Type II anti-CD20 antibodies for reducing the formation of anti-drug antibodies

Abstract

To provide Type II anti-CD20 antibodies for use in methods of treating diseases and methods for reducing formation of anti-drug antibodies in response to the administration of a therapeutic agent using the antibodies.SOLUTION: Disclosed is a Type II anti-CD20 antibody for use in a method of treating a disease in a subject, the method comprising a treatment regimen comprising: (i) administering the Type II anti-CD20 antibody to the subject, and after a certain period of time, (ii) administering a therapeutic agent to the subject, where the period of time between the administration of the Type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient for reducing the number of B-cells in the subject in response to the administration of the Type II anti-CD20 antibody, where the Type II anti-CD20 antibody is an IgG1 antibody, and at least about 40% of the N-linked oligosaccharides in the Fc region of the anti-CD20 antibody are non-fucosylated. Also disclosed is a method for reducing the formation of anti-drug antibodies in response to the administration of a therapeutic agent using the Type II anti-CD20 antibody.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to methods of treating diseases and methods of reducing the formation of anti-drug antibodies (ADA) in response to the administration of therapeutic agents. The present invention further relates to methods of treating diseases, specifically B cell proliferative disorders, and methods of reducing adverse effects in response to the administration of therapeutic agents, specifically T cell activation therapeutic agents.

Background Art

[0002] Background The number of biotechnology-derived therapeutic agents available for use in the clinical setting has increased rapidly in recent years and includes recombinant human cytokines (e.g., alpha and beta interferons, interleukin-2), cell growth factors (e.g., GM-CSF), hormones (e.g., glucagon), neuromuscular antagonists (e.g., botulinum toxin), blood products (e.g., coagulation factor VIII), recombinant receptors (e.g., etanercept), and monoclonal antibodies. Although therapeutic proteins are generally considered safe and non-toxic, antibodies to these therapeutic agents, known as anti-drug antibodies (ADA), can develop during treatment.

[0003] ADA has been observed in relation to various therapeutic agents such as erythropoietin, factor VIII, insulin, immunotoxins, and monoclonal antibodies (Schellekens and Casadevall, J Neurol (2004), 251 [Suppl 2]:II / 4-II / 9; Mossoba et al., Clin Cancer Res (2011) 17(11): 3697-3705; Hsu et al., British Journal of Dermatology (2014) 170, 261-273). ADA formation is frequent, for example, in autoimmune patients treated with TNF blockers and affects clinical outcome (Schaeverbecke et al., Rheumatology (2015) doi: 10.1093 / rheumatology / kev277).

[0004] The occurrence of ADA can affect serum concentration and the function of therapeutic agents. The presence of ADA may increase the clearance of therapeutic agents through the formation of immune complexes between therapeutic agents and antibodies (neutralizing, non-neutralizing, or both), thereby potentially reducing the half-life of therapeutic agents. Furthermore, the activity and efficacy of therapeutic agents can be reduced through the binding of antibodies to therapeutic agents. ADA may also be associated with allergic or hypersensitivity reactions and other adverse events.

[0005] Since these adverse events related to the immune response can affect the safety and efficacy profile of therapeutic drugs, the identification and development of strategies to overcome or inhibit ADA are of great importance.

[0006] For example, several protein engineering approaches for reducing the immunogenicity of protein therapeutics have been investigated, including masking or modifying protein B-cell epitopes or modifying protein T-cell epitopes. However, the clinical safety and success of these approaches have not been tested and will require a significant amount of time for evaluation. Therefore, there is an urgent need to develop new interventions using FDA-approved reagents to prevent ADA responses.

[0007] Chemotherapy-based approaches aimed at immunosuppressing the host have been reported (Mossoba et al., Clin Cancer Res (2011) 17(11): 3697-3705).

[0008] The anti-CD20 antibody rituximab has been used in combination with methotrexate and intravenous immunoglobulin to achieve resistance to enzyme replacement therapy in patients with Morbus Pompe (Mendelsohn et al., NEJM (2009) 360:2, 194-195). However, in clinical trials, pre-treatment of the host with rituximab did not inhibit the human immune response to the immunotoxin LMB-1 (Hassan et al., Clin Cancer Res (2004) 10, 16-18).

[0009] B-cell proliferative disorders represent a heterogeneous group of malignancies that include both leukemias and lymphomas. Lymphomas arise from lymphocytes and include two major categories: Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). In the United States, lymphomas of B-cell origin constitute approximately 80-85% of cases of non-Hodgkin lymphoma and are quite heterogeneous within the B-cell subset based on the genotype and phenotype expression patterns in naive B cells. For example, the B-cell lymphoma subset includes indolent, painless, and refractory diseases such as follicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as more aggressive subtypes such as mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL).

[0010] Despite the availability of various agents for the treatment of B-cell proliferative disorders, there is a continuing need to develop safe and effective therapies to prolong remission and improve the cure rate of patients.

[0011] The strategy currently under consideration is the engagement of T cells with malignant B cells. To effectively engage T cells with malignant B cells, two recent approaches have been developed. These two approaches are as follows: 1) administration of T cells engineered ex vivo to recognize tumor cells (also known as chimeric antigen receptor-modified T cell therapy [CAR-T cells]); and 2) administration of agents that activate endogenous T cells, such as bispecific antibodies (Oak and Bartlett, Expert Opin Investig Drugs (2015) 24, 715-724).

[0012] An example of the first approach was reported in the study by Maude et al., where 30 adult and pediatric patients were treated with autologous T cells transduced with a chimeric antigen receptor lentiviral vector targeting CD19 (CTL019 CAR-T cells). As a result, durable remission was obtained based on a 67% progression-free survival rate and a 78% overall survival rate at 6 months. However, all patients had cytokine release syndrome (CRS) (associated with tumor burden), and 27% of the patients had severe CRS. Central nervous system toxicity of unknown cause was also frequently observed.

[0013] In contrast, a second approach, which involves activating endogenous T cells that recognize tumor targets, can avoid this scalability hurdle and also offers competing efficacy, safety data, and potentially a longer period of efficacy. Different CD20 + In hematological malignancies, this approach is best exemplified by blinatumomab, a CD19-CD3-targeted T cell bispecific molecule that was recently approved for patients with minimal residual disease-positive acute lymphoblastic leukemia (ALL) (Bargou et al., Science (2008) 321, 974-977). This compound is composed of two single-chain Fv fragments (the so-called BiTE® format) and leads to the lysis of CD19 + cells by cytolytic T cells. The main limitation of blinatumomab is its short half-life (about 2 hours), which requires continuous infusion by pump over 4-8 weeks. Nevertheless, it has potential efficacy in patients with relapsed / refractory non-Hodgkin lymphoma (r / r NHL) and ALL who require step-up dosing (SUD) to mitigate severe cytokine release syndrome and CNS toxicity (Nagorsen and Baeuerle, Exp Cell Res (2011) 317, 1255-1260).

[0014] The CD20-CD3-targeted T cell bispecific molecule, CD20xCD3 bsAb, is another example of a next-generation B cell-targeted antibody. CD20xCD3 bsAb is a T cell bispecific (TCB) antibody that targets CD20 expressed on B cells and the CD3 epsilon chain (CD3e) present on T cells.

[0015] The mechanism of action of CD20xCD3 bsAb involves + simultaneous binding to + B cells and CD3 + T cells, leading to T cell activation and T cell-mediated killing of B cells. In the presence of B cells, whether in circulation or in tissues, pharmacologically active doses will cause T cell activation and the release of associated cytokines. CD20xCD3 bsAb has enhanced potency in preclinical models compared to competing T cell engaging agents, has an IgG-based format, and has a significantly improved half-life compared to blinatumomab.

[0016] Cytokine release is a result of T cell activation. In a phase 1 trial conducted by TeGenero (Suntharalingam et al., N Engl J Med (2006) 355, 1018 - 1028), all 6 healthy volunteers experienced a fatal and severe cytokine release syndrome (CRS) shortly after injection of an inappropriately administered T cell-stimulating superagonist anti-CD28 monoclonal antibody. More recently, in the above-mentioned study by Maude et al. on CD19-targeted, chimeric antigen receptor T cell (CAR-T cell) therapy in patients with relapsed ALL, all 30 had cytokine release, and 27% of the patients were classified as severe. CRS is a common but severe complication of CAR-T cell therapy (reviewed in Xu and Tang, Cancer Letters (2014) 343, 172 - 178).

[0017] Regarding blinatumomab, a CD19-CD3 T cell bispecific agent, severe CRS and CNS toxicity have also been frequently observed. (Klinger et al., Blood. 2012;119(26):6226-6233). In all clinical trials, neurological toxicity has occurred in approximately 50% of patients receiving blinatumomab, and the types of toxicity observed are well defined in the package insert.

[0018] Whether CNS toxicity is related to early cytokine release or T cell activation, or how it is related, is not fully understood. Similar to blinatumomab, CNS AEs (ranging from delirium to encephalopathy) have been reported in 43% (13 / 30) of patients with r / r ALL treated with CD19-targeted CAR-T cells (Maude et al., N Engl J Med (2014) 371,1507-1517; Ghorashian et al., Br J Haematol (2015) 169, 463-478). Neurotoxic effects typically occurred after the symptoms of CRS had peaked and began to resolve. However, no direct and clear association with severe CRS was found. The authors proposed that the mechanism of neurotoxicity could involve direct CAR-T cell-mediated toxicity or that it could be cytokine-mediated. In contrast, an association between severe CRS and neurotoxicity (e.g., encephalopathy) has been suggested in another study of CD19-targeted CAR-T cell therapy (Davila et al., Sci Transl Med (2014) 6, 224ra25), and is presumed to be due to general T cell activation as opposed to direct CAR-T-induced damage.

[0019] Cytokine release and / or CNS-related toxicity is particularly prominent in T cell bispecific antibodies that link CD3 + cells to tissue-restricted (i.e., non-circulating) target cells compared to other T cell bispecific antibodies that link CD3 + cells to B cells.

[0020] Accordingly, there is a need for methods to reduce or prevent such adverse effects of these promising agents that have the potential to significantly contribute to the treatment of patients with B-cell proliferative disorders such as NHL and CLL.

Summary of the Invention

[0021] The present invention is based on the surprising findings that (i) the formation of ADA in response to administration of an immunogenic therapeutic agent to a subject can be effectively and durably prevented, and (ii) cytokine release associated with administration of a therapeutic agent, specifically a T-cell activating therapeutic agent such as a CD20XCD3 bsAB, to a subject can be significantly reduced by pre-treating the subject with a type II anti-CD20 antibody, such as obinutuzumab.

[0022] Obinutuzumab is a humanized glycoengineered type II anti-CD20 mAb that binds to the CD20 antigen with high affinity and induces antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), low complement-dependent cytotoxicity (CDC) activity, and high levels of direct cell death induction. To date, the safety profile of obinutuzumab (including cytokine release) has been evaluated and managed in hundreds of patients in ongoing obinutuzumab clinical trials.

[0023] Without wishing to be bound by theory, the use of obinutuzumab (GAZYVA®) pre-treatment (GPT) should help to rapidly deplete B cells in both peripheral blood and secondary lymphoid organs, while supporting a sufficiently high exposure level of the therapeutic agent from the start of dosing to mediate tumor cell elimination and reducing the risk of highly relevant adverse events (AEs) from strong systemic T-cell activation by the (T-cell activating) therapeutic agent (e.g., CRS). In addition to supporting the safety profile of a (T-cell activating) therapeutic agent such as a CD20XCD3 bsAB, GPT should also help to prevent the formation of anti-drug antibodies (ADA) against the therapeutic molecule.

[0024] For patients, GPT needs to translate into better drug exposure with an enhanced safety profile.

[0025] Compared to other methods such as step - up dosing (SUD), GPT must be more effective in achieving the above - mentioned goals. For example, single - dose obinutuzumab should enable patients with relapsed / refractory disease to receive the full therapeutic dose of a T - cell activating therapeutic agent such as a once - determined CD20XCD3 bsAB without a time delay from step - up dosing. In contrast, in an ongoing Phase 2 trial, the blinatumomab dosing regimen for patients with r / r DLBCL incorporates a double step - up approach (i.e., 9→28→112 μg / m 2 / day), and thus takes 14 days to reach the maximum dose of 112 μg / m 2 / day (Viardot el at., Hematol Oncol (2015) 33, 242(Abstract 285)). As shown in the examples, after pre - treatment with obinutuzumab, administration of CD20XCD3 bsAB to cynomolgus monkeys was tolerated up to levels 10 - fold higher than those tolerated without GPT. In addition to a large reduction in cytokine release in peripheral blood associated with the first CD20XCD3 bsAB injection, efficient peripheral blood B - cell depletion and anti - tumor activity were observed with GPT.

[0026] Accordingly, in a first aspect, the present invention provides a method for (i) reducing the formation of anti - drug antibodies (ADA) against a therapeutic agent in a subject and / or (ii) reducing cytokine release associated with the administration of a therapeutic agent, specifically a T - cell activating therapeutic agent, in a subject, the method comprising administering a type II anti - CD20 antibody to the subject prior to the administration of the therapeutic agent. In one embodiment, the period between the administration of the type II anti - CD20 antibody and the administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to the administration of the type II anti - CD20 antibody.

[0027] In a further aspect, the present invention provides a method for treating a disease in a subject, the method comprising (i) Administration of a type II anti-CD20 antibody to a subject, and, after a certain period, consecutively (ii) Administration of a therapeutic agent to the subject comprising a treatment regimen, wherein the period between the administration of the type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to the administration of the type II anti-CD20 antibody.

[0028] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) in the subject in response to the administration of the therapeutic agent as compared to a corresponding treatment regimen without the administration of the type II anti-CD20 antibody.

[0029] In another embodiment, the treatment regimen effectively reduces cytokine release associated with the administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the type II anti-CD20 antibody. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0030] In a further aspect, the present invention provides a type II anti-CD20 antibody for use in a method for reducing the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject and / or for reducing cytokine release associated with the administration of a therapeutic agent, specifically a T cell activating therapeutic agent, in a subject, the method comprising administration of a type II anti-CD20 antibody to the subject prior to administration of the therapeutic agent.

[0031] In one embodiment, the period between the administration of the type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to the administration of the CD20 antibody.

[0032] In a further aspect, the present invention provides a type II anti-CD20 antibody for use in a method for treating a disease in a subject, the method comprising (i) Administration of a type II anti-CD20 antibody to the subject, and, after a certain period, consecutively (ii) Administration of a therapeutic agent to the subject comprising a treatment regimen that includes, wherein the period between administration of the type II anti-CD20 antibody and administration of the therapeutic agent is sufficient for the reduction in the number of B cells in the subject in response to administration of the type II anti-CD20 antibody.

[0033] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) against the therapeutic agent in the subject (in response to administration of the therapeutic agent) as compared to a corresponding treatment regimen that does not involve administration of an anti-CD20 antibody.

