2-LCDR3 domains correspond to SEQ ID NOs: 22-23-24, respectively.

[0114] The bispecific antigen-binding molecules discussed above or herein may be bispecific antibodies. In some cases, the bispecific antibodies comprise a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is of isotype IgG1. In some cases, the human IgG heavy chain constant region is of isotype IgG4. In various embodiments, the bispecific antibodies comprise a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype. In some cases, the bispecific antigen-binding molecules of the present invention may be bispecific antibodies. It includes one of the formats discussed in RFC 212,2017.

[0115] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be linked to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, a multimerization domain can be linked to the immunoglobulin C H A non-limiting example of a multimerization component is a polypeptide comprising the Fc portion of an immunoglobulin (C H 2-C H 3 domains), for example, the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotypes within each isotype group.

[0116] The bispecific antigen-binding molecules of the present invention typically comprise two multimerization domains, e.g., two Fc domains, each part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0117] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length that contains at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0118] Any bispecific antibody format or technology can be used to generate the bispecific antigen-binding molecules of the invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to generate a bispecific antigen-binding molecule. Certain exemplary bispecific formats that can be used in the context of the present invention include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knob-into-hole, common light chain (e.g., common light chain with knob-into-hole), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2 These include, but are not limited to, bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0119] In the context of the bispecific antigen-binding molecules of the present invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain with modified binding interactions (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2 or C H The modification is in the 3 region, and this modification allows the protein to function in an acidic environment (e.g., in an endosome with a pH range of about 5.5 to about 6.0). and the affinity of the Fc domain for FcRn is increased. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., 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 a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or 308 modification (e.g., 308F or 308P).

[0120] The present invention is the first C H 3 domain and second Ig C H The present invention also includes a bispecific antigen-binding molecule comprising three domains, a first and a second IgC HThe three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking that amino acid difference. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains a mutation that reduces or abolishes Protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R according to EU numbering). H 3 may further include a Y96F modification (according to IMGT, Y436F by EU). See, e.g., U.S. Patent No. 8,586,713. H Further modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I by EU), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I by IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by IMGT, EU).

[0121] In certain embodiments, the Fc domain can be a chimera that combines Fc sequences from more than one immunoglobulin isotype. For example, the chimeric Fc domain can be a human IgG1, human IgG2, or human IgG4 C. H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2, or human IgG4. HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also comprise a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein is a chimeric Fc domain that comprises, from the N-terminus to the C-terminus, an "upper hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region, combined with a "lower hinge" sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region. H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, from the N-terminus to the C-terminus, [IgG1 C H These and other examples of chimeric Fc domains that may be included in any of the antigen binding molecules of the invention are described in U.S. Publication No. 2014 / 0243504, published August 28, 2014, which is incorporated herein in its entirety. These general structural arrangements are Chimeric Fc domains and variants thereof can have altered Fc receptor binding, which in turn affects Fc effector function.

[0122] Binding properties of antigen-binding molecules The term "binding" in reference to the binding of either an antibody, immunoglobulin, antibody binding fragment, or Fc-containing protein to a given antigen, such as, for example, a cell surface protein or fragment thereof, typically refers to an interaction or association between at least two entities or molecular structures, such as an antibody-antigen interaction.

[0123] For example, binding affinities are typically around 10 as determined by surface plasmon resonance (SPR) techniques, e.g., on a BIAcore 3000 instrument, using an antigen as the ligand and an antibody, Ig, antibody binding fragment, or Fc-containing protein as the analyte (or antiligand). -7M or less, for example, about 10 -8 M or less, for example, about 10 -9 K below M D Cell-based binding strategies such as fluorescence-activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlate well with other methods such as radioligand competitive 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).

[0124] Thus, an antibody or antigen-binding protein of the invention has a K that is at least 10-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). D According to the present invention, the antibody binds to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a K value that is 10-fold lower than that of a non-specific antigen. D Although an antibody affinity corresponding to the value can be considered as undetectable binding, such an antibody can be paired with a second antigen-binding arm to produce a bispecific antibody of the invention.

[0125] "K D The term "M" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the value, the higher, i.e., the stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, i.e., a smaller K D Conversely, the term "lower affinity" or "weaker affinity" refers to a lower ability to form an interaction, i.e., a larger K DIn some situations, the higher binding affinity (or K) of a particular molecule (e.g., an antibody) to an interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., an antibody) to another interaction partner molecule (e.g., antigen Y) can be used to refer to a higher binding affinity (or K D ) is larger than K D The smaller the K value (lower, or weaker, affinity), the D (higher, or stronger, affinity), e.g., 5-fold or 10-fold higher binding affinity, as the case may be.

[0126] "k d The term "(sec-1 or 1 / sec) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. Its value is k off Also called value.

[0127] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0128] "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody binding fragment. The association equilibrium constant is k a k d It is obtained by dividing by.

[0129] "EC50" or "EC 50 The term "half maximal effective concentration" refers to the concentration of an antibody that induces a response halfway between the baseline and maximum after a specific exposure time. EC 50 The EC essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC 50The EC value is equal to the concentration of an antibody of the invention that confers half-maximal binding to cells expressing, for example, CD3 or a tumor-associated antigen (e.g., CD20), as determined, for example, by a FACS binding assay. Thus, reduced or weak binding is an EC 50 An increase in the median effective concentration (50%) is observed.

[0130] In one embodiment, the reduction in binding is an EC2 concentration that allows half-maximal binding to target cells. 50 It can be defined as an increase in antibody concentration.

[0131] In another embodiment, EC 50 The values ​​represent the concentration of the antibody of the invention that induces half-maximal depletion of target cells by T cell cytotoxicity. Thus, an increase in cytotoxicity (e.g., T cell-mediated tumor cell killing) is associated with an increase in EC 50 Or this is observed as a decrease in the median effective concentration value.

[0132] Sequence variants The antibodies and bispecific antigen-binding molecules herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention may contain an antigen-binding domain derived from any of the exemplary amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H Domain and / or V LAll framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residues are found only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or the mutated residues are found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules containing one or more antigen-binding domains obtained in this general manner are encompassed within the scope of the present invention.

[0133] pH dependent binding The present invention includes antibodies and bispecific antigen-binding molecules with pH-dependent binding properties. For example, the antibodies of the present invention may exhibit reduced binding to tumor antigens, such as CD20, at acidic pH compared to neutral pH. Alternatively, the antibodies of the present invention may exhibit enhanced binding to tumor antigens, such as CD20, at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​less than about 6.2, such as about 6.0, 5.95, 5, 9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. The term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0134] In some cases, "decreased binding ... at acidic pH compared to neutral pH" refers to the K of an antibody that binds to its antigen at neutral pH. D K value for an antibody that binds to its antigen at acidic pH D For example, an antibody or antigen-binding fragment thereof may have 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 CD20 at acidic pH compared to neutral pH" if it exhibits a ratio of 0.01 to 0.01. In certain exemplary embodiments, the acidic / neutral K D The ratio can 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.

[0135] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH compared to neutral pH. Furthermore, 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 in the antigen-binding domain (e.g., in the CDRs) with histidine residues, an antibody can be obtained that has reduced antigen binding at acidic pH compared to neutral pH.

[0136] Antibodies containing Fc variants According to certain embodiments of the invention, antibodies and bispecific antigen-binding molecules are provided that include an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides antibodies and bispecific antigen-binding molecules that include an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C H3 region, where the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), at positions 250 and 428 (e.g., L or F), 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 at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or at positions 307 or 308 (e.g., 308F, V308F), and at 434. In one embodiment, the modifications are a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or 308 modification (e.g., 308F or (includes 308 pages).

[0137] For example, the present invention includes antibodies and bispecific antigen-binding molecules comprising an Fc domain that comprises one or more pairs or groups of mutations 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 in the antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

[0138] Preparation of antigen-binding domains and construction of bispecific molecules An antigen-binding domain specific for a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD3 and CD20) can be appropriately arranged with respect to each other to produce a bispecific antigen-binding molecule of the present invention using conventional methods. (A discussion of exemplary bispecific antibody formats that can be used to construct a bispecific antigen-binding molecule of the present invention is provided elsewhere herein). In certain embodiments, one or more individual components (e.g., heavy and light chains) of a multispecific antigen-binding molecule of the present invention are derived from a chimeric antibody, a humanized antibody or a fully human antibody. Methods for producing such antibodies are well known in the art. For example, one or more heavy and / or light chains of a bispecific antigen-binding molecule of the present invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody production technique), a high affinity chimeric antibody against a particular antigen (e.g., CD3 or CD20) is first isolated with a human variable region and a mouse constant region. Antibodies are characterized and selected for desirable characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to produce fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0139] Genetically engineered animals can be used to generate human bispecific antigen-binding molecules. For example, genetically modified mice can be used that are unable to rearrange and express endogenous mouse immunoglobulin light chain variable sequences, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules that include two different heavy chains associated with the same light chain that includes a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, US2011 / 0195454). Fully human refers to an antibody, or an antigen-binding fragment thereof, or an immunoglobulin domain that includes an amino acid sequence that is encoded by DNA derived from a human sequence over the entire length of each polypeptide of the antibody, or an antigen-binding fragment thereof, or an immunoglobulin domain. In some examples, the fully human sequence is derived from an endogenous protein of a human. In other examples, the fully human protein or protein sequence includes a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any theory, chimeric proteins or sequences are generally designed to minimize the creation of immunogenic epitopes at the junctions of the component sequences, e.g., as compared to any wild-type human immunoglobulin region or domain.

[0140] biological equivalent The present invention encompasses antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein, but that retain the ability to bind to the same antigen or antigens. Such variant antibodies may contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but do not essentially possess the biological activity of the described antibodies or bispecific antigen-binding molecules. They exhibit equivalent biological activity.

[0141] The present invention includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein.Two antigen-binding proteins, or antibodies, are considered to be biologically equivalent if, for example, they are pharmaceutical equivalents or pharmaceutical options that do not show significant differences in absorption rate and degree of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions.Some antigen-binding proteins will be considered as equivalents or pharmaceutical options if their degree of absorption is equivalent but their absorption rate is not equivalent, and such differences in absorption rate are intentional and reflected in the label, are not essential for achieving effective body drug concentration for long-term use, and are considered not medically significant for the particular drug product tested.