[0034] In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen that does not involve administration of the type II anti-CD20 antibody. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0035] In a further aspect, the present invention provides the use of a type II anti-CD20 antibody in the manufacture of a medicament for (i) reducing the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject and / or (ii) reducing cytokine release associated with administration of a therapeutic agent, specifically a T cell activating therapeutic agent, in a subject, wherein the medicament is (i) administered to the subject with a type II anti-CD20 antibody, and, after a certain period, consecutively (ii) administered to the subject with the therapeutic agent in a treatment regimen that includes. wherein the period between administration of the type II anti-CD20 antibody and administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to administration of the type II anti-CD20 antibody.

[0036] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) against the therapeutic agent in the subject as compared to a corresponding treatment regimen that does not involve administration of an anti-CD20 antibody.

[0037] In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of a therapeutic agent in a subject as compared to a corresponding treatment regimen that does not involve administration of a type II anti-CD20 antibody. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0038] In yet another aspect, the present invention provides a kit for reducing (i) the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject and / or (ii) cytokine release associated with administration of a therapeutic agent, specifically a T cell activating therapeutic agent, in a subject, the kit comprising a package containing a type II anti-CD20 antibody composition and instructions for using the type II anti-CD20 antibody composition in a treatment regimen, the treatment regimen comprising (i) administration of the type II anti-CD20 antibody composition to the subject, and, after a certain period, consecutively (ii) administration of the therapeutic agent to the subject wherein the period between administration of the type II anti-CD20 antibody composition and administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to administration of the type II CD20 antibody.

[0039] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject as compared to a corresponding treatment regimen that does not involve administration of a type II anti-CD20 antibody.

[0040] In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of a therapeutic agent in a subject as compared to a corresponding treatment regimen that does not involve administration of a type II anti-CD20 antibody. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0041] In one embodiment, the kit further comprises a therapeutic agent composition.

[0042] In a further aspect, the present invention provides a therapeutic agent for use in a method of treating a disease in a subject, the method comprising​ (i) Administration of a type II anti-CD20 antibody to a subject, and, after a certain period, consecutively (ii) Administration of a therapeutic agent to the subject comprising a treatment regimen, wherein the period between the administration of the type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to the administration of the CD20 antibody.

[0043] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) in the subject in response to the administration of the therapeutic agent as compared to a corresponding treatment regimen without the administration of the type II anti-CD20 antibody.

[0044] In another embodiment, the treatment regimen effectively reduces cytokine release associated with the administration of the therapeutic agent in the subject as compared to a corresponding treatment regimen without the administration of the type II anti-CD20 antibody. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0045] In a further aspect, the present invention provides the use of a therapeutic agent in the manufacture of a medicament for the treatment of a disease in a subject, wherein the treatment (i) Administration of a type II anti-CD20 antibody to a subject, and, after a certain period, consecutively (ii) Administration of a therapeutic agent to the subject comprising a treatment regimen, wherein the period between the administration of the type II anti-CD20 antibody and the administration of the therapeutic agent is sufficient to reduce the number of B cells in the subject in response to the administration of the type II anti-CD20 antibody.

[0046] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) in the subject in response to the administration of the therapeutic agent as compared to a corresponding treatment regimen without the administration of the type II anti-CD20 antibody.

[0047] In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of a therapeutic agent in a subject as compared to a corresponding treatment regimen that does not involve administration of a type II anti-CD20 antibody. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0048] In a further aspect, the present invention provides a kit for treating a disease in a subject, comprising a therapeutic agent composition and instructions for using the therapeutic agent composition in a treatment regimen, the treatment regimen (i) administration of a type II anti-CD20 antibody to the subject, and, after a period of time, consecutively (ii) administration of the therapeutic agent composition to the subject comprising, wherein the period between administration of the type II anti-CD20 antibody and administration of the therapeutic agent composition is sufficient to reduce the number of B cells in the subject in response to administration of the type II anti-CD20 antibody.

[0049] In one embodiment, the treatment regimen effectively reduces the formation of anti-drug antibodies (ADA) against the therapeutic agent in the subject as compared to a corresponding treatment regimen that does not involve administration of a type II anti-CD20 antibody composition.

[0050] In another embodiment, the treatment regimen effectively reduces cytokine release associated with administration of a therapeutic agent in a subject as compared to a corresponding treatment regimen that does not involve administration of a type II anti-CD20 antibody composition. In such an embodiment, the therapeutic agent is preferably a T cell activating therapeutic agent.

[0051] In one embodiment, the kit further comprises a type II anti-CD20 antibody composition.

[0052] The methods, uses, type II anti-CD20 antibodies, therapeutic agents and kits of the present invention can incorporate any of the features described below alone or in combination.

[0053] In one embodiment, the type II anti-CD20 antibody comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9.

[0054] In a more specific embodiment, the type II anti-CD20 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11.

[0055] In one embodiment, the type II anti-CD20 antibody is an IgG antibody, specifically an IgG1 antibody.

[0056] In one embodiment, the type II anti-CD20 antibody is engineered such that the proportion of afucosylated oligosaccharides in the Fc region is increased as compared to an unengineered antibody. In one embodiment, at least about 40% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are afucosylated.

[0057] In a particular embodiment, the anti-CD20 antibody is obinutuzumab.

[0058] In some embodiments, particularly in related aspects of the invention involving a reduction in the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject, the therapeutic agent comprises a polypeptide.

[0059] In some embodiments, particularly in related aspects of the invention involving a reduction in the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject, the therapeutic agent comprises an antibody.

[0060] In one such embodiment, the antibody specifically binds to carcinoembryonic antigen (CEA). In one embodiment, the antibody comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 15, and HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19. In a further embodiment, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 20 and a light chain variable region sequence of SEQ ID NO: 21. In another such embodiment, the antibody specifically binds to CD3, specifically CD3 epsilon. In one embodiment, the antibody comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37. In a further embodiment, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 38 and a light chain variable region sequence of SEQ ID NO: 39.

[0061] In some embodiments, particularly in related aspects of the present invention involved in reducing the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject, the therapeutic agent comprises a cytokine.

[0062] In one such embodiment, the cytokine is interleukin-2 (IL-2).

[0063] In another such embodiment, the cytokine is a mutant human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 12).

[0064] In some embodiments, particularly in related aspects of the present invention involved in reducing the formation of anti-drug antibodies (ADA) against a therapeutic agent in a subject, the therapeutic agent comprises an immunoconjugate.

[0065] In one such embodiment, the immunoconjugate comprises: (a) an antibody comprising a heavy chain variable region that specifically binds to CEA and comprises heavy chain complementarity-determining regions (HCDRs) 1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 15, and HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising light chain CDRs (LCDRs) 1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19; and (b) a mutant human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A, and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 12).

[0066] In certain such embodiments, the therapeutic agent comprises serglifamab amunakin (CEA-IL2v).

[0067] In some embodiments, particularly in relevant aspects of the invention involved in reducing the formation of anti-drug antibodies (ADAs) against the therapeutic agent in a subject, the therapeutic agent comprises a bispecific antibody that specifically binds to CEA and CD3.

[0068] In one such embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD3 and comprises heavy chain complementarity-determining regions (HCDRs) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain CDRs (LCDRs) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37; and (ii) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CEA and comprises heavy chain complementarity-determining regions (HCDRs) 1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 15, and HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising light chain CDRs (LCDRs) 1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19 and comprises a bispecific antibody.

[0069] In certain embodiments, the therapeutic agent comprises CEA TCB.

[0070] In some embodiments, particularly in related embodiments of the invention involving a reduction in cytokine release associated with administration of a therapeutic agent in a subject, the therapeutic agent is a T cell activating therapeutic agent.

[0071] In one embodiment, the T cell activating therapeutic agent includes an antibody, specifically a multispecific (e.g., bispecific) antibody.

[0072] In one embodiment, the antibody specifically binds to an activated T cell antigen.

[0073] In one embodiment, the antibody specifically binds to an antigen selected from the group consisting of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, and CD127.

[0074] In one embodiment, the antibody specifically binds to CD3, specifically CD3ε.

[0075] In one embodiment, the antibody includes a heavy chain variable region comprising heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37.

[0076] In one embodiment, the antibody includes the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

[0077] In one embodiment, the antibody specifically binds to a B cell antigen, specifically a malignant B cell antigen.

[0078] In one embodiment, the antibody specifically binds to an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37, and CD5, particularly CD20 or CD19.

[0079] In one embodiment, the antibody specifically binds to CD20.

[0080] In one embodiment, the antibody comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9.

[0081] In one embodiment, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 10 and a light chain variable region sequence of SEQ ID NO: 11.

[0082] In one embodiment, the antibody is a multispecific antibody, specifically a bispecific antibody.

[0083] In one embodiment, the multispecific antibody specifically binds to (i) an activated T cell antigen and (ii) a B cell antigen.

[0084] In one embodiment, the multispecific antibody specifically binds to (i) CD3 and (ii) an antigen selected from CD20 and CD19.

[0085] In one embodiment, the multispecific antibody specifically binds to CD3 and CD20.

[0086] In some embodiments, particularly in related aspects of the present invention involving a reduction in cytokine release associated with the administration of a therapeutic agent in a subject, the therapeutic agent is (i) an antigen-binding portion that specifically binds to CD3 and comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37; and (ii) an antigen-binding portion that specifically binds to CD20 and comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9 and comprises a bispecific antibody.

[0087] In certain embodiments, the therapeutic agent comprises a CD20XCD3 bsAB.

[0088] In some embodiments, particularly in relevant embodiments of the invention involving a reduction in cytokine release associated with administration of the therapeutic agent in a subject, the therapeutic agent comprises T cells expressing a chimeric antigen receptor (CAR) or a CAR, specifically a CAR that specifically binds to a B cell antigen, more specifically a CAR that specifically binds to an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37, and CD5.

[0089] In some embodiments, particularly in relevant embodiments of the invention involving a reduction in cytokine release associated with administration of the therapeutic agent in a subject, the disease is a B cell proliferative disorder, specifically a CD20-positive B cell disorder. In one embodiment, the disease is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin lymphoma (HL). BRIEF DESCRIPTION OF THE DRAWINGS

[0090]

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Mode for Carrying Out the Invention

[0091] Definitions The terms in this specification are used as generally used in the art, unless otherwise specifically defined herein.

[0092] CD20 (also known as B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the human protein is characterized in the UniProt database entry P11836) is a hydrophobic transmembrane protein with an approximate molecular weight of 35 kD that is expressed in pre-B and mature B lymphocytes (Valentine, M.A. et al., J. Biol. Chem. 264 (1989) 11282-11287; Tedder, T.F., et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, D.A., et al., EMBO J. 7 (1988) 711-717; Tedder, T.F., et al., J. Immunol. 142 (1989) 2560-2568). The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron / exon splice boundaries and exhibit unique expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B-lymphocyte surface molecule that plays a role in the development and differentiation of B-cells into plasma cells. This family member is located at 11q12 within a cluster of family members. Alternative splicing of this gene results in two transcript variants that encode the same protein.

[0093] Unless otherwise indicated, the term "CD20" as used herein refers to any native CD20 derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice, rats). The term includes "full-length" and unprocessed CD20, as well as any form of CD20 resulting from intracellular processing. The term also includes naturally occurring variants of CD20, such as splice variants or allelic variants. In one embodiment, CD20 is human CD20. An exemplary amino acid sequence of human CD20 is shown in SEQ ID NO: 1.

[0094] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that can bind to CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20. In one embodiment, the degree of binding of an anti-CD20 antibody to an irrelevant, non-CD20 protein is less than about 10% of the binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD20 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20s from different species.

[0095] "Type II anti-CD20 antibody" means an anti-CD20 antibody having the binding characteristics and biological activities of a type II anti-CD20 antibody, as described in Cragg et al., Blood 103 (2004) 2738-2743; Cragg et al., Blood 101 (2003) 1045-1052, Klein et al., mAbs 5 (2013), 22-33, and summarized in Table 1 below. TIFF0007701197000001.tif91170

[0096] Examples of type II anti-CD20 antibodies include, for example, obinutuzumab (GA101), tositumomab (B1), humanized B-Ly1 antibody IgG1 (chimeric humanized IgG1 antibody disclosed in International Publication No. 2005 / 044859), 11B8 IgG1 (disclosed in International Publication No. 2004 / 035607), and AT80 IgG1.

[0097] Examples of type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, belimumab, ocrelizumab, PRO13192, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in International Publication No. 2005 / 103081), 2F2 IgG1 (disclosed in International Publication Nos. 2004 / 035607 and 2005 / 103081), and 2H7 IgG1 (disclosed in International Publication No. 2004 / 056312).

[0098] The term "humanized B-Ly1 antibody" refers to a humanized B-Ly1 antibody as disclosed in International Publication Nos. 2005 / 044859 and 2007 / 031875, obtained from the mouse monoclonal anti-CD20 antibody B-Ly1 (variable region of the mouse heavy chain: SEQ ID NO: 2 (VH); variable region of the mouse light chain: SEQ ID NO: 3) (see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerization with the human constant domain derived from IgG1 and subsequent humanization (see International Publication Nos. 2005 / 044859 and 2007 / 031875). These "humanized B-Ly1 antibodies" are disclosed in detail in International Publication Nos. 2005 / 044859 and 2007 / 031875.

[0099] As used herein, the term "cytokine" refers to a molecule that mediates and / or regulates biological or cellular functions or processes (e.g., immunity, inflammation, and hematopoiesis). As used herein, the term "cytokine" includes "lymphokine", "chemokine", "monokine", and "interleukin". Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. A particular cytokine is IL-2. As used herein, the term "cytokine" also means to include cytokine variants that contain one or more amino acid mutations in the amino acid sequence of the corresponding wild-type cytokine, such as the IL-2 variants described in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003 / 0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Patent No. 5,229,109; U.S. Patent Application Publication No. 2007 / 0036752; International Publication No. 2008 / 0034473; International Publication No. 2009 / 061853; or International Publication No. 2012 / 107417.

[0100] Unless otherwise indicated, the term "interleukin-2" or "IL-2" as used herein refers to any native IL-2 derived from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice, rats). The term includes unprocessed IL-2, as well as any form of IL-2 resulting from intracellular processing. The term also includes naturally occurring variants of IL-2, such as splice variants or allelic variants. An exemplary amino acid sequence of human IL-2 is shown in SEQ ID NO: 12. Unprocessed human IL-2 further includes an N-terminal 20 amino acid signal peptide having the sequence of SEQ ID NO: 31 that is not present in the mature IL-2 molecule. The term "interleukin-2" as used herein also means IL-2 variants that contain one or more amino acid mutations in the amino acid sequence of the corresponding wild-type cytokine, such as those described in Sauve et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Hu et al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003 / 0124678; Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al., Cancer Res 53, 2597-602 (1993) and U.S. Patent No. 5,229,109; U.S. Patent Application Publication No. 2007 / 0036752; International Publication No. 2008 / 0034473; International Publication No. 2009 / 061853; or International Publication No. 2012 / 107417.

[0101] As used herein, the term "IL-2 variant" or "variant IL-2 polypeptide" is intended to encompass any variant form of the IL-2 molecule, including full-length IL-2, truncated forms of IL-2, and forms in which IL-2 is linked to another molecule by fusion or chemical bonding, etc. "Full-length" as used in relation to IL-2 is intended to mean a mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, e.g., SEQ ID NO: 12). The various forms of the IL-2 variant are characterized by having at least one amino acid mutation that affects the interaction between IL-2 and CD25. This mutation can typically include substitution, deletion, truncation, or modification of the wild-type amino acid residue located at that position. Variants obtained by amino acid substitution are preferred. Unless otherwise indicated, the IL-2 variant can be referred to herein as an IL-2 variant peptide sequence, an IL-2 variant polypeptide, an IL-2 variant protein, or an IL-2 variant analog. In this specification, the notations of various forms of IL-2 are made with respect to the sequence shown in SEQ ID NO: 12. In this specification, various notations are used to indicate the same mutation. For example, the mutation from phenylalanine to alanine at position 42 can be indicated as 42A, A42, A42, F42A, or Phe42Ala.

[0102] As used herein, the terms "cytokine release" or "cytokine liberation" are synonymous with "cytokine storm" or "cytokine release syndrome" (abbreviated "CRS"), and refer to an increase in the levels of cytokines, particularly tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2) and / or interleukin-8 (IL-8) in the blood of a subject that causes adverse symptoms during or immediately after administration of a therapeutic agent (e.g., within 1 day). Cytokine release is a common side effect of therapeutic agents and is a type of infusion-related reaction (IRR) that is timely associated with the administration of a therapeutic agent. IRR typically occurs during or immediately after administration of a therapeutic agent, i.e., typically within 24 hours after infusion, mainly with the first infusion. In some cases, for example, after administration of CAR-T cells, CRS may occur later, e.g., several days after administration, during the proliferation of CAR-T cells. The incidence and severity typically decrease with subsequent infusions. The symptoms range from symptomatic discomfort to fatal events and include fever, chills, dizziness, hypertension, hypotension, dyspnea, restlessness, sweating, flushing, skin rash, tachycardia, headache, tumor pain, nausea, vomiting and / or organ failure.

[0103] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to reach the final construct, provided that the final construct retains the desired properties, such as reduced binding to CD25 or Fc receptors. Deletions and insertions of amino acid sequences include amino-terminal and / or carboxy-terminal deletions, as well as insertions of amino acids. Certain amino acid mutations are amino acid substitutions. For the purpose of modifying binding properties such as those of the IL-2 polypeptide or the Fc region, non-conservative amino acid substitutions, i.e., substituting one amino acid with another having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with naturally occurring amino acid derivatives or non-naturally occurring amino acids of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic methods or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods other than genetic manipulation, such as methods for modifying the side chain groups of amino acids by chemical modification, may also be useful. In this specification, various notations are used to indicate the same amino acid mutation. For example, the substitution of proline with glycine at position 329 of the Fc region is denoted as 329G, G329, G 329 , P329G, or Pro329Gly.

[0104] Unless otherwise indicated, the term "CD25" or "alpha subunit of the IL-2 receptor" as used herein refers to any native CD25 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice, rats). The term includes "full-length" and unprocessed CD25, as well as any form of CD25 resulting from intracellular processing. The term also includes naturally occurring variants of CD25, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25. The amino acid sequence of human CD25 is shown in UniProt (www.uniprot.org) accession number P01589, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000408.

[0105] As used herein, the term "high-affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor consisting of the receptor gamma subunit (the common cytokine receptor gamma subunit, gamma c , also known as CD132), the receptor beta subunit (also known as CD122 or p70), and the receptor alpha subunit (also known as CD25 or p55). In contrast, the term "intermediate-affinity IL-2 receptor" refers to an IL-2 receptor that contains only the gamma and beta subunits and does not contain the alpha subunit (see, for review, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0106] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of molecule X for its partner Y is typically represented by the dissociation constant (K D ). This dissociation constant (K D ) is the ratio of the dissociation rate constant to the association rate constant (k offand k on ) The ratio is. Therefore, equivalent affinity can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods well-known in the art. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0107] "Reduction" (and its grammatical variations such as "reduce" or "reducing"), for example, a reduction in the number of B cells or the formation of ADA or cytokine release, refers to a reduction in each quantity as measured by an appropriate method well-known in the art. For clarity, this term also includes a reduction to zero (or a value below the detection limit of the analytical method), i.e., complete disappearance or removal. Conversely, "increased" refers to an increase in each quantity.

[0108] "Regulatory T cell" or "T reg cell" means a special type of CD4 + T cell that can suppress the responses of other T cells. T reg cells are characterized by the expression of the α subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play an important role in the induction and maintenance of peripheral self-tolerance to antigens including those expressed by tumors. T reg cells require IL-2 for their function, development, and induction of their suppressive properties.

[0109] As used herein, "antigen-binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding portion can direct an entity to which it binds (e.g., a cytokine or a second antigen-binding portion) to a target site, e.g., a particular type of tumor cell or tumor stroma having an antigenic determinant. Antigen-binding portions include antibodies and fragments thereof as further defined herein. Preferred antigen-binding portions include the antigen-binding domains of antibodies, which include the heavy chain variable region and the light chain variable region of the antibody. In certain embodiments, the antigen-binding portion can include antibody constant regions as further defined herein and well known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include either of the two isotypes: κ and λ.

[0110] "Specifically binds" means that the binding is selective for the antigen and distinguishable from unwanted or non-specific interactions. The ability of an antigen-binding portion to bind to a particular antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those of skill in the art, such as surface plasmon resonance techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0111] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule to which an antigen-binding portion binds and forms an antigen-binding portion-antigen complex (e.g., a three-dimensional structure formed from a continuous stretch of amino acids or distinct regions of non-contiguous amino acids). Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, in a free state in serum, and / or in the extracellular matrix (ECM).

[0112] As used herein, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linearly linked by amino bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a chain of two or more amino acids are also included in the definition of "polypeptide", and the term "polypeptide" can be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modification of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, but does not necessarily have to be translated from a designated nucleic acid sequence. It can be produced by any method, including chemical synthesis. The polypeptides of the present invention may consist of amino acids of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more. A polypeptide can have a defined three-dimensional structure, but does not necessarily have such a structure. A polypeptide having a defined three-dimensional structure is said to be folded, and a polypeptide that does not have a defined three-dimensional structure and can take on a number of different conformations is said to be unfolded.

[0113] An "isolated" polypeptide or variant, or derivative thereof, is intended to mean a polypeptide of interest that is not in its natural environment. A specific level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are contemplated for isolation for the purposes of the present invention, such as by any suitable technique for separating, fractionating, or partially or substantially purifying native or recombinant polypeptides.

[0114] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide, after aligning the sequences and introducing gaps if necessary to obtain the maximum percent sequence identity, and assuming that any conservative substitutions are not part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the skill of the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes here, the % amino acid sequence identity value is obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was made by Genentech, Inc., and the source code was submitted with user documentation to the U.S. Copyright Office, Washington, D.C., 20559, and is registered under U.S. Copyright Registration No. TXU510087. Also, ALIGN-2 is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from its source code. The ALIGN-2 program should be compiled for use on a UNIX (registered trademark) operating system, including Digital UNIX (registered trademark) V4.0D. All sequence comparison parameters are set and not varied by the ALIGN-2 program. In the situation where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with, to, or against a given amino acid sequence B (or, it can also be said, a given amino acid sequence B with, to, or against a given amino acid sequence A having or containing a specific % amino acid sequence identity) is calculated as follows: 100 × fraction X / Y Here, X is the number of amino acid residues that have an exact match in the sequence comparison between A and B by the array alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0115] As used herein, the term "effector moiety" refers to a polypeptide, such as a protein or glycoprotein, that affects cell activity via, for example, signal transduction or other cellular pathways. Thus, the effector moiety can be associated with receptor-mediated signal transduction that transmits a signal from the outside of the cell membrane to regulate the response of a cell having one or more receptors for the effector moiety. In one embodiment, the effector moiety can induce a cytotoxic response in a cell having one or more receptors for the effector moiety. In another embodiment, the effector moiety can induce a proliferative response in a cell having one or more receptors for the effector moiety. In another embodiment, the effector moiety can induce differentiation in a cell having a receptor for the effector moiety. In another embodiment, the effector moiety can change (i.e., upregulate or downregulate) the expression of an endogenous cellular protein within a cell having a receptor for the effector moiety. Non-limiting examples of effector moieties include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. The effector moiety can associate with an antigen-binding moiety, such as an antibody, in various configurations to form an immunoconjugate.

[0116] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or blocks the function of a cell and / or causes cell death or cell destruction. Cytotoxic drugs include, but are not limited to, radioisotopes (e.g., At211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins, or enzymatically active toxins from bacteria, fungi, plants or animals (including fragments and / or variants thereof); and various antitumor or anticancer agents disclosed hereinafter.

[0117] The term "antibody" is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0118] The terms "full-length antibody", "intact antibody", and "complete antibody" are used interchangeably herein and refer to an antibody having a structure that is substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0119] "Antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabody, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. The term "antibody fragment" as used herein also encompasses single domain antibodies.

[0120] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein of approximately 150,000 daltons consisting of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also called the light chain constant region. The heavy chains of immunoglobulins can be assigned to one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and some of these are further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types called kappa (κ) and lambda (λ) based on the amino acid sequence of their constant domains. An immunoglobulin consists essentially of two Fab molecules and an Fc domain linked via the hinge region of the immunoglobulin.

[0121] The term "antigen-binding domain" refers to the part of an antibody that binds to a part or the whole of an antigen and contains a region that is complementary to a part or the whole of the antigen. The antigen-binding domain is provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0122] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have a similar structure in which each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs). For example, Kindt et al., Kuby Immunology, 6th See, e.g., ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0123] A “human antibody” is one produced by a human or human cell, or one having an amino acid sequence corresponding to that of an antibody derived from non-human origin that utilizes a human antibody repertoire or other sequences encoding human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0124] A “humanized” antibody refers to a chimeric antibody that contains amino acid residues derived from non-human HVRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may also comprise at least a portion of an antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to the humanized antibody.

[0125] As used herein, the terms “hypervariable region” or “HVR” refer to each region of an antibody variable domain that has a sequence that is hypervariable (“complementarity determining region” or “CDR”), and / or forms a structurally defined loop (“hypervariable loop”), and / or contains antigen contact residues (“antigen contact”). Generally, an antibody comprises a total of six HVRS, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein are (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3); (b) CDRs occurring at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contacts occurring at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732 - 745 (1996)); and (d) Combinations of (a), (b), and / or (c) that include HVR amino acid residues 46 - 56 (L2), 47 - 56 (L2), 48 - 56 (L2), 49 - 56 (L2), 26 - 35 (H1), 26 - 35b (H1), 49 - 65 (H2), 93 - 102 (H3), and 94 - 102 (H3). Including.

[0126] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) within the variable domain are numbered herein according to Kabat et al. supra.

[0127] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of four FR domains: namely, FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following sequences of VH (or VL): FR1 - H1 (L1) - FR2 - H2 (L2) - FR3 - H3 (L3) - FR4.

[0128] The "class" of an antibody refers to the type of constant domain or region that its heavy chain possesses. There are five major classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IGA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0129] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an IgG heavy chain can vary slightly, but typically the human IgG heavy chain Fc region is defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, an antibody produced by a host cell can undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain can include the full-length heavy chain or a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This can be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447) (numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (K447), may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). The "subunit" of the Fc domain as used herein refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain and has the ability to stably self-associate. For example, the subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0130] "Modification that promotes heterodimerization" refers to the manipulation of the peptide backbone or post-translational modification of a polypeptide, such as an immunoglobulin heavy chain, that reduces or prevents the association of polypeptides having the same polypeptide that forms a homodimer. Modifications that promote heterodimerization as used herein particularly include separate modifications applied to each of two polypeptides that desirably form a dimer, and these modifications are complementary to each other so as to promote the association of the two polypeptides. For example, modifications that promote heterodimerization can make their associations sterically or electrostatically favorable by changing the structure or charge of one or both of the polypeptides that desirably form a dimer. Heterodimerization occurs between two non-identical polypeptides, such as between two immunoglobulin heavy chains each fused to a further immunoconjugate component (e.g., an IL-2 polypeptide) that is not the same. In immunoconjugates useful in the present invention, the modification that promotes heterodimerization is in the heavy chain(s) of the immunoglobulin molecule, particularly in the Fc domain. In some embodiments, the modification that promotes heterodimerization includes amino acid mutations, particularly amino acid substitutions. In certain embodiments, the modification that promotes heterodimerization includes separate amino acid mutations, particularly amino acid substitutions, in each of two immunoglobulin heavy chains.

[0131] Similarly, a "modification that promotes the association of the first and second subunits of the Fc domain" is an operation of the peptide backbone or a post-translational modification of the subunits of the Fc domain that reduces or prevents the formation of homodimers by the association of a polypeptide containing the subunits of the Fc domain with the same polypeptide. Modifications that promote association as used herein include, in particular, separate modifications made to each of two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and these modifications are complementary to each other so as to promote the association of the two Fc domain subunits. For example, modifications that promote association can make their association sterically or electrostatically favorable by changing the structure or charge of one or both of the Fc domain subunits. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, and these polypeptides can be non-identical in that the additional components (e.g., antigen-binding portions) fused to each of the subunits are not the same. In some embodiments, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In certain embodiments, modifications that promote association include separate amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0132] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc region of an antibody, gives rise to a signaling phenomenon that stimulates receptor-bearing cells to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0133] The term "effector function", when used with respect to an antibody, refers to the biological activities attributable to the Fc region of the antibody that vary by antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and activation of B cells.

[0134] As used herein, the term "effector cell" refers to a population of lymphocytes that present effector subunit receptors, such as cytokine receptors, and / or Fc receptors on their surface, and effector cells bind via their receptors to effector subunits, such as cytokines, and / or the Fc region of an antibody, contributing to the destruction of target cells, such as tumor cells. Effector cells can mediate, for example, cytotoxic or phagocytic effects. Effector cells include, but are not limited to, CD8 + cytotoxic T cells, CD4 + helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells, and effector T cells such as macrophages / monocytes.

[0135] As used herein, terms such as "engineer," "engineered," and "engineering" are considered to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring polypeptide, a recombinant polypeptide, or a fragment thereof. Manipulations include modification of the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these approaches. In particular, "engineering" with the prefix "glyco-" and the term "glycosylation engineering" include metabolic engineering of the cellular glycosylation machinery, including genetic manipulation of the oligosaccharide synthesis pathway to achieve modification of the glycosylation of glycoproteins expressed in cells. Further, glycosylation engineering includes mutations and the effects of the cellular environment on glycosylation. In one embodiment, glycosylation engineering is modification of glycosyltransferase activity. In certain embodiments, engineering results in modification of glucosaminyltransferase activity and / or fucosyltransferase activity. Glycosylation engineering can be used to obtain a "host cell having increased GnTIII activity" (e.g., a host cell engineered to express increased levels of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), a "host cell having increased ManII activity" (e.g., a host cell engineered to express increased levels of one or more polypeptides having α-mannosidase II (ManII) activity), or a "host cell having decreased α(1,6) fucosyltransferase activity" (e.g., a host cell engineered to express decreased levels of α(1,6) fucosyltransferase).