[0142] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or efficacy.

[0143] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times compared to therapy continued without switching between the reference product and the biological product without an expected increase in risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy.

[0144] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mode of action for a condition or condition of use, to the extent that such mechanism is known.

[0145] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals where the concentration of the antibody or its metabolites is measured as a function of time in blood, plasma, serum or other biological fluids, (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data, (c) in vivo studies in humans or other mammals where the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0146] Biologically equivalent variants of the exemplary antibodies and bispecific antigen-binding molecules shown herein can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antigen-binding proteins can include variants of the exemplary antibodies and bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation characteristics of the molecule, for example mutations that eliminate or remove glycosylation.

[0147] Therapeutic Formulation and Delivery The present invention provides pharmaceutical compositions comprising the 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 migration, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles ( Excipient formulations include lipids (cationic or anionic), including LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA complexes, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures including carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0148] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), by infusion or bolus injection, and can be administered together with other biologically active agents. Administration can be systemic or local. In one embodiment, the therapeutic protein of the present invention is administered via intravenous infusion.

[0149] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device facilitates application when delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily 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. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0150] A number of reusable pen and autoinjector delivery devices have utility for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (Trademark) (Novo Nordisk, Copenhagen, Denmark), BD (Trademark) pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN (Trademark), OPTIPEN PRO (Trademark), OPTIPEN Examples of disposable pen delivery devices having application in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), to name just a few. ), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park IL).

[0151] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used, see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0152] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, the injectable preparations may be prepared by dissolving, suspending or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium that is conventionally used for injection. The aqueous medium for injection may be, for example, physiological saline, glucose-containing isotonic solution, and other auxiliary agents, which may be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As the oily medium, for example, sesame oil, soybean oil, etc. may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injections thus prepared are preferably filled into suitable ampoules.

[0153] Therapeutic Uses of Antigen-Binding Molecules The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising a therapeutic protein. The therapeutic composition may comprise any of the antibodies or bispecific antigen-binding molecules disclosed herein and a pharma- ceutically acceptable carrier or diluent. As used herein, the term "subject in need thereof" refers to a human or non-human animal that exhibits one or more symptoms or signs of cancer (e.g., a subject that develops a tumor or a subject that suffers from any of the cancers described herein below).

[0154] The antibodies and bispecific antigen-binding molecules of the present invention (and therapeutic compositions comprising them) are particularly useful for the treatment of any disease or disorder in which stimulating, activating and / or targeting immune responses is beneficial. In particular, the antibodies or bispecific antigen-binding molecules of the present invention can be used for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by, for example, CD20, PSMA, MUC16, STEAP2, BCMA expression or activity or the proliferation of CD20+, PSMA+, MUC16+, STEAP2+ or BCMA+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved includes the killing of cells expressing such antigens in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0155] The antigen binding molecules of the invention may be used to treat primary and / or metastatic tumors arising in, for example, the brain and meninges, head and neck, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and adnexa, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, kidney, 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, including, but not limited to, 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 glioma, breast cancer (e.g., ductal or intraductal carcinoma, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, (renal) oncocytoma, (renal) transitional cell carcinoma, urothelial carcinoma, (bladder) adenocarcinoma, or (bladder) small cell carcinoma. According to certain embodiments of the invention, the antibodies or bispecific antibodies are useful for treating patients suffering from refractory or treatment-resistant cancer, such as castration-resistant prostate cancer.

[0156] In some embodiments, the antigen binding molecule is a bispecific anti-CD3 x anti-CD20 antibody useful for treating CD20-expressing cancers 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 myeloma. 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.

[0157] Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy. Among the heterogeneous group of NHLs, 85-90% are of B-cell origin, including follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and several other B-NHLs. Anti-CD20 antibodies in combination with chemotherapy are the standard of care for the treatment of B-NHL, but despite initial responses, many patients relapse and often experience progressively shorter response periods and poorer outcomes in subsequent lines of treatment. Thus, in some embodiments, the antigen-binding molecule is a bispecific anti-CD3 x anti-CD20 that binds to CD3+ T cells and CD20+ B cells and targets CD20+ tumor cells via T cell-mediated cytotoxicity. In some cases, the anti-CD3xCD20 bispecific antibody is for the treatment of a B cell cancer (eg, NHL) in a subject who has previously failed treatment with an anti-CD20 monospecific antibody.

[0158] For patients who do not respond completely to CAR-T therapy, the outcome is generally poor and there are no standard treatment options.Therefore, in some cases, the anti-CD3xCD20 bispecific antibody of the present invention is for the treatment of B-cell cancer (e.g., NHL, such as DLBCL) in subjects who have failed or are not responsive to previous CAR-T therapy (e.g., anti-CD19 CAR-T therapy).

[0159] 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, renal cancer, bladder cancer, colorectal cancer, and gastric cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0160] In some embodiments, the antigen binding molecule is a bispecific anti-CD3 x anti-MUC16 antibody useful for treating MUC16-expressing cancers, including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, cholangiocarcinoma mass forming type, cervical adenocarcinoma, and adenocarcinoma of the gastric tract. In some embodiments, the cancer is ovarian cancer.

[0161] In some embodiments, the antigen binding molecule is a bispecific anti-CD3 x anti-STEAP2 antibody useful for treating STEAP2-expressing cancers, including prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, uterine cancer, and ovarian cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0162] In some embodiments, the antigen binding molecule is a bispecific anti-CD3 x anti-BCMA antibody useful for treating multiple myeloma or other B-cell or plasma cell cancers, such as Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and BCMA-expressing cancers including 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.

[0163] Combination therapy The present invention provides methods comprising administering a pharmaceutical composition comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents that can be administered in combination with or in combination with the antigen-binding molecules of the present invention include, for example, anti-tumor agents (e.g., chemotherapeutic agents including melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine (Treanda), or any other known to be effective in treating plasma cell neoplasms in a subject). In certain embodiments, the second therapeutic agent is a regimen including radiation therapy or hematopoietic stem cell transplantation. In certain embodiments, the second therapeutic agent can be an immunomodulatory agent. 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 may be a histone deacetylase inhibitor, such as panobinostat (Farydak). In certain embodiments, the second therapeutic agent may 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 a bispecific antigen-binding molecule as disclosed herein) may also be administered as part of a therapeutic plan that includes one or more therapeutic combinations selected from monoclonal antibodies other than those described herein that may interact with different antigens on the plasma cell surface, bispecific antibodies with one arm that binds to an antigen on the tumor cell surface and the other arm that binds to an antigen on a T cell, antibody drug conjugates, bispecific antibodies conjugated to an anti-tumor agent, checkpoint inhibitors, such as those that target PD-1 or CTLA-4, or combinations thereof. In certain embodiments, the checkpoint inhibitor may 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-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 can be used in conjunction with the antibodies of the invention are described above.

[0164] The present invention also includes therapeutic combinations comprising any of the antigen-binding molecules described herein and one or more inhibitors 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, uroplakin, where the inhibitor is an aptamer, antisense molecule, ribozyme, siRNA, peptibody, nanobody or antibody fragment (e.g., Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or other engineered molecules such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units). The antigen-binding molecules of the present invention can also be administered and / or co-formulated in combination with antiviral drugs, antibiotics, analgesics, and / or NSAIDs. The antigen binding molecules of the present invention may also be administered as part of a treatment regimen that also includes radiation therapy and / or conventional chemotherapy.

[0165] The additional therapeutically active ingredient(s) may be administered immediately prior to, simultaneously with, or immediately following administration of the antigen-binding molecule of the present invention. (For purposes of this disclosure, such a dosing regimen will be considered as administering the antigen-binding molecule "in combination" with the additional therapeutically active ingredient.

[0166] The present invention includes pharmaceutical compositions in which an antigen-binding molecule of the invention is co-formulated with one or more of the additional therapeutically active ingredient(s) described elsewhere herein. EXAMPLES

[0167] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0168] Example 1: Clinical evaluation and dose escalation of bispecific antibodies The clinical trial described below is an open-label, multicenter, Phase 1 study investigating 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.

[0169] OBJECTIVES:The primary objectives of this trial were to assess the safety, tolerability, and dose-limiting toxicities (DLTs) of intravenously (IV) administered REGN1979 and to investigate the antitumor activity of REGN1979 in expanded cohorts including diffuse large B-cell lymphoma (DLBCL) after failure of chimeric antigen receptor T-cell (CAR-T) therapy, aggressive lymphoma (excluding DLBCL after failure of CAR-T therapy), follicular lymphoma (FL) grades 1-3a, and chronic lymphocytic leukemia (CLL). Secondary objectives of this trial were: (1) to characterize the pharmacokinetic (PK) profile of REGN1979; (2) to assess the immunogenicity of REGN1979; and (3) to investigate the preliminary antitumor activity of REGN1979 administered to patients with CD20+ B-cell malignancies (non-Hodgkin's lymphoma [NHL] or chronic lymphocytic leukemia [CLL] previously treated with anti-CD20 antibody therapy). and (4) to investigate the preliminary antitumor activity of REGN1979 in the dose escalation portion of the trial. Minimal residual disease (MRD) assessment was performed in patients with CLL. Exploratory objectives of this trial were to evaluate the mechanism of action, observed toxicities, and biomarkers that may correlate with potential antitumor activity, including but not limited to: (1) assessment of cytokine profiling and inflammatory markers (e.g., C-reactive protein [CRP]), (2) peripheral blood B- and T-cell subsets and immunophenotype, and (3) gene expression changes in peripheral blood.

[0170] Study Design: Patients were assigned to dose-level (DL) cohorts consisting of an initial starting dose followed by a higher step-up dose for the second and subsequent dose administrations. Patients were enrolled based on indication (NHL or CLL). Each DL had two cohorts (one for each indication), with 3–6 patients per NHL cohort and 1–6 patients per CLL cohort. Patients with small lymphocytic lymphoma (SLL) were enrolled in the CLL group and followed the NHL assessment.

[0171] Patients who initially demonstrated clinical benefit and then relapsed or progressed, or had a suboptimal response to treatment, could be retreated with REGN1979 at the highest DL deemed acceptable at the time of relapse or progression.

[0172] Patients underwent a screening procedure to determine eligibility within 28 days prior to the first dose of REGN1979. Patients were enrolled sequentially based on indication (NHL or CLL), in the order of confirmation of eligibility by the sponsor, until each cohort was filled according to protocol criteria.

[0173] Each DL had separate, independent dose-escalation cohorts for NHL and CLL. Each DL consisted of an initial dose and subsequent doses that were higher than the starting dose if tolerated.

[0174] Dose escalation followed a conventional 3+3 dose escalation design for patients with NHL, with 3–6 patients planned per cohort based on observed toxicities.

[0175] Dose escalation followed a modified 3+3 with an accelerated titration component for patients with CLL. Based on observed toxicities, 1 to 6 patients were planned per cohort.

[0176] Once the dose escalation phase was completed and the recommended dose for further trials in patients with NHL was determined, three expansion cohorts would be initiated: 1) patients with DLBCL after failure of CAR-T therapy (20 patients), 2) aggressive lymphoma (excluding DLBCL after failure of CAR-T therapy) (40 patients, of which 20 patients were to be enrolled in aggressive lymphoma cohort 1 [160 mg step-up dose] and cohort 2 [80 mg step-up dose], respectively), and 3) patients with relapsed / refractory follicular lymphoma grade 1-3a (60 patients, of which 30 patients were to be enrolled in cohort 1 [80 mg step-up dose] and cohort 2 [160 mg step-up dose], respectively). The weekly step-up dose of REGN1979 for each of these expansion cohorts (except for aggressive lymphoma cohort 2 and follicular lymphoma cohort 1) was set at 160 mg once weekly, followed by Q2W maintenance with 320 mg of REGN1979. In aggressive lymphoma cohort 2 and follicular lymphoma cohort 1, the weekly step-up dose of REGN1979 was 80 mg, and the Q2W maintenance dose of REGN1979 was 160 mg. For patients assigned to the 80 mg step-up dose, patients received 80 mg once weekly during a 4-week run-in period after the initial dose escalation, followed by 160 mg of REGN1979 once weekly. Eight additional weekly doses were administered, and ongoing 160 mg Q2W treatment was administered with the option to discontinue treatment after the patient had a sustained response for at least 9 months after initial demonstration of CR.For patients assigned to the 160 mg step-up dose, patients received 160 mg weekly during a 4-week run-in period after the initial dose escalation, followed by eight additional weekly doses and ongoing 320 mg Q2W treatment with the option to discontinue treatment after the patient had a sustained response for at least 9 months after initial demonstration of CR.

[0177] The utility of a rituximab induction dose before the first dose of REGN1979 was trialed 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 induction cohort, REGN1979 was administered using step-up doses of 160 mg during weekly treatment and 320 mg during Q2W maintenance treatment. An additional 24 patients were then treated with the optimal dosing schedule and dose, and together with the 6 patients in the rituximab induction group who were treated with the optimal dose, a total of 30 patients were studied for safety and tolerability.

[0178] The first DL of the dose escalation portion required a 48-h waiting period between the first study drug administrations for the first 3 patients within the same indication. Subsequent patients in the first DL were not treated on the same day, regardless of indication. In subsequent cohorts, the first infusions for the first 3 patients were administered at least 24 hours apart if no unexpected toxicity was observed in the previous cohort or within the cohort.

[0179] After each cohort of patients was enrolled, treated, and the DLT observation period was completed, a decision was made to open a subsequent DL cohort for enrollment (or expand an ongoing DL cohort) after both the sponsor and investigator(s) had reviewed the safety data.

[0180] The DLT observation period was defined as the first 28 days of treatment, which in this study corresponds to the run-in period. During run-in, patients were treated with four weekly doses of REGN1979.

[0181] To be evaluable for a DLT, an individual patient must have received at least the first two doses of REGN1979 (day 1 of week 1 ["initial or primary dose"] and day 1 of week 2 ["secondary and subsequent doses"]) or experienced a DLT. Patients enrolled on DL11 or above must have received at least the first three doses of REGN1979 (the initial dose in week 1, the intermediate or secondary dose in week 2, and the tertiary or step-up dose in week 3) or experienced a DLT. Additionally, patients must have been evaluated for at least 28 days since the first dose and at least 21 days since the second dose.

[0182] Dose escalation and cohorts for NHL and CLL patients are shown in Table 1 below. [Table 1]

[0183] In this trial, each dose level consists of an initial REGN1979 dose followed by a step-up dose. For cohorts DL11 and above, secondary doses were added before reaching the step-up dose (see Table 1). The initial (primary) dose, intermediate (secondary) dose (if applicable), and first dose of the step-up (tertiary) dose are each optionally split over at least 2 days. All patients in the highest dose cohorts at DL7 and above received split doses (e.g., sub-doses) of the initial (primary) and intermediate (secondary) doses. Patients in the highest dose cohorts at DL11 and above received split doses (e.g., sub-doses) of the initial (primary), intermediate (secondary), and first step-up (tertiary) doses, followed by the step-up dose (maximum weekly dose).

[0184] Duration of the trial: Treatment duration is 9 months. Patients will be treated with up to 24 doses of REGN1979 - 4 weekly doses during a 4-week lead-in period, followed by 8 additional weekly doses, and 12 or more doses administered twice monthly (Q2W) during an ongoing maintenance period with the option to discontinue treatment 9 months after patients achieve a complete response. Patients may be followed for efficacy and safety for up to 15 months after the end of treatment.

[0185] Study Population: Assuming full enrollment in DL17 and all expansion cohorts, up to 370 patients are planned at approximately 15 sites in the United States and Germany. Up to 204 patients will be enrolled in DL1 for both indications (NHL and CLL) during the dose escalation period. 7 will be enrolled in the dose escalation cohort. Up to 100 patients will be enrolled in the disease-specific expansion cohort, consisting of 90 patients with NHL (20 patients with DLBCL after CAR-T failure, 40 patients with aggressive lymphoma [excluding DLBCL after CAR-T failure], 30 patients with FL grade 1-3a), and 10 patients with CLL. Up to 42 patients will be enrolled in the rituximab induction cohort to determine the optimal dose regimen. Once the optimal rituximab induction dose regimen is determined, an additional 24 patients will be enrolled in the rituximab induction expansion and combined with the 6 patients from the above rituximab induction group who were treated with the optimal dose regimen and dose. Thus, a total of 30 patients will be evaluated at the optimal rituximab induction dose regimen and dose.

[0186] Patients must have documented CD20+ B-cell malignancies, active disease unresponsive to previous therapy, no standard treatment options available, and treatment with anti-CD20 antibodies may be appropriate. Patients with NHL must have been previously treated with CD20-directed antibody therapy.

[0187] Inclusion Criteria: Patients must meet the following criteria to be eligible for inclusion in the study. 1. Documented CD20+ B-cell malignancy with active disease unresponsive to prior therapy, no standard treatment options available, and in whom treatment with an anti-CD20 antibody may be appropriate: B-NHL confirmed by the 2007 NCI Working Group criteria (Cheson 2007, Appendix 2), and Patients with CLL-small lymphocytic lymphoma (SLL) confirmed by the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) Working Group criteria, 2008 (Hallek 2008, Appendix 3) were enrolled in the CLL group and followed the NHL assessment. NOTE - Patients with a CD20 negative lymph node (NHL) biopsy performed as standard of care immediately prior to enrollment may remain eligible for the study if the patient has previously documented CD20+ disease and has been previously treated with rituximab or other CD20-directed antibody therapy within approximately 6 months. Individual cases may be discussed with the medical monitor. 2. Patients with NHL must have been previously treated with anti-CD20 antibody therapy. Patients with CLL do not need to have been previously treated with anti-CD20 antibody therapy if the patient has failed either a BTK inhibitor or a PI3K inhibitor and the treating physician deems it appropriate to enter the patient into a phase 1 trial. For inclusion in the expansion cohort for FL grade 1-3a, patients must have received at least two prior lines of systemic therapy, including an anti-CD20 antibody and an alkylating agent. For inclusion in the disease-specific expansion cohort enrolling patients with DLBCL after failure of CAR-T therapy, patients must have recovered from the toxicity of lymphodepleting therapy and CAR-T infusion. The prior CAR-T therapy does not need to be the most recent line of treatment prior to study enrollment. 3. All patients (B-cell NHL and CLL) must have at least one bidimensionally measurable lesion ≥1.5 cm) documented by CT or MRI scan if CT scan is not feasible. 4. Patients with CLL must have a white blood cell (WBC) count of ≦200×109 / L. 5. Age ≥ 18 years 6.Eastern Cooperative Oncology Group (ECOG) performance status ≤ 1 7. Life expectancy of at least 6 months 8. Adequate bone marrow function as documented by: a. Platelet count ≥ 75 x 109 / L, b. Hb level ≥ 9 g / dL, c. ANC ≥ 1 x 109 / L, Note: Patients with cell counts below the above thresholds may be considered for enrollment if, in the opinion of the investigator, the reason is due to bone marrow infiltration from the underlying disease. Investigators must discuss eligibility with the sponsor and receive written approval for enrollment. 9. Adequate organ function as described by: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 x ULN Total bilirubin ≦1.5×ULN NOTE: Patients with Gilbert syndrome do not need to meet this requirement unless their total bilirubin has changed from baseline. Creatinine clearance calculated by Cockcroft-Gault formula ≥ 50mL / min NOTE: Patients may be considered for enrollment if, in the opinion of the investigator, the abnormal test results are due to an underlying condition. In such cases, the investigator should discuss eligibility with the sponsor and receive written approval for enrollment. NOTE: Patients with borderline creatinine clearance by Cockcroft-Gault may be considered for enrollment if their measured creatinine clearance (based on 24-hour urine or other reliable method) is ≥ 50 mL / min. 10. Willingness to undergo mandatory tumor biopsy pre-treatment if, in the investigator's opinion, the patient has accessible lesions that can be biopsied without significant risk to the patient. 11. Willing and able to comply with clinic visits and study-related procedures. 12. Provide signed informed consent.