[0136] The terms "host cell, host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. A host cell includes "transformants" and "transformed cells", including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parental cell and may include mutations. Mutant progeny having the same function or biological activity as that screened or selected in the originally transformed cell are included herein. A host cell is any type of cell line that can be used to produce a protein for use in the present invention. In one embodiment, the host cell is engineered to enable the production of an antibody having modified oligosaccharides. In certain embodiments, the host cell is engineered to express increased levels of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments, the host cell is further engineered to express increased levels of one or more polypeptides having α-mannosidase II (ManII) activity. Host cells include cultured cells, such as mammalian cultured cells, for example, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, and further include cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0137] As used herein, the term "polypeptide having GnTIII activity" refers to a polypeptide capable of catalyzing the addition of an N-acetylglucosamine (GlcNAc) residue to the β-linked mannoside of the trimannosyl core of an N-linked oligosaccharide. This includes fusion polypeptides that exhibit an enzymatic activity similar but not necessarily identical to that of β(1,4)-N-acetylglucosaminyltransferase III, also known as β-1,4-mannosylglycoprotein 4-β-N-acetylglucosaminyltransferase (EC 2.4.1.144) (by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)), which is measured, with or without dose-dependence, in certain biological assays. When dose-dependence exists, it need not be identical to that of GnTIII, but is substantially similar to the dose-dependence at a given activity compared to GnTIII (i.e., the candidate polypeptide will exhibit greater activity or activity no more than about 25-fold, preferably no more than about 10-fold, most preferably no more than about 3-fold that of GnTIII). In certain embodiments, a polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi-resident polypeptide. In particular, the Golgi localization domain is the localization domain of mannosidase II or GnTI, most specifically the localization domain of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of α1,6 core fucosyltransferase. Methods for generating such fusion polypeptides and using them to produce antibodies having increased effector function are disclosed in International Publication No. WO 2004 / 065540, U.S. Provisional Patent Application No. 60 / 495,142, and U.S. Patent Application Publication No. 2004 / 0241817, the entire contents of which are hereby expressly incorporated by reference.

[0138] As used herein, the term "Golgi localization domain" refers to the amino acid sequence of a Golgi resident polypeptide that serves to anchor a polypeptide in the Golgi complex. Generally, the localization domain includes the amino-terminal "tail" of the enzyme.

[0139] As used herein, the term "polypeptide having ManII activity" refers to a polypeptide capable of catalyzing the hydrolysis of terminal 1,3- and 1,6-linked α-D-mannose residues in the branched GlcNAcMan5GlcNAc2 mannose intermediate of N-linked oligosaccharides. This includes polypeptides that exhibit enzymatic activity similar but not necessarily identical to that of Golgi α-mannosidase II, also known as mannosyl oligosaccharide 1,3-1,6-α-mannosidase II (EC 3.2.1.114) (by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)).

[0140] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that leads to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which an antibody or a fragment thereof containing an Fc region specifically binds generally via the protein moiety that is the N-terminus of the Fc region. As used herein, the term "increased / decreased ADCC" refers to an increase / decrease in the number of target cells lysed within a predetermined time by the mechanism of ADCC as defined above at a predetermined antibody concentration in the medium surrounding the target cells, and / or a decrease / increase in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a predetermined number of target cells within a predetermined time by the mechanism of ADCC. The increase / decrease in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell that has not been manipulated, using the same standard production, purification, formulation, and storage methods (which are well known to those skilled in the art). For example, an increase in ADCC mediated by an antibody produced by a host cell engineered to modify the pattern of glycosylation (e.g., to express a glycosyltransferase, GnTIII, or other glycosyltransferase) by the methods described herein is relative to ADCC mediated by the same antibody produced by the same type of unengineered host cell.

[0141] An "antibody having increased / decreased antibody-dependent cell-mediated cytotoxicity (ADCC)" means an antibody having increased / decreased ADCC as determined by any suitable method well known to those skilled in the art. One acceptable in vitro ADCC assay is as follows: 1) The assay uses target cells known to express a target antigen recognized by the antigen-binding region of the antibody; 2) The assay uses, as effector cells, human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors; 3) The assay is performed according to the following protocol: i) Isolate PBMCs using standard density centrifugation procedures and resuspend them at 5x10 6Suspend in RPMI cell culture medium at cells / ml; ii) Grow target cells by standard tissue culture methods, harvest from the exponential growth phase with a viability exceeding 90%, wash in RPMI cell culture medium, and label with 100 microcuries of 51 Cr, wash twice with cell culture medium, and resuspend in cell culture medium at a density of 10 5 cells / ml; iii) Transfer 100 microliters of the above final target cell suspension to each well of a 96-well microtiter plate; iv) Serially dilute the antibody in cell culture medium from 4000 ng / ml to 0.04 ng / ml, add 50 microliters of the resulting antibody solution to the target cells in the 96-well microtiter plate, and test three times at various antibody concentrations covering the entire above concentration range. v) For the maximum release (MR) control, add 50 microliters of a 2% (v / v) aqueous solution of a non-ionic detergent (Nonidet, Sigma, St. Louis) to three additional wells in the plate containing the labeled target cells, instead of the antibody solution (in item iv above); vi) For the spontaneous release (SP) control, add 50 microliters of RPMI cell culture medium to three additional wells in the plate containing the labeled target cells, instead of the antibody solution (in item iv above); vii) Then, centrifuge the 96-well microtiter plate at 50 x g for 1 minute and incubate at 4 °C for 1 hour; viii) Add 50 microliters of the PBMC suspension (in item i above) to each well such that the effector:target cell ratio is 25:1, and place the plate in an incubator at 37 °C in a 5% CO2 atmosphere for 4 hours; ix) Recover the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter; x) Calculate the percentage of specific lysis for each antibody concentration according to the formula (ER - MR) / (MR - SR)×100, where ER is the average radioactivity quantified for that antibody concentration (see item ix above), MR is the average radioactivity quantified for the MR control (see item v above) (see item ix above), and SR is the average radioactivity quantified for the SR control (see item vi above) (see item ix above). 4) "Increased / decreased ADCC" is defined as either an increase / decrease in the maximum percentage of specific lysis observed within the tested antibody concentration range described above, and / or a decrease / increase in the antibody concentration required to achieve half of the maximum percentage of specific lysis observed within the tested antibody concentration range described above. The increase / decrease in ADCC is relative to ADCC mediated by the same antibody produced by the same type of host cell, produced using the same standard production, purification, formulation, and storage methods (which are well known to those skilled in the art) but not manipulated, and measured by the above assay.

[0142] As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one effector moiety, such as a cytokine, and an antigen-binding moiety, such as an antibody. In certain embodiments, the immunoconjugate comprises one or fewer effector moieties. Certain immunoconjugates useful in the present invention consist essentially of an antibody linked by one effector moiety and one or more peptide linkers. Certain immunoconjugates according to the present invention are fusion proteins, i.e., the components of the immunoconjugate are linked by peptide bonds.

[0143] As used herein, the term "monoclonal antibody" means an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variants that may occur naturally, e.g., those containing mutations, or that may arise during the production of the monoclonal antibody preparation and that are present generally in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and these and other exemplary methods for making monoclonal antibodies are described herein.

[0144] As used herein, terms such as "first," "second," "third," etc. with respect to antigen-binding portions, etc. are used for convenience of distinction when there are two or more of each type of portion. The use of such terms is not intended to impart a particular order or orientation unless specifically stated otherwise.

[0145] The terms "multispecificity" and "bispecificity" mean that an antigen-binding molecule is capable of specifically binding to at least two different antigenic determinants. Typically, a bispecific antigen-binding molecule comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can bind simultaneously to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0146] As used herein, the term "valence" means the presence of a specific number of antigen-binding sites within an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" means the presence of one (and no more than one) antigen-binding site specific for the antigen within the antigen-binding molecule.

[0147] "Antigen-binding site" refers to the site of an antigen-binding molecule that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody contains amino acid residues derived from complementarity-determining regions (CDRs). A natural immunoglobulin molecule typically has two antigen-binding sites, and a Fab molecule typically has a single antigen-binding site.

[0148] As used herein, a "T cell activation therapeutic agent" refers to a therapeutic agent that can induce T cell activation in a subject, particularly a therapeutic agent designed to induce T cell activation in a subject. Examples of T cell activation therapeutic agents include bispecific antibodies that specifically bind to activation T cell antigens such as CD3 and target cell antigens such as CD20 or CD19. Further examples include chimeric antigen receptors (CARs) that include a T cell activation domain and an antigen-binding portion that specifically binds to a target cell antigen such as CD20 or CD19.

[0149] As used herein, an "activation T cell antigen" refers to an antigen determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, that can induce or enhance T cell activation upon interaction with an antigen-binding molecule. Specifically, the interaction between an activation T cell antigen and an antigen-binding molecule can induce T cell activation by triggering the signal transduction cascade of the T cell receptor complex. An exemplary activation T cell antigen is CD3.

[0150] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The T cell activation bispecific antigen-binding molecules and T cell activation therapeutic agents used in the present invention can induce T cell activation. Suitable assays for measuring T cell activation are well known in the art described herein.

[0151] As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of target cells, such as tumor cells or intratumoral cells such as cells of the tumor stroma.

[0152] As used herein, "B cell antigen" refers to an antigenic determinant presented on the surface of B lymphocytes, particularly malignant B lymphocytes (in which case the antigen is also referred to as a "malignant B cell antigen").

[0153] As used herein, "T cell antigen" refers to an antigenic determinant presented on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0154] "Fab molecule" refers to a protein consisting of the VH and CH1 domains of the heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain") of an immunoglobulin.

[0155] "Chimeric antigen receptor" or "CAR" means a genetically engineered receptor protein comprising an antigen-binding portion, such as a single-chain variable fragment (scFv) of a target antibody, a transmembrane domain, an intracellular T cell activation signaling domain (e.g., the CD3 zeta chain of the T cell receptor), and optionally one or more intracellular co-stimulatory domains (e.g., CD28, CD27, CD137 (4-1BB), Ox40). CARs mediate antigen recognition, T cell activation, and - in the case of second-generation CARs - co-stimulation that increases T cell functionality and persistence. See, for review, Jackson et al., Nat Rev Clin Oncol (2016) 13, 370-383.

[0156] "B-cell proliferative disorder" means a disease in which the number of a patient's B cells is increased compared to the number of B cells in a healthy subject, and in particular, a disease in which the increase in the number of B cells is the cause or characteristic of the disease. "CD20-positive B-cell proliferative disorder" is a B-cell proliferative disorder in which B cells, specifically malignant B cells (in addition to normal B cells), express CD20. Exemplary B-cell proliferative disorders include non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), as well as several types of multiple myeloma (MM) and Hodgkin lymphoma (HL).

[0157] "Fused" means that a plurality of components (e.g., Fab molecules and Fc domain subunits) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0158] "Anti-drug antibody" or "ADA" refers to an antibody that binds to a therapeutic agent and can affect the serum concentration and function of the therapeutic agent in a subject. The presence of ADA can increase the clearance of the therapeutic agent by forming immune complexes between the therapeutic agent and the antibody (neutralizing, non-neutralizing, or both), thereby potentially decreasing the half-life of the therapeutic agent. Furthermore, the activity and efficacy of the therapeutic agent can be decreased via binding of the antibody to the therapeutic agent (especially when neutralizing ADA). ADA can also be associated with allergic or hypersensitivity reactions and other adverse events.

[0159] "An effective amount of" a drug refers to the amount necessary to bring about a physiological change in the cell or tissue to which it is administered.

[0160] The "therapeutically effective amount" of a drug, e.g., a pharmaceutical composition, refers to an effective amount at a dose and for a period of time necessary to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of a drug eliminates, reduces, delays, minimizes, or prevents the harmful effects of a disease.

[0161] The term "therapeutic agent" refers to an active ingredient of a pharmaceutical composition, for example, which is administered to a subject in an attempt to alter the natural course of a disease in the subject being treated and which can be administered for prophylaxis or during the course of a clinical pathology. An "immunotherapeutic agent" refers to a therapeutic agent that is administered to a subject in an attempt to restore or enhance the subject's immune response to a tumor, for example.

[0162] The term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0163] The term "pharmaceutical composition" refers to a preparation in a form in which the biological activity of the active ingredient contained therein is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition will be administered.

[0164] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0165] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated and can be carried out for prophylaxis or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of progression of a disease, improving or alleviating a disease state, and achieving remission or improving the prognosis. In some embodiments, the methods of the invention are used to delay the onset of a disease or slow the progression of a disease.

[0166] The term "accompanying document" is used to refer to instructions customarily included in the commercial package of a therapeutic product that contain information on indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0167] "CD3", unless otherwise specified, refers to any native CD3 derived from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice, rats). The term includes "full-length" and unprocessed CD3, as well as any form of CD3 resulting from intracellular processing. The term also includes naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, specifically the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO: 115. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank number BAB71849.1. See also SEQ ID NO: 116.

[0168] Unless otherwise indicated, "CD19" refers to the B-lymphocyte antigen CD19, also known as B-lymphocyte surface antigen B4 or T-cell surface antigen Leu-12, and includes any native CD19 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" and unprocessed CD19, as well as any form of CD19 resulting from intracellular processing. The term also includes naturally occurring variants of CD19, such as splice variants or allelic variants. In one embodiment, CD19 is human CD19. An exemplary amino acid sequence of human CD19 is shown in UniProt (www.uniprot.org) accession number P15391 (version 174), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_001770.5, and SEQ ID NO: 117.

[0169] Unless otherwise indicated, "cancer fetal antigen" or "CEA" (also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)) refers to any native CEA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term includes "full-length" and unprocessed CEA, as well as any form of CEA resulting from intracellular processing. The term also includes naturally occurring variants of CEA, such as splice variants or allelic variants. In one embodiment, CEA is human CEA. The amino acid sequence of human CEA is shown in UniProt (www.uniprot.org) accession number P06731, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2.

[0170] Unless otherwise indicated, "fibroblast activation protein" or "FAP" (also known as seprase) refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed FAP as well as any form of FAP resulting from intracellular processing. The term also encompasses variants of naturally occurring FAP, such as splice variants or allelic variants. In one embodiment, FAP is human FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884, or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2.

[0171] A "crossover" Fab molecule (also denoted as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., substituted for each other), i.e., a crossover Fab molecule comprises a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1 in the N-terminal to C-terminal direction), and a peptide chain consisting of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL in the N-terminal to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light and heavy chains are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0172] In contrast, a "conventional" Fab molecule means a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain consisting of a variable domain and a constant domain (VH-CH1 in the N-terminal to C-terminal direction) and a light chain consisting of a variable domain and a constant domain (VL-CL in the N-terminal to C-terminal direction).

[0173] Type II anti-CD20 antibody The CD20 molecule (also referred to as human B lymphocyte-restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein expressed on the surface of malignant and non-malignant pre-B and mature B lymphocytes and has been described in detail (Valentine, M.A., et al., J. Biol. Chem. 264 (1989) 11282-11287; and Einfeld, D.A., et al., EMBO J. 7 (1988) 711-717; Tedder, T.F., et al., Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 208-212; Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-1980; Tedder, T.F., et al., J. Immunol. 142 (1989) 2560-2568).

[0174] CD20 is highly expressed by more than 90% of B-cell non-Hodgkin lymphoma (NHL) (Anderson, K.C., et al., Blood 63 (1984) 1424-1433), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other normal tissues (Tedder, T.F., et al., J, Immunol. 135 (1985) 973-979).

[0175] There are two different types of anti-CD20 antibodies that differ significantly in their CD20 binding modes and biological activities (Cragg, M.S., et al., Blood 103 (2004) 2738-2743; and Cragg, M.S., et al., Blood 101 (2003) 1045-1052). Type I anti-CD20 antibodies mainly utilize complement to kill target cells, while type II antibodies act mainly by direct induction of cell death.

[0176] Type I and type II anti-CD20 antibodies and their properties are reviewed, for example, in Klein et al., mAbs 5 (2013), 22-33. Type II anti-CD20 antibodies do not localize CD20 to lipid rafts, show low CDC activity, exhibit only about half the binding capacity to B cells compared to type I anti-CD20 antibodies, and induce homotypic aggregation and direct cell death. In contrast, type II antibodies localize CD20 to lipid rafts, show high CDC activity, exhibit full binding capacity to B cells, and show very weak induction of homotypic aggregation and direct cell death.

[0177] Obinutuzumab and tositumomab (CAS number 192391-48) are examples of type II anti-CD20 antibodies, while rituximab, ofatumumab, belimumab, ocrelizumab, PRO131921, and ublituximab are examples of type I anti-CD20 antibodies.

[0178] According to the present invention, the anti-CD20 antibody is a type II anti-CD20 antibody. In one embodiment according to the present invention, the type II anti-CD20 antibody can reduce the number of B cells in a subject. In one embodiment, the type II anti-CD20 antibody is an IgG antibody, specifically an IgG1 antibody. In one embodiment, the type II anti-CD20 antibody is a full-length antibody. In one embodiment, the type II anti-CD20 antibody comprises an Fc region, particularly an IgG Fc region, or more specifically an IgG1 Fc region. In one embodiment, the type II anti-CD20 antibody is a humanized B-Ly1 antibody. In particular, the type II anti-CD20 antibody is a humanized IgG class type II anti-CD20 antibody having the binding specificity of a mouse B-Ly1 antibody (Poppema and Visser, Biotest Bulletin 3, 131-139 (1987); SEQ ID NO: 2 and 3).

[0179] In one embodiment, the type II anti-CD20 antibody comprises a heavy chain variable region comprising heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9. In particular, FR1, FR2, and FR3 of the heavy chain variable region framework region (FR) of the type II anti-CD20 antibody are human FR sequences encoded by the VH1_10 human germline sequence, the heavy chain variable region FR4 of the anti-CD20 antibody is a human FR sequence encoded by the JH4 human germline sequence, FR1, FR2, and FR3 of the light chain variable region FR of the type II anti-CD20 antibody are human FR sequences encoded by the VK_2_40 human germline sequence, and the light chain variable region FR4 of the anti-CD20 antibody is a human FR sequence encoded by the JK4 human germline sequence. In one embodiment, the type II anti-CD20 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11.

[0180] In certain embodiments, the type II anti-CD20 antibody is obinutuzumab (recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101. The trade names are GAZYVA® or GAZYVARO®. This replaces all previous versions (e.g., Vol. 25, No. 1, 2011, p.75-76), which were previously known as ofatumumab (recommended INN, WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p. 124). In one embodiment, the type II anti-CD20 antibody is tositumomab.

[0181] The type II anti-CD20 antibodies useful in the present invention can be engineered to have increased effector function compared to the corresponding unengineered antibody. In one embodiment, an antibody engineered to have increased effector function has at least 2-fold, at least 10-fold, or at least 100-fold increased effector function compared to the corresponding unengineered antibody. The increase in effector function can include, but is not limited to, an increase in Fc receptor binding, an increase in C1q binding and complement-dependent cytotoxicity (CDC), an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), an increase in antibody-dependent cell phagocytosis (ADCP), an increase in cytokine secretion, an increase in immune complex-mediated antigen uptake by antigen-presenting cells, an increase in binding to NK cells, an increase in binding to macrophages, an increase in binding to monocytes, an increase in binding to polymorphonuclear cells, an increase in direct signaling to induce apoptosis, an increase in cross-linking of the target-binding antibody, an increase in dendritic cell maturation, or an increase in T cell priming.

[0182] In one embodiment, the increase in effector function is one or more selected from the group consisting of an increase in Fc receptor binding, an increase in CDC, an increase in ADCC, an increase in ADCP, and an increase in cytokine secretion. In one embodiment, the increase in effector function is an increase in binding to activated Fc receptors. In one such embodiment, the binding affinity for the activated Fc receptor is increased by at least 2-fold, particularly at least 10-fold, compared to the binding affinity of the corresponding non-engineered antibody. In certain embodiments, the activated Fc receptor is selected from the group of FcγRIIIa, FcγRI, and FcγRIIa. In one embodiment, the activated Fc receptor is FcγRIIIa, particularly human FcγRIIIa. In another embodiment, the increase in effector function is an increase in ADCC. In one such embodiment, the ADCC is increased by at least 10-fold, particularly at least 100-fold, compared to the ADCC mediated by the corresponding non-engineered antibody. In yet another embodiment, the increase in effector function results in an increase in binding to activated Fc receptors and an increase in ADCC.

[0183] The increase in effector function can be measured by methods well known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry) TMNon-radioactive cytotoxicity assays (see Cell Technology, Inc., Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998). Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard measurement means such as a BIAcore instrument (GE Healthcare), and such Fc receptors can be obtained by recombinant expression. According to certain embodiments, the binding affinity for the activated Fc receptor is measured by surface plasmon resonance using a BIACORE® T100 instrument (GE Healthcare) at 25°C. Alternatively, the binding affinity of an antibody for an Fc receptor may be evaluated using a cell line known to express the particular Fc receptor, such as an NK cell expressing the FcγIIIa receptor. A C1q binding assay can also be performed to determine whether an antibody can bind to C1q and thereby have CDC activity. See, for example, the C1q and C3c binding ELISAs of WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, et al., Blood 101:1045-1052 (2003); and Cragg, and Glennie, Blood 103:2738-2743 (2004)).

[0184] The increase in effector function can result, for example, from glycan engineering of the Fc region or introduction of amino acid mutations in the Fc region of the antibody. In one embodiment, the anti-CD20 antibody is engineered by introducing one or more amino acid mutations in the Fc region. In certain embodiments, the amino acid mutations are amino acid substitutions. In even more specific embodiments, the amino acid substitutions are at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region. Further suitable amino acid mutations are described, for example, in Shields et al., J Biol Chem 9(2), 6591-6604 (2001); U.S. Patent No. 6,737,056; International Publication No. WO 2004 / 063351 and International Publication No. WO 2004 / 099249. The mutant Fc region can be prepared by deletion, substitution, insertion, or modification of amino acids using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, and the like. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0185] In another embodiment, the type II anti-CD20 antibody is engineered by modification of glycosylation in the Fc region. In certain embodiments, the type II anti-CD20 antibody is engineered such that the proportion of afucosylated oligosaccharides in the Fc region is increased compared to the non-engineered antibody. The increase in the proportion of afucosylated oligosaccharides in the Fc region of the antibody results in the antibody having an increased effector function, particularly increased ADCC.

[0186] In a more specific embodiment, at least about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 40% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are non-fucosylated. In one embodiment, at least about 40% to about 80% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are non-fucosylated. In one embodiment, at least about 40% to about 60% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are non-fucosylated. The non-fucosylated oligosaccharides may be of the hybrid type or the complex type.

[0187] In another specific embodiment, the type II anti-CD20 antibody is engineered such that the proportion of bisected oligosaccharides in the Fc region is increased compared to the non-engineered antibody. In a more specific embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 40% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are bisected. In one embodiment, at least about 40% to about 80% of the N-linked oligosaccharides in the Fc region of the anti-CD20 antibody are bisected. In one embodiment, at least about 40% to about 60% of the N-linked oligosaccharides in the Fc region of the type II anti-CD20 antibody are bisected. The bisected oligosaccharides may be of the hybrid type or the complex type.

[0188] In yet another specific embodiment, the anti-CD20 antibody is engineered such that the proportion of bisected, non-fucosylated oligosaccharides in the Fc region is increased as compared to the non-engineered antibody. In a more specific embodiment, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, preferably at least about 15%, more preferably at least about 25% of the N-linked oligosaccharides in the Fc region of the anti-CD20 antibody are bisected and non-fucosylated. The bisected, non-fucosylated oligosaccharides may be of the hybrid or complex type.

[0189] The oligosaccharide structure in the antibody Fc region can be analyzed by methods well known in the art, such as MALDI TOF mass spectrometry, as described in Umana et al., Nat Biotechnol 17, 176-180 (1999) or Ferrara et al., Biotechn Bioeng 93, 851-861 (2006). The percentage of non-fucosylated oligosaccharides is the amount of oligosaccharides lacking fucose residues relative to all oligosaccharides identified by MALDI TOF MS in a sample treated with N-glycosidase F that are attached to Asn297 (e.g., complex, hybrid, and high mannose structures). Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 can also be located between positions 294 and 300, i.e., approximately ±3 amino acids upstream or downstream of position 297, due to minor sequence variations in the antibody. The percentage of bisected, or bisected non-fucosylated oligosaccharides is determined similarly.

[0190] In one embodiment, the type II anti-CD20 antibody is engineered to have modified glycosylation in the Fc region compared to the unengineered antibody by producing the antibody in a host cell in which the activity of one or more glycosyltransferases is modified. Glycosyltransferases include β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), β(1,4)-galactosyltransferase (GalT), β(1,2)-N-acetylglucosaminyltransferase I (GnTI), β(1,2)-N-acetylglucosaminyltransferase II (GnTII), and α(1,6)-fucosyltransferase. In certain embodiments, the type II anti-CD20 antibody is engineered such that the proportion of afucosylated oligosaccharides in the Fc region is increased compared to the unengineered antibody by producing the antibody in a host cell having increased β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In even more certain embodiments, the host cell further has increased α-mannosidase II (ManII) activity. The methodology of glycoengineering that can be used to engineer the antibodies useful in the present invention is described in detail in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342 (U.S. Patent No. 6602684; European Patent No. 1071700); International Publication No. 2004 / 065540 (U.S. Patent Application Publication No. 2004 / 0241817; European Patent No. 1587921), International Publication No. 03 / 011878 (U.S. Patent Application Publication No. 2003 / 0175884), the entire contents of each of which are incorporated herein by reference in their entirety. Antibodies glycoengineered using this methodology are referred to herein as GlycoMab.

[0191] Generally, to generate a cell line for the production of an anti-TNC A2 antibody having an altered glycosylation pattern, any type of cultured cell line containing the cell lines described herein can be used. Specific cell lines include CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, and other mammalian cells. In certain embodiments, the host cell is engineered to express increased levels of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In certain embodiments, the host cell is engineered to express increased levels of one or more polypeptides having α-mannosidase II (ManII) activity. In certain embodiments, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi resident polypeptide. In particular, the Golgi localization domain is the Golgi localization domain of mannosidase II. Methods for generating such fusion polypeptides and using them to produce antibodies having increased effector function are disclosed in Ferrara et al., Biotechn Bioeng 93, 851-861 (2006) and International Publication No. WO 2004 / 065540, the entire contents of which are hereby expressly incorporated by reference.

[0192] A host cell that contains a coding sequence of an antibody useful for the present invention and / or a coding sequence of a polypeptide having glycosyltransferase activity and expresses a biologically active gene product can be identified by, for example, DNA-DNA or DNA-RNA hybridization; the presence or absence of "marker" gene function; evaluating the level of transcription measured by the expression of each mRNA transcript in the host cell; or detection of the gene product measured by an immunoassay or by its biological activity - by methods well known in the art. GnTIII or ManII activity can be detected, for example, by using a lectin that binds to the biosynthetic product of GnTIII or ManII, respectively. An example of such a lectin is the E4-PHA lectin that preferentially binds to oligosaccharides containing bisecting GlcNAc. The biosynthetic product of a polypeptide having GnTIII or ManII activity (i.e., a specific oligosaccharide structure) can also be detected by mass spectrometry of the oligosaccharides released from glycoproteins produced by cells expressing the polypeptide. Alternatively, a functional assay that measures an increased effector function, such as an increase in Fc receptor binding mediated by antibodies produced by cells engineered with a polypeptide having GnTIII or ManII activity, can be used.

[0193] In another embodiment, the anti-CD20 antibody is engineered such that, by producing the antibody in a host cell having reduced α(1,6)-fucosyltransferase activity, the proportion of afucosylated oligosaccharides in the Fc region is increased compared to the non-engineered antibody. The host cell having reduced α(1,6)-fucosyltransferase activity may be a cell in which the α(1,6)-fucosyltransferase gene is disrupted or otherwise inactivated, e.g., knocked out (see Yamane-Ohnuki et al., Biotech Bioeng 87, 614 (2004); Kanda et al., Biotechnol Bioeng, 94(4), 680-688 (2006); Niwa et al., J Immunol Methods 306, 151-160 (2006)).

[0194] Other examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch Biochem Biophys 249, 533-545 (1986); US Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11). Alternatively, antibodies useful in the present invention may be glycoengineered such that the fucose residues in the Fc region are reduced, for example, by reducing or inactivating the activity of the GDP-fucose transporter protein in the host cell used for antibody production, according to the techniques disclosed in European Patent No. 1176195 (A1), International Publication Nos. 03 / 084570, 03 / 085119, and US Patent Application Publication Nos. 2003 / 0115614, 2004 / 093621, 2004 / 110282, 2004 / 110704, 2004 / 132140, US Patent No. 6946292 (Kyowa).

[0195] The carbohydrate-engineered antibodies useful in the present invention can also be produced in an expression system that produces modified glycoproteins, as taught in WO 03 / 056914 (GlycoFi, Inc.) or WO 2004 / 057002 and WO 2004 / 024927 (Greenovation).

[0196] Therapeutic agent The present invention relates to various therapeutic agents, particularly those that are immunogenic in a subject (i.e., have the ability to induce an immune response in a subject) and / or activate T cells in a subject. Such therapeutic agents include, for example, recombinant proteins.

[0197] In one embodiment, the therapeutic agent induces the formation of ADA in a subject when administered to the subject in a treatment regimen not involving the administration of a type II anti-CD20 antibody. In one embodiment, the therapeutic agent induces the release of cytokines in a subject when administered to the subject in a treatment regimen not involving the administration of a type II anti-CD20 antibody. In one embodiment, the therapeutic agent induces the formation of ADA and the release of cytokines in a subject when administered to the subject in a treatment regimen not involving the administration of a type II anti-CD20 antibody.

[0198] In one embodiment, the therapeutic agent is a biological agent. In one embodiment, the therapeutic agent comprises a polypeptide, particularly a recombinant polypeptide. In one embodiment, the therapeutic agent comprises a polypeptide that is not naturally occurring in a subject and / or is immunogenic in a subject. In one embodiment, the therapeutic agent should be administered systemically. In one embodiment, the therapeutic agent should be administered by infusion, particularly intravenous infusion.