[0188] Exclusion Criteria: Patients who meet any of the following criteria will be excluded from the study. 1. Known or suspected CNS involvement by primary central nervous system (CNS) lymphoma or non-primary CNS NHL 2. History or current relevant CNS pathology, such as: Epilepsy, seizures, paresis, aphasia, stroke, severe brain injury, cerebellar disease, organic brain syndrome, psychosis, or Evidence of inflammatory lesions and / or vasculitis on cerebral MRI 3. Standard antineoplastic chemotherapy (non-biologic) within 5 half-lives or 28 days, whichever is shorter, prior to the first dose of investigational drug. 4. Standard radiotherapy within 14 days of the first dose of study drug. NOTE - Palliative radiotherapy to symptomatic nodes / lesions is permitted if the irradiated lesion(s) or lymph node(s) are not included as target lesions in the tumor assessment. 5. Allogeneic stem cell transplantation 6. Treatment with rituximab, alemtuzumab or other investigational or marketed biologic agents within 12 weeks prior to the first dose of study drug. NOTE: For patients with aggressive lymphoma who require immediate treatment, the washout period may be reduced to 28 days. This requires discussion with and approval by the Sponsor in writing. 7. Immunosuppressive therapy (excluding biologics) within 28 days of the first dose of investigational drug. 8. Treatment with an investigational non-biologic agent within 28 days of the first dose of study drug. 9. History of allergic reactions due to compounds of similar chemical or biological composition to the investigational drug. 10. History of hypersensitivity to any compound of the tetracycline antibiotic group. 11. Concurrent active malignancy for which the patient is receiving treatment. 12. Have a confirmed active bacterial, viral, fungal, mycobacterial or other infection or a significant episode of infection requiring hospitalization or treatment with IV anti-infectives within 4 weeks of first dose. 13. Any significant cardiovascular disease (e.g., New York Heart Association class III or IV heart disease, myocardial infarction within the past 6 months, unstable angina or unstable angina) and / or significant pulmonary disease (e.g., history of obstructive pulmonary disease and symptomatic bronchospasm), including, but not limited to, Without limitation, evidence of significant intercurrent illness or medical condition that may interfere with the conduct of the trial or place the patient at significant risk. NOTE - Patients with a history of cardiac disease should be evaluated by echo or multi-gated acquisition scan (MUGA) prior to the first dose of REGN1979 to ensure adequate cardiac reserve and function. 14. Ongoing systemic corticosteroid treatment, excluding use of corticosteroids for other (non-neoplastic and non-immunosuppressive) indications with up to 10 mg / day of prednisone or equivalent. 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+) who have controlled their infection (serum hepatitis B virus DNA below the limit of detection and receiving hepatitis B antiviral therapy) are permitted in consultation with the physician managing their infection. 16. Hypersensitivity to both allopurinol and rasburicase has been documented. 17. Pregnant or lactating women. 18. Females of childbearing potential who are unwilling to practice highly effective contraception before their first investigational drug treatment, during the study, and for at least 6 months after the last dose.* Highly effective contraceptive measures include stable use of (estrogen- and progestogen-containing) hormonal contraceptives (oral, intravaginal, transdermal) or progestogen-only hormonal contraceptives (oral, injectable, implantable) associated with the suppression of ovulation initiated for at least two menstrual cycles prior to screening, intrauterine device, intrauterine hormone-releasing system, bilateral tubal ligation, vasectomized partner, and / or sexual abstinence†,‡. *Postmenopausal women must be amenorrhea for at least 12 months to be considered for childbearing potential. Pregnancy testing and contraception are not required for women with a documented hysterectomy or tubal ligation. †Sexual abstinence is considered highly effective only if it is defined as abstinence from heterosexual intercourse for the entire period of risk associated with the investigational treatment. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial and the patient's preferred usual lifestyle. ‡Cyclic abstinence (calendar, symptothermal, or postovulatory), withdrawal (withdrawal), spermicide only, and lactational amenorrhea method (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used together. 19.Reception of live vaccines within 28 days of first dose of investigational drug 20. Clinical site study team members and / or their immediate families, unless prior approval by the Sponsor.

[0189] Treatment: REGN1979 was supplied as a liquid in sterile, single-use vials. Each vial contained REGN1979 at a concentration of 2 mg / mL. Detailed preparation and administration instructions were provided to the site in the pharmacy manual. Diluents were provided for preparation of the REGN1979 study drug.

[0190] Patients received REGN1979 once weekly during a 4-week run-in period, followed by eight additional weekly doses at their assigned cohort dose, then Q2W doses until progression.

[0191] In the rituximab induction cohort and expansion only, a single dose of rituximab (375 mg / m 2) was administered 1 day prior to the first dose of REGN1979 [i.e., study day (-1)]. REGN1979 was initiated on day 1 of week 1, and the duration of REGN1979 treatment was 9 months. Patients were treated with up to 24 doses of REGN1979: 4 weekly doses during a 4-week run-in period, followed by 8 additional weekly doses, and maintenance until progression. More than 12 doses were administered Q2W during the period. In the rituximab induction cohort, REGN1979 was administered using step-up doses of 160 mg during weekly treatment and 320 mg during Q2W maintenance treatment. Dose groups with step-up doses of REGN1979 treatment less than 160 mg once weekly and REGN1979 maintenance treatment less than 320 mg Q2W could also be evaluated. An additional 24 patients were then evaluated with the optimal dose schedule and dose, and together with the 6 patients in the rituximab induction group who were treated with the optimal dose schedule and dose, a total of 30 patients were studied for safety and tolerability.

[0192] Endpoint PRIMARY ENDPOINTS: The primary endpoints were safety (specifically, adverse events [AEs] and DLTs) to determine the maximum tolerated dose (MTD) and / or optimal biological dose (OBD) as the recommended phase 2 dose (RP2D) of REGN1979, and efficacy as measured by objective response rate (ORR) in the expansion cohort of patients with DLBCL after failure of CAR-T therapy, aggressive lymphoma (excluding DLBCL after failure of CAR-T therapy) expansion cohorts 1 and 2, the FL grade 1-3a expansion cohort, and the CLL expansion cohort.

[0193] Secondary Endpoints: Secondary endpoints were: Pharmacokinetics: REGN1979 concentration ·Immunogenicity: Anti-REGN1979 antibody Antitumor activity: -Objective response rate (ORR) Tumor response assessment according to the Revised Response Criteria for Malignant Lymphoma of the NCI-International Working Group (NCI-WG) Tumor response assessment according to the International Workshop on Chronic Lymphocytic Leukemia Guidelines for the Diagnosis and Treatment of CLL For patients enrolled in the NHL expansion cohort, tumor response assessment by Lugano classification -Progression-free survival (PFS) and overall survival (OS) -Minimal Residual Disease (MRD) in patients with CLL

[0194] Exploratory endpoints included: Pharmacodynamic (PD) measures including: -B and T cell subsets and phenotypes Circulating cytokine levels CRP -Changes in gene expression in peripheral blood

[0195] Procedures and Assessment Baseline Procedures: Brain MRI, electrocardiogram (ECG), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV), and coagulation. Safety Procedures: Medical history, physical examination, symptom assessment, performance status evaluation, laboratory tests, vital signs, AEs, and concomitant medications.

[0196] Efficacy procedures: Tumor assessment including CT or MRI scan, 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) scan, bone marrow aspirate and biopsy, lymph node and / or tumor biopsy, peripheral blood sample (for patients with CLL only). Blood samples for PK and anti-drug antibody (ADA) assessment were collected. Biomarker samples were collected to monitor changes in cytokine production, serum levels of proinflammatory cytokines, and changes in lymphocyte subsets and activation status. Furthermore, these samples allowed genetic analysis of tumor or somatic cells for variations that influence the clinical course of the underlying disease or modulate side effects of treatment.

[0197] Statistical Plan: The study design was based on a traditional 3+3 design with 3–6 patients per DL for patients with NHL and a modified 3+3 design with an accelerated titration component with 1–6 patients per DL for patients with CLL. The exact number of patients enrolled would depend on the number of patients (NHL and CLL) observed with protocol-defined DLTs and treatment-related toxicity of grade ≥2, acute effects (excluding relevant laboratory abnormalities) resolving to ≤grade 1 or baseline (for CLL in the accelerated titration component) within 72 hours, and the need to expand currently defined DLs or open additional cohorts at lower DLs.

[0198] Patient enrollment is ongoing, with up to 370 patients planned. Up to 204 patients will be enrolled in the dose escalation cohort by DL17 for both indications (NHL and CLL) during the dose escalation period. Up to 100 patients, including 90 patients with NHL (20 patients with DLBCL after CAR-T failure, 40 patients with aggressive lymphoma [excluding DLBCL after CAR-T failure], and 30 patients with FL grade 1-3a), and 10 patients with CLL, will be enrolled in the disease-specific expansion cohort. Up to 42 patients will be enrolled in the rituximab induction cohort to determine the optimal dose regimen. An additional 24 patients will be enrolled in the rituximab induction expansion and combined with 6 patients from the above rituximab induction cohort who were treated with the optimal dose regimen and dose. Thus, a total of 30 patients will be evaluated at the optimal rituximab induction dose regimen and dose.

[0199] Data are aggregated using descriptive statistics only. In general, data are aggregated by DLBCL and indication (NHL or CLL). Within the NHL indication, data are also aggregated by DLBCL after CAR-T failure, aggressive lymphoma (excluding DLBCL after CAR-T failure) cohorts 1 and 2, and FL grade 1-3a subgroups and doses. Within the NHL indication, data are also aggregated by indolent and aggressive NHL subgroups. Demographic and baseline characteristics are aggregated descriptively by group.

[0200] Safety aggregation and analysis was performed in the safety analysis set (SAF). The primary analysis of safety was based on treatment-emergent AEs (TEAEs). This analysis constituted the basis on which conclusions were drawn regarding the safety profile of REGN1979. All AEs reported in this study were coded using the currently available version of the ICH Medical Dictionary for Regulatory Activities (MedDRA®). Coding was low-level. Verbatim text, preferred term (PT), and primary organ system class (SOC) were listed. Analyses of efficacy and baseline variables were performed in the efficacy analysis set (FAS). Efficacy analyses for expansion cohorts including DLBCL after CAR-T failure, aggressive lymphoma (excluding DLBCL after CAR-T failure) cohorts 1 and 2, FL grades 1-3a, and CLL will be conducted separately after all patients in each cohort have completed the 24-week visit or discontinued from the study prior to this time.

[0201] Results: Results for patients receiving maximum weekly doses of 5-320 mg indicate a low incidence of CRS. Serum concentrations of REGN1979 in patients receiving maximum weekly doses of as little as 12 mg were observed to approach or exceed serum concentration levels demonstrated to be effective in the Raji tumor xenograft mouse model (data not shown). Patients receiving maximum weekly doses of as little as 40 mg maintained serum concentrations above the minimum concentration levels (2000 μg / L) demonstrated to be effective in the Raji tumor xenograft mouse model (data not shown). Additionally, numerous partial and complete responses were observed in patients treated at these levels as shown in Tables 2-9 below. The accumulated REGN1979 safety and PK experience during the DLT evaluation period for cohort 13N (27,000mcg REGN1979) indicates that the management algorithm for CRS or IRR reactions (i.e., incremental dose escalation, split dosing during the first few weeks of REGN1979 administration, and premedication with corticosteroids) proved effective in preventing severe CRS or IRR despite dose escalation in the sequential dosing cohort. Split dosing benefited patient safety in weeks 1-4 (available data), with little overall incidence of severe CRS / IRR observed. Notably, the dosing strategy discussed herein provides a safer strategy for titrating doses to levels above 80 mg and even 160 mg or higher, with milder events occurring in weeks 3 and 4 as the higher doses reached and exceeded the desired serum concentrations discussed above. Figure 1 shows the incidence of CRS / IRR in patients receiving doses up to a maximum of 320 mg. To date, no patients have discontinued due to CRS / IRR adverse events. [Table 2]

[0202] After data cutoff (Table 2), two additional evaluable patients demonstrated complete responses (CR), one at 40 mg and one at 320 mg. [Table 3]

[0203] Two of the three 80 mg patients in CR had failed CAR T-cell therapy. All complete responses described in Table 3 were complete metabolic responses.

[0204] All CRs at the 80 mg, 160 mg, and 320 mg doses were ongoing CRs on investigational treatment, indicating durability of response. [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

[0205] In general, widespread activity observed in patients with heavily pretreated relapsed / refractory B-NHL treated with REGN1979, including those who had progressed after prior CAR T-cell therapy, included the following: (i) FL grade 1-3a: at a dose of ≥5 mg, 12 / 13 (92.3%) ORR, 8 / 13 CR (61.5%); (ii) DLBCL: at doses of 80–160 mg, 4 / 6 (66.7%) ORR (all CR), with 2 patients achieving CR after failure of CD19-directed CAR T-cell therapy; (iii) MCL: 3 / 3 responses including 1 CR at doses ≥ 5 mg; (iv) MZL: 3 / 5 responses including 2 CRs at doses ≥5 mg; (v) Tolerability in patients with B-NHL has been demonstrated up to doses of 320 mg once weekly, with no DLTs observed in patients with B-NHL. (vi) the majority of adverse events were mild to moderate in severity; (vii) infection was reported in 49.4% of patients (14.8% grade 3-4, including 2 deaths (2.5%)); (viii) No patients discontinued treatment due to CRS or neurological adverse events; of 96 initially treated patients, only 7 patients experienced grade 3 CRS. (ix) Dexamethasone did not inhibit the cytotoxicity of REGN1979, had a modest effect on upregulating T cell activation, and inhibited cytokine release.

[0206] Example 2: Clinical evaluation of bispecific antibodies The clinical trial described below is an open-label, multicenter, Phase 2 study to assess the antitumor activity and safety of REGN1979, an anti-CD20 × anti-CD3 bispecific antibody, in patients with relapsed or refractory follicular lymphoma.

[0207] OBJECTIVE: The primary objective of this trial is to evaluate the antitumor activity of single-agent REGN1979, as measured by objective response rate (ORR) according to the Lugano Classification of Responses in Malignant Lymphomas (Cheson, 2014) by independent central review, in patients with follicular lymphoma (FL) that has relapsed or is refractory to at least two prior lines of systemic therapy including an anti-CD20 antibody and an alkylating agent. The secondary objectives of this study are (1) to assess the antitumor activity of single-agent REGN1979 in patients with relapsed or refractory FL, including (a) ORR by Lugano classification (Cheson, 2014) assessed by institutional investigator assessment, (b) complete response (CR) rate by Lugano classification assessed by third-party central review and institutional investigator assessment, (c) progression-free survival (PFS) by Lugano classification assessed by third-party central review and institutional investigator assessment, (d) overall survival (OS), and (e) Lugano classification by third-party central review and institutional investigator assessment. (1) to evaluate the safety and tolerability of REGN1979, as measured by the duration of response (DOR) by classification, (f) disease control rate (DCR) by Lugano classification assessed by independent central review and institutional investigator assessment, (g) duration of disease control (DDC) by Lugano classification assessed by independent central review and institutional investigator assessment, (1) to evaluate the safety and tolerability of REGN1979, (2) to evaluate the pharmacokinetics (PK) of REGN1979, (3) to evaluate the immunogenicity of REGN1979, as measured by a validated instrument, the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC). To assess the impact of REGN1979 on quality of life as measured by the EuroQoL 5 Dimensions 3 Levels (EQ-5D-3L) and the QLQ-C30.

[0208] Study Design: The trial will consist of a screening period of up to 28 days, a total treatment period of up to 98 weeks including once-weekly (QW) dosing for 12 weeks followed by once-every-2-weeks (Q2W) dosing for up to 86 weeks, and a post-treatment follow-up period of 96 weeks.

[0209] REGN1979 is administered as a single agent intravenously (IV) at an initial (primary) dose of 1 mg, followed by an intermediate (secondary) dose of 20 mg, followed by a nominal (tertiary and maximum weekly) dose of 80 mg on a 12 QW treatment regimen followed by 80 mg of REGN1979 Q2W.

[0210] Enrollment will follow an open-label, single-arm design.

[0211] The screening period begins with the signing of the informed consent form (ICF) and ends when the patient is determined to be eligible for the trial and has begun treatment, or when the patient is determined to be ineligible and designated as a screen failure.

[0212] The treatment period begins with the first dose of REGN1979 and consists of 12 QW infusions of REGN1979, followed by 86 weeks of Q2W dosing, for a total of 98 weeks of study drug administration, unless the patient discontinues study treatment due to disease progression, initiation of subsequent lymphoma treatment, adverse event (AE), or other reason.

[0213] The post-treatment follow-up period is 96 weeks after the last dose of study treatment. All patients will be followed for vital status every 12 weeks until death, loss to follow-up, withdrawal of patient consent for follow-up, or termination of the study by the sponsor, whichever occurs first. For patients who discontinue study treatment for reasons other than disease progression, initiation of subsequent lymphoma treatment, 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 treatment, or withdrawal of patient consent for follow-up, whichever occurs first.

[0214] Study Duration: The study duration for each patient, excluding the screening period, will be approximately 194 weeks unless the patient experiences disease progression or initiates subsequent treatment, or until death, loss to follow-up, withdrawal of patient consent for follow-up, or termination of the study by the sponsor. End of the study is defined as the last visit of the last patient.

[0215] Study Population: Up to 481 patients will be enrolled at up to 100 sites. The study population will consist of patients aged 18 years or older with previously treated FL grades 1-3a that have relapsed or are refractory to at least two prior lines of systemic therapy including anti-CD20 antibodies and alkylating agents. Central nervous system histopathological confirmation of FL diagnosis will be required prior to enrollment. Patients with FL grade 3b are ineligible. Refractory disease is defined as lack of response to standard dosing regimens or progression within 6 months of last treatment.

[0216] Inclusion Criteria: Patients must meet the following criteria to be eligible for inclusion in the trial: 1. Age 18 or older 2. Central nervous system histopathological confirmation of FL grade 1-3a diagnosis must be obtained prior to study enrollment. Patients with FL grade 3b are ineligible. Subclassification of follicular lymphoma is based on the World Health Organization (WHO) classification (Swerdlow, 2017). 3. Disease must have relapsed or be refractory to ≥ 2 prior lines of systemic therapy, including an anti-CD20 antibody and an alkylating agent. Patients must require treatment for FL at the time of study enrollment, in the opinion of the investigator. 4. Measurable disease on cross-sectional images (defined as at least one bidimensionally measurable lymph node disease ≥ 1.5 cm in greatest transverse diameter (GTD), regardless of short-axis diameter) recorded by imaging (computed tomography [CT], or magnetic resonance imaging [MRI]). 5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. 6. Adequate bone marrow function as documented below: (a) Platelet count ≥ 50 x 10 9 / L. Patients may not have received a platelet transfusion within 7 days prior to the first dose of REGN1979 to meet platelet eligibility criteria; (b) hemoglobin ≥ 9.0 g / dL; (c) absolute neutrophil count (ANC) ≥ 1.0 × 10 9 / L. Patients may not have received granulocyte colony-stimulating factor within 2 days prior to the first dose of REGN1979 to meet ANC eligibility criteria. 7. Adequate liver function: (a) total bilirubin ≤ 1.5 x upper limit of normal (ULN) (≤ 3 x ULN if due to lymphomatous infiltration of the liver); (b) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 x ULN (≤ 5 x ULN if due to lymphomatous infiltration of the liver); (c) alkaline phosphatase (ALP) ≤ 2.5 x ULN (≤ 5 x ULN if due to lymphomatous infiltration of the liver). Note - Patients with AST > 2.5 x ULN and / or ALT > 2.5 x ULN and total bilirubin > 1.5 x ULN are excluded, regardless of the presence of lymphomatous infiltration of the liver; patients with known Gilbert's syndrome do not need to meet this total bilirubin requirement unless values ​​are unchanged from baseline levels. 8. Serum creatinine ≤ 1.5 x ULN or creatinine clearance ≥ 50 mL / min calculated by the Cockcroft-Gault equation. NOTE: Patients with a calculated creatinine clearance < 50 mL / min may be considered for enrollment if their measured creatinine clearance (based on a 24-hour urine collection or other reliable method) is ≥ 50 mL / min. 9. Willingness to undergo a tumor biopsy at baseline. If the investigator determines that a baseline tumor biopsy cannot be safely obtained, the sponsor may grant an exception to the biopsy requirement only after discussion with and approval by the Medical Monitor. 10. Ability to understand the purpose and risks of the clinical trial and provide signed and dated informed consent and authorization to use protected health information (subject to national and local subject privacy regulations). 11. Willing and able to comply with clinic visits and study-related procedures. 12. Provide informed consent signed by the study patient or legal representative. 13. Able to understand and complete clinical trial-related questionnaires.