[0199] In one embodiment, the therapeutic agent comprises an antigen-binding polypeptide. In one embodiment, the therapeutic agent comprises a polypeptide selected from the group consisting of an antibody, an antibody fragment, an Fc domain, and an immunoconjugate. In one embodiment, the therapeutic agent comprises a polypeptide selected from the group consisting of an antibody, an antibody fragment, an antigen receptor or an antigen-binding fragment thereof, and a receptor ligand or a receptor-binding fragment thereof. In one embodiment, the therapeutic agent comprises an antibody. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a polyclonal antibody. In one embodiment, the antibody is a human antibody. In one embodiment, the antibody is a humanized antibody. In one embodiment, the antibody is a chimeric antibody. In one embodiment, the antibody is a full-length antibody. In one embodiment, the antibody is an IgG-class antibody, specifically an IgG1 subclass antibody. In one embodiment, the antibody is a recombinant antibody.

[0200] In certain embodiments, the therapeutic agent comprises an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); also see International Publication No. 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. See also U.S. Pat. No. 5,869,046 for a discussion of Fab and F(ab’)2 fragments that include salvage receptor binding epitope residues and that have increased in vivo half-lives. In one embodiment, the antibody fragment is a Fab fragment or an scFv fragment.

[0201] A diabody is an antibody fragment that has two antigen-binding sites and can be bivalent or bispecific. See, for example, European Patent No. 404,097; International Publication No. WO 93 / 11,61; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0202] A single-domain antibody is an antibody fragment that comprises all or part of the heavy-chain variable domain of an antibody, or all or part of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA; for example, see U.S. Patent No. 6,248,516 (B1)).

[0203] Antibody fragments can be made by a variety of techniques, including, but not limited to, proteolysis of intact antibodies, as described herein, and production by recombinant host cells (e.g., E. coli or phage).

[0204] In certain embodiments, the therapeutic agent comprises a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0205] In certain embodiments, the therapeutic agent comprises a humanized antibody. Typically, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody comprises one or more variable domains wherein the HVRs, such as CDRs (or a portion thereof) are derived from a non-human antibody and the FRs (or a portion thereof) are derived from a human antibody sequence. A humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived) to, for example, restore or improve antibody specificity or affinity.

[0206] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337; 7,527,791; 6,982,321; and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing grafting of specificity-determining regions (SDRs)); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "guided selection" approach to FR shuffling).

[0207] Human framework regions that can be used for humanization are not limited, but include framework regions selected using the "best fit" method (see, for example, Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, for example, Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatic mutated) framework regions or human germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions from FR library screening (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0208] In certain embodiments, the therapeutic agent comprises a human antibody. Human antibodies can be produced using a variety of techniques well known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0209] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present episomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also see, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe the XENOMOUSE TM technology, U.S. Pat. No. 5,770,429, which describes the HuMab® technology, U.S. Pat. No. 7,041,870, which describes the K-M MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, which describes the VelociMouse® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0210] Human antibodies can also be made by hybridoma-based methods. Human myelomas and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described in, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (triosoma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0211] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0212] The antibodies contained in the therapeutic agent can be isolated by screening a combinatorial library of antibodies having the desired activity(ies). For example, various methods for creating a phage display library and screening a combinatorial library of antibodies having the desired binding properties are known in the art. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0213] In certain phage display methods, the repertoires of VH genes and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then antigen-binding phages are screened as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to an immunogen without the need to construct hybridomas. Instead, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), a naive repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without any immunization. Finally, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), naive libraries can also be synthetically generated by cloning stem cell-derived unrearranged V gene segments, encoding hypervariable CDR3 regions using PCR primers containing random sequences, and achieving rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0214] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or fragments of human antibodies.

[0215] In certain embodiments, the therapeutic agent includes a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different sites. In certain embodiments, the binding specificities are for different antigens. In certain embodiments, the binding specificities are for different epitopes on the same antigen. Bispecific antibodies can also be used to localize a cytotoxic agent to cells expressing an antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0216] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two sets of immunoglobulin heavy-chain-light-chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)) and "knob-in-hole" technology (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies also include engineering the electrostatic steering effect for making Fc-heterodimeric molecules of antibodies (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and can be made, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol. 147: 60 (1991).

[0217] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "Octopus antibodies" (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576).

[0218] Antibodies or fragments herein also include "dual action FAb" or "DAF" that include antigen-binding sites that bind two different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0219] "Crossmab" antibodies are also included herein (see, e.g., International Publication Nos. 2009080251, 2009080252, 2009080253, and 2009080254).

[0220] Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261, and 2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain contains two single-chain Fv (scFv) fragments, each of which has a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes can be specific for different cell types such that when each scFv is bound to its cognate epitope, two different cell types of cells are brought into proximity or linked. One particular embodiment of this approach includes an scFv that recognizes a cell surface antigen expressed by an immune cell, such as the CD3 polypeptide on a T cell, linked to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0221] Since it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic expression system well-known in the art, such as a CHO cell line. However, specific purification techniques (see, e.g., European Patent No. 1691833) will be required to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography and the polypeptide is eluted with a gradient of imidazole concentration. This eluate is further purified using anion exchange chromatography and the polypeptide is eluted with a gradient of sodium chloride concentration. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species.

[0222] Antibodies with three or more valences are also conceivable. For example, trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991).

[0223] In certain embodiments, the antibody included in the therapeutic agent is known in the art and can be further modified to include additional non - proteinaceous moieties that are readily available. Moieties suitable for derivatizing the antibody include, but are not limited to, water - soluble polymers. Non - limiting examples of water - soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly - 1,3 - dioxolane, poly - 1,3,6 - trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n - vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene (prolypropylene) oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when multiple polymers are attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used in therapy under defined conditions.

[0224] The therapeutic agent may also include an antibody conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins or fragments thereof of bacterial, fungal, plant or animal origin), or radioisotopes.

[0225] In one embodiment, the therapeutic agent comprises an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs including, but not limited to, maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0425235 (B1)); auristatins such as DE and DF (MMAE and MMAF) which are monomethyl auristatin drug moieties (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecene; and CC1065.

[0226] In another embodiment, the therapeutic agent comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof (including, but not limited to) diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, saporin, dianthin protein, pokeweed antiviral protein (PAPI, PAPII, and PAP-S), cucurbitacin, crocin, bryodin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0227] In other embodiments, the therapeutic agent comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 、and radioisotopes of Lu. When used for detection, the radioactive conjugate may contain a radioactive atom for scintigraphy, such as tc 99m or I 123 、or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0228] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and biactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for the conjugation of radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug intracellularly. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0229] In some embodiments, the therapeutic agent is a monoclonal antibody, such as, but not limited to, alemtuzumab (LEMTRADA®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), pertuzumab (OMNITARG®, 2C4), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), abciximab (REOPRO®), adalimumab (HUMIRA®), apolizumab, aselizumab, atorizumab, bapineuzumab, basiliximab (SIMULECT®), babiximab, belimumab (BENLYSTA®), briankinumab, canakinumab (ILARIS®), certolizumab pegol (CIMZIA®), cidfusituzumab, cidtuzumab, sizatumumab, clazakizumab, crenezumab, daclizumab (ZENAPAX®), daratumumab, denosumab (PROLIA®, XGEVA®), eculizumab (SOLIRIS®), efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, golimumab (SIMPONI®), ipilimumab, imgatuzumab, infliximab (REMICADE®), labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucatumumab, lulizumab pegolpegol), lumretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab, muronomab, natalizumab (TYSABRI®), necitumumab (PORTRAZZA®), nimotuzumab (THERACIM®), nolovizumab, numavizumab, olokizumab, omalizumab (XOLAIR®), ocrelizumab (also known as MetMAb), palivizumab (SYNAGIS®), pascolizumab, pecfusituzumab, pectuzumab, pembrolizumab (KEYTRUDA®), pacilizumab, priliximab, ralivizumab, ranibizumab (LUCENTIS®), reslivizumab, reslizumab, resyvizumab, robatumumab, rontalizumab, rovelizumab, ruplizumab, sarilumab, secukinumab, seribantumab, sifalimumab, sibrotuzumab, siltuximab (SYLVANT®), siplizumab, sontuzumab, tadocizumab, talizumab, tefibazumab, tocilizumab (ACTEMRA®), toralizumab, tucusituzumab, umavizumab, urtoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), visilizumab, zanolimumab, zalutumumab may be included.

[0230] In one embodiment, the therapeutic agent comprises an antibody adapted for the treatment of cancer. In one embodiment, the therapeutic agent comprises an antibody adapted for the treatment of autoimmune diseases. In one embodiment, the therapeutic agent is an immunotherapeutic agent. In one embodiment, the therapeutic agent is adapted for the treatment of cancer. In some embodiments, particularly in relevant aspects of the present invention involving a reduction in cytokine release associated with administration of the therapeutic agent in a subject, the cancer is a B cell proliferative disorder. In one embodiment, the cancer is a CD20-positive B cell proliferative disorder. In one embodiment, the cancer is selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin lymphoma (HL). In one embodiment, the therapeutic agent is an immunotherapeutic agent.

[0231] In one embodiment, the therapeutic agent is an immunosuppressive agent. In one embodiment, the therapeutic agent is adapted for the treatment of autoimmune diseases.

[0232] Although not wishing to be bound by theory, enhancing T cell stimulation by promoting co-stimulatory molecules or inhibiting negative co-stimulatory molecules is thought to promote tumor cell death and thereby treat cancer or delay cancer progression. In some embodiments, the therapeutic agent may comprise an agonist to a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule may comprise CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist to the co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the therapeutic agent may comprise an antibody that targets GITR. In some embodiments, the antibody that targets GITR is TRX518. In some embodiments, the therapeutic agent may comprise an antagonist to an inhibitory co-stimulatory molecule. In some embodiments, the inhibitory co-stimulatory molecule may comprise CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the antagonist to the inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase.

[0233] In some embodiments, the therapeutic agent may comprise an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab, formerly known as lambrolizumab), and CT-011 (pidilizumab). MDX-1106-04, ONO-4538, BMS-936558, or MDX-1106, also known as nivolumab, is an anti-PD-1 antibody described in WO 2006 / 121168. MK-3475, also known as pembrolizumab or (formerly) lambrolizumab, is an anti-PD-1 antibody described in WO 2009 / 114335. hBAT, hBAT-1, or CT-011, also known as pidilizumab, is an anti-PD-1 antibody described in WO 2009 / 101611.

[0234] In some embodiments, the therapeutic agent may comprise an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In one embodiment, the therapeutic agent can comprise AMP-224, also known as B7-DCIg, a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0235] In some embodiments, the therapeutic agent may comprise an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. The antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO 2010 / 077634. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007 / 005874. MEDI4736 is an anti-PD-L1 monoclonal antibody described in WO 2011 / 066389 and US Patent Application Publication No. 2013 / 034559. In one embodiment, the anti-PD-L1 antibody is atezolizumab.

[0236] In some embodiments, the therapeutic agent can include an antibody against CTLA-4 (also known as CD152), such as a blocking antibody. In some embodiments, the therapeutic agent can include ipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In some embodiments, the therapeutic agent can include tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the therapeutic agent can include an antibody against B7-H3 (also known as CD276), such as a blocking antibody. In some embodiments, the therapeutic agent can include MGA271. In some embodiments, the therapeutic agent can include an antagonist against TGF beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

[0237] In some embodiments, the therapeutic agent can include an agonist against CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the therapeutic agent can include urelumab (also known as BMS-663513). In some embodiments, the therapeutic agent can include a ligand of CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In some embodiments, the therapeutic agent can include an agonist against CD40, such as an activating antibody. In some embodiments, the therapeutic agent can include CP-870893. In some embodiments, the therapeutic agent can include an agonist against OX40 (also known as CD134), such as an activating antibody. In some embodiments, the therapeutic agent can include an anti-OX40 antibody (such as AgonOX). In some embodiments, the therapeutic agent can include a ligand of OX40, such as OX40L. In some embodiments, the therapeutic agent can include an agonist against CD27, such as an activating antibody. In some embodiments, the therapeutic agent can include CDX-1127.

[0238] In some embodiments, the therapeutic agent can include T cells that express a chimeric antigen receptor (CAR) (e.g., cytotoxic T cells or CTLs). In some embodiments, the therapeutic agent can include T cells that include a dominant negative TGF beta receptor, such as a dominant negative TGF beta type II receptor.

[0239] In some embodiments, the therapeutic agent may include an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate includes mertansine or monomethyl auristatin E (MMAE). In some embodiments, the therapeutic agent may include an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, the therapeutic agent may include trastuzumab emtansine (T-DM1, ado-trastuzumab emtansine, or also known as KADCYLA®). In some embodiments, the therapeutic agent may include DMUC5754A. In some embodiments, the therapeutic agent may include an antibody-drug conjugate that targets the endothelin B receptor (EDNBR), for example, an antibody against EDNBR conjugated with MMAE (also known as DEDN6526A). In some embodiments, the therapeutic agent may include gemtuzumab ozogamicin (MYLOTARG®). In some embodiments, the therapeutic agent may include inotuzumab ozogamicin. In some embodiments, the therapeutic agent may include bivatuzumab mertansine. In some embodiments, the therapeutic agent may include cantuzumab mertansine. In some embodiments, the therapeutic agent may include cantuzumab ravtansine. In some embodiments, the therapeutic agent may include brentuximab vedotin (ADECTRIS®). In some embodiments, the therapeutic agent may include pinatuzumab vedotin. In some embodiments, the therapeutic agent may include polatuzumab vedotin. In some embodiments, the therapeutic agent may include glembatumumab vedotin. In some embodiments, the therapeutic agent may include lorvotuzumab mertansine.In some embodiments, the therapeutic agent may include tacatuzumab tetraxetan. In some embodiments, the therapeutic agent may include vandortuzumab vedotin (DSTP3086S). In some embodiments, the therapeutic agent may include ibritumomab tiuxetan (ZEVALIN®).

[0240] In some embodiments, the therapeutic agent may include an antibody against angiopoietin 2 (also known as Ang2). In some embodiments, the therapeutic agent may include MEDI3617.

[0241] In some embodiments, the therapeutic agent may include an antibody that targets CSF-1R (also known as M-CSFR or CD115). In some embodiments, the therapeutic agent may include IMC-CS4 (LY3022855). In some embodiments, the therapeutic agent may include emactuzumab.

[0242] In some embodiments, the therapeutic agent may include a cytokine. In some embodiments, the therapeutic agent may include an interferon, such as interferon alpha or interferon gamma. In some embodiments, the therapeutic agent may include Roferon-A (also known as recombinant interferon alpha-2a). In some embodiments, the therapeutic agent may include GM-CSF (recombinant human granulocyte macrophage colony-stimulating factor, rhu GM-CSF, sargramostim, or LEUKIN E®). In some embodiments, the therapeutic agent may include aldesleukin (PROLEUKIN®). In some embodiments, the therapeutic agent may include IL-12. In some embodiments, the therapeutic agent may include IL-10.

[0243] In some embodiments, the therapeutic agent may include an IL-2 fusion protein. In some embodiments, the therapeutic agent may include tucotuzumab celmoleukin. In some embodiments, the therapeutic agent may include darleukin. In some embodiments, the therapeutic agent may include teleukin.