[0217] Exclusion Criteria: Patients who meet any of the following criteria will be excluded from the study. 1. Known involvement by primary central nervous system (CNS) lymphoma or non-primary CNS NHL (suspected CNS lymphoma should be evaluated with mandatory head CT or MRI plus lumbar puncture as indicated). Treatment with systemic anti-lymphoma therapy within 2.5 half-lives or 28 days prior to the first dose of study drug, whichever is shorter. 3. History of allogeneic stem cell transplant. 4. Previous treatment with chimeric antigen receptor T-cell (CAR-T) therapy. 5. Ongoing systemic corticosteroid treatment with more than 10 mg prednisone or anti-inflammatory equivalent per day within 72 hours of starting study drug. 6. History of neurodegenerative conditions or CNS movement disorders. History of uncontrolled seizure disorder, defined as seizures within 12 months prior to study enrollment. 7. Vaccination with a replication-capable vector within 28 days prior to the first investigational drug administration. 8. Another malignancy other than FL in the past 5 years, excluding non-melanoma skin cancer that has been treated with potentially curative therapy, cervical intraepithelial neoplasia, or other tumors that have been deemed effectively treated with definitive local control and curative intent. 9. Evidence of any significant intercurrent illness or condition that may interfere with the conduct of the trial 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 past 6 months, unstable angina or unstable angina) and / or significant pulmonary disease (e.g., history of obstructive pulmonary disease and symptomatic bronchospasm). 10. Cardiac ejection fraction <40% by echocardiogram or multi-gated acquisition (MUGA) scan. 11. Infection requiring hospitalization or treatment with IV anti-infectives within 2 weeks of first dose of study drug. 12. Uncontrolled infection due to human immunodeficiency virus (HIV), hepatitis B or C infection, or other uncontrolled infection, except: (a) patients with HIV who have controlled infection (undetectable viral load either spontaneously or in response to stable antiviral therapy and CD4 count greater than 350 cells / microliter) are allowed, (b) patients with hepatitis B (HepBsAg+) who have controlled infection (serum hepatitis B virus DNA polymerase chain reaction [PCR] below the limit of detection and receiving hepatitis B antiviral therapy) are allowed, and (c) patients with hepatitis C virus antibody-positive (HCV Ab+) who have controlled infection (HCV RNA undetectable by PCR either spontaneously or in response to a previous successful course of anti-HCV treatment) are allowed. 13. History of severe allergic reaction due to compounds with similar chemical or biological composition as the investigational product or excipients. A severe allergic reaction is defined for this purpose as one requiring hospitalization and / or treatment with epinephrine. 14. Hypersensitivity to both allopurinol and rasburicase has been documented. 15. Members of the clinical site investigational team or their immediate families, unless prior approval by the Sponsor. 16. Women with a positive serum β-hCG pregnancy test at the screening visit. If positive, pregnancy must be ruled out by ultrasound for the patient to be eligible. 17. Patients who have been committed to the institution by order issued by either a judicial or administrative authority. 18. Pregnant or lactating women. 19. Women* or men of childbearing potential who are unwilling to practice highly effective contraception before the first dose / initiation of first treatment, throughout the study, and for at least 6 months after the last dose. Highly effective contraceptive measures include (a) stable use of (estrogen- and progestogen-containing) hormonal contraceptives (oral, intravaginal, transdermal) or progestogen-only hormonal contraceptives (oral, injectable, implantable) associated with the suppression of ovulation initiated for at least two 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. † , ‡ Includes: Postmenopausal women must be amenorrhea for at least 12 months to be considered childbearing potential. Pregnancy testing and contraception are not required for women with a documented hysterectomy or tubal ligation. † Sexual abstinence is considered highly effective only if it is defined as abstinence from heterosexual intercourse for the entire period of risk associated with the investigational drug. The reliability of sexual abstinence needs to be evaluated in relation to the duration of the clinical trial and the subject's preferred usual lifestyle. ‡ Periodic abstinence (calendar, symptothermal, or postovulatory methods), withdrawal (withdrawal), spermicide only, and lactational amenorrhea method (LAM) are not acceptable methods of contraception. Female condoms and male condoms should not be used together.

[0218] Treatment: REGN1979 is administered by IV infusion at an initial dose of 1 mg in week 1, an intermediate dose of 20 mg in week 2, and a nominal dose of 80 mg or 160 mg during subsequent doses. For the initial, intermediate, and first nominal doses (primary, secondary, and tertiary doses, respectively), treatment is split into two separate infusions, each administered over 4 hours every 2 days, preferably consecutive but no more than 3 days apart (e.g., day 1 of week 1 and day 2 of week 1). Subsequent treatments (maximum weekly dose, e.g., 320 mg) may be administered as a single infusion or two separate infusions, administered over 1-4 hours depending on tolerability. The study treatment consists of 12 QW doses followed by 86 weeks of Q2W dosing for a total of 98 weeks of study drug administration.

[0219] Endpoints: The primary endpoint of this trial is ORR from the first dose to 194 weeks after the first dose in patients with FL that has relapsed or is refractory to at least two prior lines of systemic therapy including an anti-CD20 antibody and an alkylating agent, as measured by the Lugano Classification of Responses in Malignant Lymphoma (Cheson, 2014) and by third-party central review. Secondary endpoints were: (1) ORR from the first dose to 194 weeks after the first dose by Lugano classification assessed by the site investigator's assessment; (2) CR rate from the first dose to 194 weeks after the first dose by Lugano classification assessed by third-party central review and site investigator's assessment; (3) PFS from the first dose to 194 weeks after the first dose by Lugano classification assessed by third-party central review and site investigator's assessment; (4) OS from the first dose to 194 weeks after the first dose; and (5) OS from the first dose to 194 weeks after the first dose by third-party central review and site investigator's assessment. (5) DOR from the first dose to 194 weeks after the first dose as assessed by Lugano classification by independent investigator assessment; (6) DCR from the first dose to 194 weeks after the first dose as assessed by Lugano classification by independent central review and by site investigator assessment; (7) DDC from the first dose to 194 weeks after the first dose as assessed by Lugano classification by independent central review and by site investigator assessment; (8) incidence and severity of treatment-emergent adverse events (TEAEs) from the first dose to 194 weeks after the first dose; and (9) change in patient-reported outcome scores from the first dose to 194 weeks after the first dose as measured by the validated instruments EORTC QLQ-C30 and EQ-5D-3L.

[0220] Procedures and assessment: Computed tomography (CT) or magnetic resonance imaging was performed on all patients. Using MRI, 18Disease will be assessed radiologically by F-fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. Tumor response according to the Lugano classification criteria will be adjudicated by independent central radiology review. Bone marrow aspiration, bone marrow biopsy, lymph node and / or tumor biopsies will be performed and samples will be evaluated histologically and may be used for other studies including immunohistochemistry. Safety will be assessed by assessment of vital signs, physical examination, Eastern Cooperative Oncology Group (ECOG) performance status, electrocardiogram (ECG), incidence of AEs, and reporting of concomitant medications. Laboratory evaluations will include complete blood count with differential, blood chemistry values, serum immunoglobulin G (IgG), serum pregnancy test (if applicable), ferritin, and C-reactive protein (CRP). Blood samples for PK and anti-drug antibody (ADA) assessment will be collected. Peripheral blood samples will be collected to assess biomarker changes (e.g., cytokine production, serum levels of proinflammatory cytokines, and changes in lymphocyte subsets and activation status). Additionally, these samples will allow genetic analysis of tumor or somatic cells for variations that influence the clinical course of the underlying disease or modulate side effects of treatment. Quality of life assessment will be performed using self-administered EORTC QLQ-C30 and EQ-5D-3L questionnaires.

[0221] Statistical Plan: This trial is designed to evaluate the efficacy and safety of REGN1979 for patients with FL that has relapsed or is refractory to at least two prior lines of systemic therapy. Analysis of the primary efficacy endpoint will be performed after all patients have completed the 28-week study treatment period up to assessment and have undergone tumor response assessment or withdrawn from the study. Sample Size Justification - A single-stage exact binomial design has been employed for the primary endpoint of ORR. Two-sided 95% confidence intervals for the observed ORR were calculated based on a sample size of 100. With 100 patients, if the observed ORR is at least 60%, 66%, 70%, and 75%, the lower limit of the 95% CI excludes ORRs of 49%, 55%, 60%, and 65%, respectively, i.e., ORR is significantly different from 49%, 55%, 60%, and 65%, as shown in Table 10 below. [Table 10] With a sample size of 100 patients, if the true treatment effect of REGN1979 is 64%, 70%, 75%, or 80%, the probability of the observed lower limit of the 95% CI is 82%, 83%, 89%, or 91%, excluding 49%, 55%, 60%, or 65%, respectively. The sample size is increased by an additional 10% to account for patients who prematurely withdraw from the trial. Thus, the total sample size is 112 patients.

[0222] Statistical methods: Demographic and baseline characteristics will be summarized descriptively. The primary efficacy outcome measure is ORR by Lugano classification based on independent central review. ORR and two-sided 95% confidence intervals will be summarized. Patients not evaluable for best overall response will be considered non-responders. Secondary efficacy outcomes of ORR determined by investigator review by Lugano classification and CR rate and DCR by site investigator assessment and independent central review by Lugano classification will be summarized with two-sided 95% confidence intervals. Other secondary efficacy outcomes including DOR, DDC, PFS, and OS will be summarized by median and its 95% confidence interval using the Kaplan-Meier method by Lugano classification. Disease control rates will be summarized with two-sided confidence intervals. Quality of life, measured by the validated instruments EORTC QLQ-C30 and EQ-5D-3L, will be summarized by descriptive statistics. Safety observations and measurements, including drug exposures, AEs, laboratory data, vital signs and ECOG performance status, will be summarized and presented in tables and lists.

[0223] Interim Analysis: The interim analysis will be conducted after the first 50 patients have completed tumor assessment at Week 28 or have withdrawn from the study earlier. ORRs and associated 95% confidence intervals will be aggregated. As the primary objective of this interim analysis is to characterize the point estimates and precision of the point estimates in ORR, there are no hypothesis tests associated with this interim analysis. Therefore, type I error correction will not be applied to this planned interim analysis. For other efficacy endpoints, two-sided 95% confidence intervals will also be presented.

[0224] Additional efficacy investigations of REGN1979 may be included in this trial or in additional trials that include patients with: (a) follicular lymphoma (grades 1-3a) as third line or higher (3L+) therapy, (b) follicular lymphoma (grades 1-3a) suitable for full dose chemotherapy as second line or higher (2L+) therapy, (c) follicular lymphoma (grades 1-3a) not suitable for full dose chemotherapy as 2L+ therapy, (d) follicular lymphoma (grades 1-3a) not previously treated and suitable for full dose chemoimmunotherapy, (e) follicular lymphoma (grades 1-3a) not previously treated and suitable for full dose chemoimmunotherapy, (f) follicular lymphoma (grades 1-3a) suitable for full dose chemoimmunotherapy as first line (1L) therapy against standard of care. (g) patients with follicular lymphoma (grades 1–3a) not eligible for full-dose chemoimmunotherapy as 1L therapy versus standard of care, (h) patients with follicular lymphoma (grades 1–3a) not eligible for full-dose chemoimmunotherapy as 2L+ therapy versus standard of care, (i) patients with follicular lymphoma (grades 1–3a) not eligible for full-dose chemoimmunotherapy as 2L+ therapy versus standard of care, and / or (j) patients with follicular lymphoma in combination with standard of care.

[0225] Additional efficacy investigations of REGN1979 may be included in this trial or in additional trials that include patients with: (a) de novo or transformed diffuse large B-cell lymphoma (DLBCL) as a 3L+ therapy; (b) patients with DLBCL after failure of CAR-T therapy; (c) patients with DLBCL eligible for autologous HSCT as a 2L+ therapy; (d) patients with DLBCL ineligible for HSCT as a 2L+ therapy; (e) patients with previously untreated and poor molecular prognostic factors; (f) previously untreated patients with DLBCL with poor molecular prognostic factors (non-germinal center B cell type, double hit or triple hit) and not suitable for full-dose chemoimmunotherapy, (g) patients with DLBCL who are CAR-T naive, (h) patients with DLBCL with a maximum weekly dose of 320 mg, (i) patients with DLBCL in combination with standard of care, (j) patients with DLBCL suitable for full-dose chemoimmunotherapy as 1L therapy against standard of care, (k) patients with DLBCL not suitable for full-dose chemoimmunotherapy as 1L therapy against standard of care, (l) patients with DLBCL eligible for auto-HSCT as 2L+ therapy against standard of care, and / or (m) patients with DLBCL not suitable for auto-HSCT as 2L+ therapy against standard of care.

[0226] Additional efficacy investigations of REGN1979 may be included in this or additional clinical trials that include patients with: (a) mantle cell lymphoma (MCL) after BTK inhibitor failure as 2L+ therapy, (b) marginal zone lymphoma (MZL) as 2L+ therapy, and / or (c) lymphoblastic lymphoma, lymphoplasmocytic lymphoma, lympho- ... Patients with myeloma, Burkitt lymphoma, or other B-NHL subtypes.

[0227] Additional efficacy investigations of REGN1979 may be included in this or additional trials that include (a) patients with CD20+ B-cell malignancies who received a single dose of rituximab 1 day prior to the first dose of REGN1979. This rituximab induction cohort and expansion only will include patients who received a single dose of rituximab (375 mg / m2) 1 day prior to the first dose of REGN1979 [i.e., Study Day (-1)]. 2 ) will be administered. REGN1979 will begin on day 1 of week 1, resulting in a 9-month treatment period for REGN1979. Patients will be treated with up to 24 doses of REGN1979: 4 weekly doses during a 4-week lead-in period, followed by 8 additional weekly doses, and 12 doses Q2W during a 6-month maintenance period. In the first part of this rituximab lead-in cohort, REGN1979 will be administered using an 80 mg step-up dose. Once the optimal dose regimen is identified, one additional dose group of 6 patients will be evaluated with a step-up dose of 320 mg REGN1979 at the optimal dose regimen. Dose groups with step-up doses of 80 mg to 320 mg REGN1979 can also be evaluated. An additional 24 patients will then be evaluated with this optimal dosing regimen and optimal dose, and together with the six patients in the rituximab induction group who were treated with the optimal dose, a total of 30 patients will be assessed for safety and tolerability.

[0228] In any of the combination trials with standard of care, combinations may include REGN1979 and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), ICE (ifosfamide, carboplatin, and etoposide), Gem-Ox (gemcitabine and oxaliplatin), lenalidomide, or lenalidomide and rituximab.

[0229] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the 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 be included within the scope of the appended claims.

Claims

1. 1. A method of administering a therapeutic protein to a subject in a dosing regimen to reduce adverse effects of cytokine release syndrome or infusion-related reactions, comprising: administering fractions of a primary dose (D1) of said therapeutic protein during week 1 of said dosing regimen, said primary dose comprising 10 mg or less of said therapeutic protein, a first dose fraction (F1D1) constituting 40%-60% of the total primary dose and administered to said subject on day 1 of week 1, and a second dose fraction (F2D1) constituting the remaining 40%-60% of the total primary dose and administered to said subject 12-96 hours after administration of said F1D1; administering a fraction of a secondary dose (D2) of said therapeutic protein during a second week of said dosing regimen, said secondary dose being less than or equal to half of the maximum weekly dose of said therapeutic protein, a first dose fraction (F1D2) comprising 40-60% of the total secondary dose and a second dose fraction (F2D2) comprising the remaining 40%-60% of said total secondary dose, said F2D2 being administered to said subject 12-96 hours after administration of said F1D2 during the second week of said dosing regimen; and administering said maximum weekly dose of said therapeutic protein to said subject as a single dose during subsequent weeks of said dosing regimen.

2. The method of claim 1, wherein the F2D1 is administered to the subject 24 to 96 hours after administration of the F1D1.

3. The method of claim 1, wherein the F2D1 is administered to the subject 18 to 72 hours after administration of the F1D1.

4. The method of any one of claims 1 to 3, wherein the F2D2 is administered to the subject 24 to 96 hours after administration of the F1D2.

5. The method of any one of claims 1 to 3, wherein the F2D2 is administered to the subject 18 to 72 hours after administration of the F1D2.

6. 6. The method of any one of claims 1 to 5, wherein the subsequent week is week 3 of the dosing regimen.

7. 6. The method of any one of claims 1 to 5, wherein the subsequent week is week 4 of the dosing regimen.

8. 6. The method of any one of claims 1 to 5, wherein the subsequent week is week 14 of the dosing regimen.

9. 6. The method of any one of claims 1 to 5, wherein the subsequent week is any one of weeks 4 to 36 of the dosing regimen.

10. moreover, administering a fraction of a tertiary dose (D3) of the therapeutic protein during week 3 of the dosing regimen, wherein the tertiary dose is greater than or equal to half of the maximum weekly dose of the therapeutic protein and less than or equal to the maximum weekly dose of the therapeutic protein, a first dose fraction (F1D3) comprising 40%-60% of the total tertiary dose and a second dose fraction (F2D3) comprising the remaining 40%-60% of the total tertiary dose, and wherein the F2D3 is administered to the subject 12-96 hours after administration of the F1D3 during week 3 of the dosing regimen; administering said maximum weekly dose of said therapeutic protein to said subject once during subsequent weeks of said dosing regimen; 6. The method of claim 1, further comprising administering the compound as a dose.

11. The method of claim 10, wherein the F2D3 is administered to the subject 24 to 96 hours after administration of the F1D3.

12. The method of claim 10, wherein the F2D3 is administered to the subject 18 to 72 hours after administration of the F1D3.

13. 13. The method of any one of claims 10-12, wherein the subsequent week is week 4 of the dosing regimen.

14. 13. The method of any one of claims 10-12, wherein the subsequent week is week 14 of the dosing regimen.

15. 13. The method of any one of claims 10-12, wherein the subsequent week is any one of weeks 4 to 36 of the dosing regimen.

16. 13. The method of any one of claims 10-12, wherein the tertiary dose is administered as a single dose in weeks 4-12 of the dosing regimen.

17. 1. A method of administering a therapeutic protein to a subject in a dosing regimen to reduce adverse effects of cytokine release syndrome or infusion-related reactions, comprising: administering fractions of a primary dose (D1) of said therapeutic protein during week 1 of said dosing regimen, said primary dose comprising 10 mg or less of said therapeutic protein, a first dose fraction (F1D1) constituting 40%-60% of the total primary dose and administered to said subject on day 1 of week 1, and a second dose fraction (F2D1) constituting the remaining 40%-60% of the total primary dose and administered to said subject 12-96 hours after administration of said F1D1; administering a fraction of a secondary dose (D2) of said therapeutic protein during a second week of said dosing regimen, said secondary dose being equal to a maximum weekly dose of said therapeutic protein, a first dose fraction (F1D2) comprising 40%-60% of the total secondary dose and a second dose fraction (F2D2) comprising the remaining 40%-60% of said total secondary dose, said F2D2 being administered to said subject 12-96 hours after administration of said F1D2 during the second week of said dosing regimen; and administering said maximum weekly dose of said therapeutic protein to said subject as a single dose during subsequent weeks of said dosing regimen.

18. 18. The method of any one of claims 1-17, wherein the maximum weekly dose of the therapeutic protein is administered to the subject as a single dose for 1-8 weeks, 1-12 weeks, or 1-16 weeks during the weekly phase of the dosing regimen.

19. 19. The method of any one of claims 1-18, further comprising administering said maximum weekly dose of said therapeutic protein to said subject as a single dose once every two weeks during a maintenance phase of said dosing regimen following completion of the weekly phase of said dosing regimen.