[0244] In some embodiments, the therapeutic agent may include an IL-10 fusion protein. In some embodiments, the therapeutic agent may include dekavil. In some embodiments, the therapeutic agent may include a TNF fusion protein. In some embodiments, the therapeutic agent may include fibromun.

[0245] In some embodiments, the therapeutic agent may include a bispecific antibody. In some embodiments, the therapeutic agent may include a bispecific antibody, for example, but not limited to, duligotuzumab, MM-111, MM141, TF2, ABT-981, ABT-122, LY3164530, SAR156597, GSK2434735, ozoralizumab, ALX-0761, ALX-0061, ALX-0141, ACE910, etc.

[0246] In some embodiments, the therapeutic agent may include a bispecific antibody that can bind to T cells and target cells, such as tumor cells. In some embodiments, the therapeutic agent may include a bispecific antibody that specifically binds to CD3 on T cells and a target cell antigen. In some embodiments, the therapeutic agent may include a bispecific T cell engager (BiTE®). In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD19. In one embodiment, the bispecific antibody is blinatumomab (BLINCYTO®). In one embodiment, the bispecific antibody is AFM11. In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and EpCAM. In one embodiment, the bispecific antibody is catumaxomab (REVOMAB®). In one embodiment, the bispecific antibody is solitomab (AMG 110, MT110). In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and Her2. In one embodiment, the bispecific antibody is ertumaxomab. In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and PSMA. In one embodiment, the bispecific antibody is BAY2010112 (AMG212, MT112). In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CEA. In one embodiment, the bispecific antibody is MEDI565 (AMG211, MT111). In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD33. In one embodiment, the bispecific antibody is AMG330. In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD123. In one embodiment, the bispecific antibody is MGD006. In one embodiment, the bispecific antibody is XmAb® 14045. In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD38. In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and gpA33. In one embodiment, the bispecific antibody is MGD007. In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD20.In one embodiment, the bispecific antibody is XmAb®13676. In one embodiment, the bispecific antibody is REGN1979. In one embodiment, the bispecific antibody is FBTA05 (Lymphomun).

[0247] In some embodiments, the therapeutic agent may include a bispecific antibody against CD30 and CD16A. In one embodiment, the bispecific antibody is AFM13. In some embodiments, the therapeutic agent may include a bispecific antibody against DR5 and FAP. In some embodiments, the therapeutic agent may include a bispecific antibody against Ang2 and VEGF. In one embodiment, the bispecific antibody is vanucizumab.

[0248] In some embodiments, the therapeutic agent may include an Fc domain. In some embodiments, the therapeutic agent may include a fusion protein comprising an Fc domain.

[0249] In some embodiments, the therapeutic agent may include a recombinant receptor or a fragment thereof. In some embodiments, the receptor is a T cell receptor. In some embodiments, the receptor is a TNF receptor. In some embodiments, the therapeutic agent may include etanercept (ENBREL®). In some embodiments, the receptor is a VEGF receptor. In some embodiments, the therapeutic agent may include aflibercept (ziv-aflibercept) (ZALTRAP®). In some embodiments, the therapeutic agent may include aflibercept (EYLEA®). In some embodiments, the receptor is an IL-1 receptor. In some embodiments, the therapeutic agent may include rilonacept (ARCALYST®). In some embodiments, the therapeutic agent may include IMCgp100. In some embodiments, the therapeutic agent may include a chimeric antigen receptor (CAR). In some embodiments, the therapeutic agent may include a factor IX-Fc fusion protein. In some embodiments, the therapeutic agent may include a factor VIII-Fc fusion protein. In some embodiments, the therapeutic agent may include a CTLA-4-Fc fusion protein, such as belatacept, abatacept (ORENCIA®). In one embodiment, the therapeutic agent may include romiplostin.

[0250] In some embodiments, the therapeutic agent may include a recombinant receptor ligand, such as a TNF receptor ligand.

[0251] In some embodiments, the therapeutic agent may include an agent, such as a generic, biosimilar or non-equivalent biological version of an antibody named herein.

[0252] In one embodiment, the therapeutic agent does not include obinutuzumab.

[0253] T cell activation therapeutic agent The T cell activation therapeutic agents for which the present invention may be useful, and in particular, embodiments of the present invention involved in reducing cytokine release associated with administration of the therapeutic agent to a subject, will be described in more detail below.

[0254] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to an activated T cell antigen. In one embodiment, the therapeutic agent may comprise an antibody that specifically binds to an antigen selected from the group consisting of CD3, CD28, CD137 (also known as 4-1BB), CD40, CD226, OX40, GITR, CD27, HVEM, and CD127.

[0255] In one embodiment, the therapeutic agent comprises an antibody that specifically binds to CD3, specifically CD3ε.

[0256] In one embodiment, the therapeutic agent is antibody H2C (PCT Publication No. WO2008 / 119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl 7, 45-50 (1992) and U.S. Patent No. 6054297), antibody FN18 (Nooij et al., Eur J Immunol 19, 981-984 (1986)), antibody SP34 (Pessano et al., EMBO J 4, 337-340 (1985)), antibody OKT3 (Kung et al., Science 206, 347-349 (1979)), antibody WT31 (Spits et al., J Immunol 135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol 129, 1451-1457 (1982)), antibody 7D6 (Coulie et al., Eur J Immunol 21, 1703-1709 (1991)) or antibody Leu-4, or an antibody capable of competing for binding with those antibodies. In some embodiments, the therapeutic agent also includes an antibody that specifically binds to CD3 as described in International Publication No. WO2005 / 040220, International Publication No. WO2005 / 118635, International Publication No. WO2007 / 042261, International Publication No. WO2008 / 119567, International Publication No. WO2008 / 119565, International Publication No. WO2012 / 162067, International Publication No. WO2013 / 158856, International Publication No. WO2013 / 188693, International Publication No. WO2013 / 186613, International Publication No. WO2014 / 110601, International Publication No. WO2014 / 145806, International Publication No. WO2014 / 191113, International Publication No. WO2014 / 047231, International Publication No. WO2015 / 095392, International Publication No. WO2015 / 181098, International Publication No. WO2015 / 001085, International Publication No. WO2015 / 104346, International Publication No. WO2015 / 172800, International Publication No. WO2016 / 020444 or International Publication No. WO2016 / 014974.

[0257] In one embodiment, the therapeutic agent may include an antibody that specifically binds to a B cell antigen, specifically a malignant B cell antigen. In one embodiment, the therapeutic agent may include an antibody that specifically binds to an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37, and CD5, particularly CD20 or CD19.

[0258] In some embodiments, the therapeutic agent may include an antibody selected from rituximab, ofatumumab, obinutuzumab, ocaratuzumab, belimumab, and ublituximab.

[0259] In some embodiments, the therapeutic agent may include a multispecific antibody, specifically a bispecific antibody. In some embodiments, the therapeutic agent may include a bispecific antibody that can bind to T cells and target cells, such as tumor cells. In some embodiments, the target cells are B cells, specifically malignant B cells. In some embodiments, the therapeutic agent may include a bispecific antibody that specifically binds to (i) an activated T cell antigen and (ii) a B cell antigen. In some embodiments, the therapeutic agent may include a bispecific antibody that specifically binds to CD3 on T cells and a target cell antigen. In some embodiments, the target cell antigen is a B cell antigen, specifically a malignant B cell antigen. In some embodiments, the therapeutic agent may include a bispecific T cell engager (BiTE®).

[0260] In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD20. In one embodiment, the bispecific antibody is XmAb®13676. In one embodiment, the bispecific antibody is REGN1979. In one embodiment, the bispecific antibody is FBTA05 (Lymphomun).

[0261] In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD19. In one embodiment, the bispecific antibody is blinatumomab (BLINCYTO®). In one embodiment, the bispecific antibody is AFM11. In one embodiment, the bispecific antibody is MGD011 (JNJ-64052781).

[0262] In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD38. In one embodiment, the bispecific antibody is XmAb® 13551, XmAb® 15426, or XmAb® 14702.

[0263] In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and BCMA. In one embodiment, the bispecific antibody is BI836909.

[0264] In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD33. In one embodiment, the bispecific antibody is AMG330.

[0265] In some embodiments, the therapeutic agent may include a bispecific antibody against CD3 and CD123. In one embodiment, the bispecific antibody is MGD006. In one embodiment, the bispecific antibody is XmAb® 14045. In one embodiment, the bispecific antibody is JNJ-63709178.

[0266] In some embodiments, the therapeutic agent may include a recombinant receptor or a fragment thereof. In some embodiments, the receptor is a T cell receptor (TCR). In some embodiments, the therapeutic agent may include a chimeric antigen receptor (CAR).

[0267] In some embodiments, the therapeutic agent may include T cells (e.g., cytotoxic T cells or CTLs) that express a chimeric antigen receptor (CAR). In some embodiments, the therapeutic agent may include T cells that express a recombinant T cell receptor (TCR).

[0268] In one embodiment, the therapeutic agent may include a chimeric antigen receptor (CAR) that specifically binds to a B cell antigen, specifically a malignant B cell antigen. In one embodiment, the therapeutic agent may include a CAR that specifically binds to an antigen selected from the group consisting of CD20, CD19, CD22, ROR-1, CD37, and CD5, particularly CD20 or CD19.

[0269] In some embodiments, the therapeutic agent may include a CAR directed to CD19, or T cells that express a CAR directed to CD19. In some embodiments, the therapeutic agent may include KTE-C19, CTL019, JCAR-014, JCAR-015, JCAR-017, BPX-401, UCART19.

[0270] In some embodiments, the therapeutic agent may include a CAR directed to CD22, or T cells that express a CAR directed to CD22. In some embodiments, the therapeutic agent may include JCAR-018 or UCART22.

[0271] In some embodiments, the therapeutic agent may include an agonist to a T cell activation costimulatory molecule. In some embodiments, the T cell activation costimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist to the T cell activation costimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the therapeutic agent may include an antibody that targets GITR. In some embodiments, the antibody that targets GITR is TRX518.

[0272] In some embodiments, the therapeutic agent can include an agonist for CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the therapeutic agent can include urelumab (also known as BMS-663513). In some embodiments, the therapeutic agent can include a ligand for CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as 4-1BBL. In some embodiments, the therapeutic agent can include an agonist for CD40, such as an activating antibody. In some embodiments, the therapeutic agent can include CP-870893. In some embodiments, the therapeutic agent can include an agonist for OX40 (also known as CD134), such as an activating antibody. In some embodiments, the therapeutic agent can include an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the therapeutic agent can include a ligand for OX40, such as OX40L. In some embodiments, the therapeutic agent can include an agonist for CD27, such as an activating antibody. In some embodiments, the therapeutic agent can include CDX-1127.

[0273] Specific therapeutic agents (i) Reduction in the formation of anti-drug antibodies (ADA) The therapeutic agents described below are particularly useful in the context of the present invention, particularly in relevant embodiments of the present invention that are involved in reducing the formation of anti-drug antibodies (ADA) against the therapeutic agent in a subject.

[0274] In some embodiments, the therapeutic agent includes an antibody that specifically binds to a carcinoembryonic antigen (CEA).

[0275] In one embodiment, an antibody that specifically binds to CEA comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 15, and HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19. In a further embodiment, an antibody that specifically binds to CEA comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 21. In a further embodiment, an antibody that specifically binds to CEA comprises the heavy chain variable region sequence of SEQ ID NO: 20 and the light chain variable region sequence of SEQ ID NO: 21.

[0276] In one embodiment, an antibody that specifically binds to CEA comprises a heavy chain variable region comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 136, HCDR2 of SEQ ID NO: 137, and HCDR3 of SEQ ID NO: 138; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 139, LCDR2 of SEQ ID NO: 140, and LCDR3 of SEQ ID NO: 141. In a further embodiment, an antibody that specifically binds to CEA comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 142, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 143. In a further embodiment, an antibody that specifically binds to CEA comprises the heavy chain variable region sequence of SEQ ID NO: 142 and the light chain variable region sequence of SEQ ID NO: 143.

[0277] In one embodiment, an antibody that specifically binds to CEA is a full-length antibody. In one embodiment, an antibody that specifically binds to CEA is an antibody of the human IgG class, specifically an antibody of the human IgG1 class. In one embodiment, an antibody that specifically binds to CEA is an antibody fragment, specifically a Fab molecule or a scFv molecule, more specifically a Fab molecule. In one embodiment, an antibody that specifically binds to CEA is a humanized antibody.

[0278] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to fibroblast activation protein (FAP). In one embodiment, the antibody that specifically binds to FAP comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 26. In a further embodiment, the antibody that specifically binds to FAP comprises the heavy chain variable region sequence of SEQ ID NO: 25 and the light chain variable region sequence of SEQ ID NO: 26.

[0279] In one embodiment, the antibody that specifically binds to FAP is a full-length antibody. In one embodiment, the antibody that specifically binds to FAP is an antibody of the human IgG class, specifically an antibody of the human IgG1 class. In one embodiment, the antibody that specifically binds to FAP is an antibody fragment, specifically a Fab molecule or a scFv molecule, more specifically a Fab molecule. In one embodiment, the antibody that specifically binds to FAP is a human antibody.

[0280] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to CD3, specifically CD3 epsilon. In one embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region comprising the heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, the HCDR2 of SEQ ID NO: 33, and the HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising the light chain CDR (LCDR) 1 of SEQ ID NO: 35, the LCDR2 of SEQ ID NO: 36, and the LCDR3 of SEQ ID NO: 37. In a further embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In a further embodiment, the antibody that specifically binds to CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

[0281] In one embodiment, the antibody that specifically binds to CD3 is a full-length antibody. In one embodiment, the antibody that specifically binds to CD3 is an antibody of the human IgG class, specifically an antibody of the human IgG1 class. In one embodiment, the antibody that specifically binds to CD3 is an antibody fragment, specifically a Fab molecule or a scFv molecule, more specifically a Fab molecule. In certain embodiments, the antibody that specifically binds to CD3 is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., substituted for each other). In one embodiment, the antibody that specifically binds to CD3 is a humanized antibody.

[0282] In some embodiments, the therapeutic agent comprises a cytokine. In one embodiment, the cytokine is selected from the group consisting of GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. In one embodiment, the cytokine is IL-2, specifically human IL-2. The sequence of wild-type human IL-2 is shown in SEQ ID NO: 12.

[0283] In one embodiment, the therapeutic agent comprises a mutant IL-2 polypeptide having a reduced binding affinity for the α-subunit of the IL-2 receptor as compared to wild-type IL-2. The α-subunit (also known as CD25) forms a heterotrimeric high-affinity IL-2 receptor together with the β and γ subunits (also known as CD122 and CD132, respectively), while a dimer receptor consisting of only the β and γ subunits is called an intermediate-affinity IL-2 receptor. A mutant IL-2 polypeptide with reduced binding to the α-subunit of the Il-2 receptor is a regulatory T (T reg) It has a reduced ability to induce IL-2 signaling in cells, induces less activation-induced cell death (AICD) in T cells, and has a reduced toxicity profile in vivo compared to wild-type IL-2 polypeptide (see, for example, International Publication No. WO 2012 / 107417, which is hereby incorporated by reference in its entirety).