20. 19. The method of any one of claims 1-18, further comprising administering said maximum weekly dose of said therapeutic protein to said subject as a single dose once every three weeks during a maintenance phase of said dosing regimen following completion of the weekly phase of said dosing regimen.

21. 19. The method of any one of claims 1-18, further comprising administering said maximum weekly dose of said therapeutic protein to said subject as a single dose once every four weeks during a maintenance phase of said dosing regimen following completion of the weekly phase of said dosing regimen.

22. The method of any one of claims 19 to 21, wherein the maintenance phase is for at least 24 weeks.

23. 23. The method of claim 22, wherein the maintenance phase is 24 weeks.

24. The method according to any one of claims 1 to 23, wherein the first dose (D1) is 1 mg.

25. The method according to any one of claims 1 to 24, wherein the secondary dose (D2) is 20 mg.

26. 26. The method of any one of claims 10-16 or 18-25, wherein the tertiary dose is 40 mg.

27. 26. The method of any one of claims 10-16 or 18-25, wherein the tertiary dose is 80 mg.

28. 26. The method of any one of claims 10-16 or 18-25, wherein the tertiary dose is 160 mg.

29. 26. The method of any one of claims 10-16 or 18-25, wherein the tertiary dose is 320 mg.

30. 30. The method of any one of claims 1 to 29, wherein the F1D1 constitutes 50% of the total primary dose and the F2D1 constitutes 50% of the total primary dose.

31. 31. The method of any one of claims 1 to 30, wherein the F1D2 constitutes 50% of the total secondary dose and the F2D2 constitutes 50% of the total secondary dose.

32. 32. The method of any one of claims 10-16 or 18-31, wherein the F1D3 constitutes 50% of the total tertiary dose and the F2D3 constitutes 50% of the total tertiary dose.

33. 33. The method of any one of claims 1-32, wherein the maximum weekly dose of the therapeutic protein is between 5 mg and 320 mg.

34. 34. The method of claim 33, wherein 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, 320 mg, 480 mg, or 640 mg.

35. 35. The method of claim 34, wherein the maximum weekly dose is 80 mg.

36. 35. The method of claim 34, wherein the maximum weekly dose is 160 mg.

37. 35. The method of claim 34, wherein the maximum weekly dose is 320 mg.

38. 38. The method of any one of claims 1 to 37, wherein each dose or dose fraction is administered to the subject over a period of 1 to 6 hours.

39. The method of any one of claims 1 to 38, wherein the subject has been diagnosed with cancer, such as a hematological cancer.

40. 40. The method of claim 39, wherein the cancer is a B cell malignancy.

41. 41. The method of claim 40, wherein the B cell malignancy is a CD20+ B cell malignancy.

42. 42. The method of claim 40 or 41, wherein the cancer is 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, Burkitt's lymphoma, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma, or Waldenstrom's macroglobulinemia.

43. The method of any one of claims 1 to 38, wherein the subject has been diagnosed with follicular lymphoma (FL).

44. 44. The method of claim 43, wherein the FL is grade 1-3a.

45. The method of any one of claims 1 to 38, wherein the subject has been diagnosed with diffuse large B-cell lymphoma (DLBCL).

46. 46. ​​The method of claim 45, wherein the subject has failed a previous CAR-T therapy.

47. The method of any one of claims 1 to 38, wherein the subject has been diagnosed with mantle cell lymphoma (MCL).

48. 48. The method of claim 47, wherein the subject has failed previous Bruton's tyrosine kinase (BTK) inhibitor therapy.

49. The method of any one of claims 1 to 38, wherein the subject has been diagnosed with marginal zone lymphoma (MZL).

50. 40. The method of claim 39, wherein 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 glioma, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal cell carcinoma, oncocytoma, transitional cell carcinoma, urothelial carcinoma, bladder adenocarcinoma, or bladder small cell carcinoma.

51. The method of any one of claims 1 to 50, wherein the subject is an adult human.

52. The method of any one of claims 1 to 51, wherein the therapeutic protein is an antibody or an antigen-binding fragment thereof.

53. 53. The method of claim 52, wherein the antibody is a fully human antibody.

54. 54. The method of claim 52 or 53, wherein the antibody is a bispecific antibody or an antigen-binding fragment thereof.

55. 55. The method of claim 54, wherein a first antigen-binding arm of the bispecific antibody or antigen-binding fragment binds to a T cell antigen.

56. 56. The method of claim 55, wherein the T cell antigen is CD3.

57. 56. The method of claim 55, wherein the T cell antigen is CD28.

58. 58. The method of any one of claims 54 to 57, wherein the second antigen-binding arm of the bispecific antibody or antigen-binding fragment binds to a tumor cell antigen.

59. 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, EGF RvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, 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-12 5), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAM 59. The method of claim 58, wherein the polypeptide is selected from the group consisting of E, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

60. 60. The method of claim 59, wherein the tumor cell antigen is CD20.

61. 61. The method of claim 60, wherein the bispecific antibody is an anti-CD20 x anti-CD3 antibody.

62. The method of claim 61, wherein the anti-CD20xanti-CD3 antibody is REGN1979, or an antibody comprising the HCVR / LCVR of REGN1979, or an antibody comprising the CDRs of REGN1979.

63. 60. The method of claim 59, wherein the tumor cell antigen is BCMA.

64. 64. The method of claim 63, wherein the bispecific antibody is an anti-BCMA x anti-CD3 antibody.

65. 60. The method of claim 59, wherein the tumor cell antigen is PSMA.

66. 66. The method of claim 65, wherein the bispecific antibody is an anti-PSMA x anti-CD3 antibody.

67. 60. The method of claim 59, wherein the tumor cell antigen is MUC16.

68. 68. The method of claim 67, wherein the bispecific antibody is an anti-MUC16 x anti-CD3 antibody.

69. 60. The method of claim 59, wherein the tumor cell antigen is STEAP2.

70. 70. The method of claim 69, wherein the bispecific antibody is an anti-STEAP2 x anti-CD3 antibody.

71. 71. The method of any one of claims 1-70, wherein the therapeutic protein is maintained at a serum concentration of about 2000 mcg / L or greater following administration of the maximum weekly dose for the duration of the dosing regimen.

72. 72. The method of claim 71, wherein the therapeutic protein is maintained at a serum concentration of about 2600 mcg / L or greater after administration of the maximum weekly dose for the duration of the dosing regimen.

73. 73. The method of claim 72, wherein the therapeutic protein is maintained at an average serum concentration of at least about 3700 mcg / L following administration of the maximum weekly dose for the duration of the dosing regimen.

74. 74. The method of any one of claims 1-73, wherein the therapeutic protein is administered to the subject in combination with a second agent selected from a steroid, an antihistamine, acetaminophen, a nonsteroidal anti-inflammatory drug (NSAID), an IL-6 antagonist, or an IL-6R antagonist.

75. 75. The method of claim 74, wherein the steroid is dexamethasone or the NSAID is indomethacin.

76. 76. The method of claim 75, wherein the dexamethasone is administered to the subject about 1 to 3 hours prior to the first dose fraction (F1D1).

77. 76. The method of claim 75, wherein the dexamethasone is administered to the subject about 1-3 hours prior to the F1D1, the F2D1, the F1D2, and the F2D2.

78. 76. The method of claim 75, wherein the dexamethasone is administered to the subject about 1-3 hours prior to the F1D1, the F2D1, the F1D2, the F2D2, the F1D3, and the F2D3.

79. 75. The method of claim 74, wherein the IL-6 antagonist is an anti-IL-6 antibody or the IL-6R antagonist is an anti-IL-6R antibody.

80. The method of claim 79, wherein the anti-IL-6R antibody is sarilumab.

81. 81. The method of any one of claims 74-80, wherein administration of the second agent is excluded after the first administration of the maximum weekly dose during the dosing regimen.

82. 82. The method of any one of claims 1-81, wherein the therapeutic protein is administered to the subject in combination with a second therapeutic agent.

83. the second therapeutic agent is selected from the group consisting of rituximab, obinutuzumab, cyclophosphamide, doxorubicin, vincristine, prednisone, prednisolone, bendamustine, lenalidomide, chlorambucil, ibritumomab tiuxetan, idelalisib, copanlisib, duvelisib, etoposide, methylprednisolone, cytarabine, cisplatin, mesna, ifosfamide, mitoxantrone, and procal.

83. The method of claim 82, comprising at least one of the following: basin.

84. 83. The method of claim 82, wherein the second therapeutic agent comprises a combination of cyclophosphamide, doxorubicin, vincristine, and prednisone.

85. 83. The method of claim 82, wherein the second therapeutic agent comprises a combination of ifosfamide, cisplatin, and etoposide.

86. 83. The method of claim 82, wherein the second therapeutic agent comprises a combination of gemcitabine and oxaliplatin.

87. 83. The method of claim 82, wherein the second therapeutic agent comprises a combination of lenalidomide and rituximab.

88. 83. The method of claim 82, wherein the second therapeutic agent is lenalidomide.

89. 1. A method of treating B cell cancer in a subject, comprising: selecting a subject diagnosed with B cell cancer; Administering to the subject a therapeutic protein according to the method of any one of claims 1 to 88.

90. 90. The method of claim 89, wherein the subject has previously been treated with an anti-cancer therapy.

91. 91. The method of claim 89 or 90, wherein the subject is refractory to or has relapsed after a previous treatment.

92. 90. The method of claim 89, wherein the subject has previously been treated with an anti-CD20 antibody therapy.

93. The method of any one of claims 89-92, wherein the subject has previously been treated with a CAR-T therapy.

94. 94. The method of any one of claims 89-93, wherein 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.

95. 95. The method of any one of claims 1 to 94, wherein the incidence of grade 3 CRS and IRR is less than 10%.

96. 96. The method of claim 95, wherein the incidence of grade 3 CRS and IRR is less than 7.5% or less than 7%.

97. 97. The method of claim 95 or 96, wherein the maximum weekly dose is equal to or greater than 80 mg.

98. 98. The method of any one of claims 1 to 97, wherein any dose administered as a single dose is administered within one hour.

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