[0284] In more specific embodiments, the mutant IL-2 polypeptide comprises three amino acid substitutions at positions corresponding to residues 42, 45, and 72 of human IL-2. In even more specific embodiments, the mutant IL-2 polypeptide is a human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A, and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 12). In one embodiment, the mutant IL-2 polypeptide further comprises an amino acid mutation at the position corresponding to position 3 of human IL-2 that eliminates the O-glycosylation site of IL-2. In one embodiment, the amino acid mutation that eliminates the O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2 is an amino acid substitution selected from the group consisting of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In particular, the further amino acid mutation is an amino acid substitution that replaces a threonine residue with an alanine residue. A specific mutant IL-2 polypeptide useful in the present invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45, and 72 of human IL-2. The specific amino acid substitutions are T3A, F42A, Y45A, and L72G. This mutant IL-2 polypeptide does not show detectable binding to CD25, shows a reduced ability to induce apoptosis in T cells, a reduced ability to induce IL-2 signaling in Treg cells, and a reduced toxicity profile in vivo (see, for example, International Publication No. WO 2012 / 107417, which is hereby incorporated by reference in its entirety). However, it activates IL-2 signaling in effector cells, induces the proliferation of effector cells, and retains the ability to generate IFN-γ as a secondary cytokine by NK cells.

[0285] The IL-2 or mutant IL-2 polypeptide according to any of the above embodiments may include further mutations that provide further advantages such as increased expression or stability. For example, the cysteine at position 125 may be substituted with a neutral amino acid such as serine, alanine, threonine, or valine, resulting in C125S IL-2, C125A IL-2, C125T IL-2, or C125V IL-2, respectively, as described in U.S. Patent No. 4,518,584. As described therein, it is also possible to delete the N-terminal alanine residue of IL-2 to generate mutants such as des-A1 C125S or des-A1 C125A. Alternatively, or in combination, the IL-2 mutant may include a mutation in which the methionine that normally occurs at position 104 of wild-type human IL-2 is substituted with a neutral amino acid such as alanine (see U.S. Patent No. 5,206,344). The resulting mutants, e.g., des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants can be found in U.S. Patent No. 5,116,943 and Weiger et al., Eur J Biochem 180, 295-300 (1989)), may be used in combination with the specific IL-2 mutations described herein.

[0286] Accordingly, in certain embodiments, the IL-2 or mutant IL-2 polypeptide includes a further amino acid mutation at the position corresponding to residue 125 of human IL-2. In one embodiment, the further amino acid mutation is the amino acid substitution C125A.

[0287] In certain embodiments, the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule, specifically a human full-length IL-2 molecule. In one embodiment, the mutant IL-2 polypeptide includes a polypeptide sequence that is at least 80%, at least 85%, or at least 90% identical to the sequence of SEQ ID NO: 12.

[0288] In certain embodiments, the variant IL-2 polypeptide comprises the polypeptide sequence of SEQ ID NO: 13.

[0289] In some embodiments, the therapeutic agent comprises an immunoconjugate. Certain immunoconjugates are described in International Publication No. WO 2012 / 107417 and International Publication No. WO 2012 / 146628 (each incorporated herein by reference in its entirety).

[0290] In one embodiment, the immunoconjugate comprises an antibody that specifically binds to CEA as described herein, and a variant IL-2 polypeptide as described herein. In one embodiment, the antibody is a full-length antibody.

[0291] In one embodiment, the therapeutic agent is (i) an antibody of the human IgG1 subclass comprising a heavy chain variable region that specifically binds to CEA and comprises heavy chain CDR (HCDR) 1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 15, and HCDR3 of SEQ ID NO: 16; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19; and (ii) a variant human IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A, and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 12) comprising an immunoconjugate.

[0292] In one embodiment, the immunoconjugate comprises an antibody that specifically binds to FAP as described herein, and a variant IL-2 polypeptide as described herein. In one embodiment, the antibody is a full-length antibody.

[0293] In one embodiment, the therapeutic agent is (i) an antibody of the human IgG1 subclass that specifically binds to FAP and comprises the heavy chain variable region sequence of SEQ ID NO: 25; and the light chain variable region sequence of SEQ ID NO: 26; and (ii) A mutant human IL-2 polypeptide comprising amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 12) comprising an immunoconjugate.

[0294] In one embodiment, the immunoconjugate comprises one or fewer mutant IL-2 polypeptides. In one embodiment, the mutant IL-2 polypeptide is fused to the carboxy-terminal amino acid of one of the antibody heavy chains, optionally via a linker peptide. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n or G4(SG4) n peptide linkers, where n is generally a number between 1 and 10, typically a number between 2 and 4. One embodiment is that the linker peptide is (G4S)3.

[0295] In one embodiment, the immunoconjugate comprises a polypeptide having a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 22, a polypeptide having a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 23, and a polypeptide having a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 24.

[0296] In one embodiment, the immunoconjugate comprises a polypeptide comprising the sequence of SEQ ID NO: 22, a polypeptide comprising the sequence of SEQ ID NO: 23, and a polypeptide comprising the sequence of SEQ ID NO: 24.

[0297] In one embodiment, the immunoconjugate is cergutuzumab amunaleukin (see WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Recommended INN: List 75, 2016, pre-publication copy, which is incorporated herein by reference in its entirety).

[0298] In one embodiment, the immunoconjugate comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 28, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 29.

[0299] In one embodiment, the immunoconjugate comprises a polypeptide comprising the sequence of SEQ ID NO: 27, a polypeptide comprising the sequence of SEQ ID NO: 28, and a polypeptide comprising the sequence of SEQ ID NO: 29.

[0300] In one embodiment, the therapeutic agent comprises a bispecific antibody. Particular bispecific antibodies are described in PCT Publication No. WO 2013 / 026833 and WO 2014 / 131712, PCT Application No. PCT / EP2016 / 073171 (each of which is incorporated herein by reference in its entirety).

[0301] In one embodiment, the bispecific antibody comprises an antibody that specifically binds to CEA as described herein and an antibody that specifically binds to CD3 as described herein. In one embodiment, the bispecific antibody comprises a first antibody that specifically binds to CD3 as described herein and second and third antibodies that specifically bind to CEA as described herein. In one embodiment, the first antibody is a crossover Fab molecule as described herein, and the second and first antibodies are each conventional Fab molecules. In one embodiment, the bispecific antibody further comprises an Fc domain as described herein. The bispecific antibody can have an antibody format as described herein and can comprise an antigen-binding portion as described herein. The bispecific antibody can include modifications to the Fc region and / or antigen-binding portion as described herein.

[0302] In one embodiment, the therapeutic agent is (i) A first antigen-binding portion comprising a heavy-chain variable region that specifically binds to CD3 and comprises heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light-chain variable region that comprises light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37, wherein the first antigen-binding portion is a crossover Fab molecule in which either the variable or constant region of the Fab light chain and the Fab heavy chain, particularly the constant region, is exchanged; (ii) Second and third antigen-binding portions that specifically bind to CEA and comprise a heavy-chain variable region that comprises heavy-chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 15, and HCDR3 of SEQ ID NO: 16; and a light-chain variable region that comprises light-chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 17, LCDR2 of SEQ ID NO: 18, and LCDR3 of SEQ ID NO: 19, wherein the second and third antigen-binding portions are Fab molecules, particularly conventional Fab molecules; (iii) An Fc domain consisting of first and second subunits capable of stable association A bispecific antibody comprising wherein the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, and the first antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the third antigen-binding portion is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0303] In one embodiment, the first antigen-binding portion that specifically binds to CD3 comprises the heavy-chain variable region sequence of SEQ ID NO: 38 and the light-chain variable region sequence of SEQ ID NO: 39. In one embodiment, the second and third antigen-binding portions that specifically bind to CEA comprise the heavy-chain variable region sequence of SEQ ID NO: 20 and the light-chain variable region sequence of SEQ ID NO: 21.

[0304] In one embodiment, the antigen-binding portion and the Fc region are fused to each other by a peptide linker, particularly by the peptide linkers of SEQ ID NO: 42 and SEQ ID NO: 43. In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 40, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 41, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 42, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 43.

[0305] In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 40, a polypeptide comprising the sequence of SEQ ID NO: 41, a polypeptide comprising the sequence of SEQ ID NO: 42, and a polypeptide comprising the sequence of SEQ ID NO: 43. (CEA TCB)

[0306] In one embodiment, the therapeutic antibody is (i) a first antigen-binding portion that specifically binds to CD3 and comprises a heavy-chain variable region comprising heavy-chain CDR (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light-chain variable region comprising light-chain CDR (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37, wherein the first antigen-binding portion is a first antigen-binding portion that is a crossover Fab molecule in which either the variable region or the constant region of the Fab light chain and the Fab heavy chain, particularly the variable region, is exchanged; (ii) a heavy chain variable region that specifically binds to CEA and comprises heavy chain CDR (HCDR) 1 of SEQ ID NO: 136, HCDR2 of SEQ ID NO: 137, and HCDR3 of SEQ ID NO: 138; and a light chain variable region that comprises light chain CDR (LCDR) 1 of SEQ ID NO: 139, LCDR2 of SEQ ID NO: 140, and LCDR3 of SEQ ID NO: 141, wherein the second and third antigen-binding portions are the second and third antigen-binding portions each being a Fab molecule, particularly a conventional Fab molecule; (iii) a bispecific antibody comprising an Fc domain consisting of first and second subunits capable of stable association, wherein the second antigen-binding portion is fused to the N-terminus of the Fab heavy chain of the first antigen-binding portion at the C-terminus of the Fab heavy chain, and the first antigen-binding portion is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third antigen-binding portion is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0307] In one embodiment, the first antigen-binding portion that specifically binds to CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39. In one embodiment, the second and third antigen-binding portions that specifically bind to CEA comprise the heavy chain variable region sequence of SEQ ID NO: 142 and the light chain variable region sequence of SEQ ID NO: 143.

[0308] In one embodiment, the antigen-binding portion and the Fc region are fused to each other by a peptide linker, particularly by the peptide linkers of SEQ ID NO: 145 and SEQ ID NO: 146.

[0309] In one embodiment, in the constant domain CL of the second and third Fab molecules of (ii), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat). In the constant domain CH1 of the second and third Fab molecules of (ii), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat).

[0310] In one embodiment, the bispecific antibody comprises a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 144, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 145, a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 146, and a polypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 147.

[0311] In one embodiment, the bispecific antibody comprises a polypeptide comprising the sequence of SEQ ID NO: 144, a polypeptide comprising the sequence of SEQ ID NO: 145, a polypeptide comprising the sequence of SEQ ID NO: 146, and a polypeptide comprising the sequence of SEQ ID NO: 147.

[0312] (ii) Reduction of cytokine release The therapeutic agents described below are particularly useful in the context of the present invention, particularly in relevant embodiments of the present invention that are involved in reducing cytokine release associated with the administration of therapeutic agents in a subject.

[0313] Aspects of the invention that are involved in reducing cytokine release associated with administration of a therapeutic agent in a subject are particularly useful in connection with a therapeutic agent that activates T cells in a subject (a T cell activating therapeutic agent), i.e., a therapeutic agent having the ability to induce T cell activation in a subject. Such therapeutic agents include, for example, T cell antigens (particularly activating T cell antigens), or antibodies directed to T cells modified with a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR). Aspects of the invention that are involved in reducing cytokine release associated with administration of a therapeutic agent in a subject are particularly useful in connection with a B cell targeted T cell activating therapeutic agent.

[0314] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to CD3, specifically CD3 epsilon.

[0315] In one embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region comprising heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37. In a further embodiment, the antibody that specifically binds to CD3 comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 38, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39. In a further embodiment, the antibody that specifically binds to CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

[0316] In one embodiment, an antibody that specifically binds to CD3 comprises a heavy chain variable region comprising heavy chain HVR 1 (H1-HVR) of SEQ ID NO: 120, H2-HVR of SEQ ID NO: 121, and H3-HVR of SEQ ID NO: 122; and a light chain variable region comprising light chain HVR 1 (L1-HVR) of SEQ ID NO: 123, L2-HVR of SEQ ID NO: 124, and L3-HVR of SEQ ID NO: 125. In a further embodiment, an antibody that specifically binds to CD3 comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 126, and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 127. In a further embodiment, an antibody that specifically binds to CD3 comprises the heavy chain variable region sequence of SEQ ID NO: 126 and the light chain variable region sequence of SEQ ID NO: 127.

[0317] In one embodiment, the antibody that specifically binds to CD3 is a full-length antibody. In one embodiment, the antibody that specifically binds to CD3 is an antibody of the human IgG class, specifically an antibody of the human IgG1 class. In one embodiment, the antibody that specifically binds to CD3 is an antibody fragment, specifically a Fab molecule or an scFv molecule, more specifically a Fab molecule. In certain embodiments, the antibody that specifically binds to CD3 is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., substituted for one another). In one embodiment, the antibody that specifically binds to CD3 is a humanized antibody.

[0318]

[0319] In one embodiment, the therapeutic agent comprises a multispecific antibody, specifically a bispecific antibody. In one embodiment, the multispecific antibody specifically binds to (i) an activated T cell antigen and (ii) a B cell antigen. Certain bispecific antibodies are described in PCT Publication No. WO 2016 / 020309 and PCT Application No. PCT / EP2016 / 073041 and PCT Publication No. WO 2015 / 095392 (each of which is incorporated herein by reference in its entirety).In one embodiment, the bispecific antibody specifically binds to CD3 and CD20. In one embodiment, the bispecific antibody comprises an antigen-binding portion that specifically binds to CD20 and an antigen-binding portion that specifically binds to CD3. In one embodiment, the bispecific antibody comprises a first antigen-binding portion that specifically binds to CD3 and second and third antigen-binding portions that specifically bind to CD20. In one embodiment, the first antigen-binding portion is a crossover Fab molecule and the second and first antigen-binding portions are conventional Fab molecules, respectively. In one embodiment, the bispecific antibody further comprises an Fc domain. The bispecific antibody can have the antibody formats described herein and can include the antigen-binding portions described herein. The bispecific antibody can include modifications to the Fc region and / or antigen-binding portions described herein.

[0320] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD3 and comprises heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37; and (ii) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD20 and comprises heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, HCDR2 of SEQ ID NO: 5, and HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 7, LCDR2 of SEQ ID NO: 8, and LCDR3 of SEQ ID NO: 9 and comprises a bispecific antibody.

[0321] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region sequence of SEQ ID NO: 38 that specifically binds to CD3; and a light chain variable region sequence of SEQ ID NO: 39; and (ii) an antigen-binding portion comprising a heavy chain variable region sequence of SEQ ID NO: 10 that specifically binds to CD20; and a light chain variable region sequence of SEQ ID NO: 11 and comprises a bispecific antibody.

[0322] In certain embodiments, the therapeutic agent is a) a first Fab molecule that specifically binds to a first antigen; b) a second Fab molecule that specifically binds to a second antigen, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted with each other; c) a third Fab molecule that specifically binds to the first antigen; and d) an Fc domain consisting of first and second subunits capable of stable association and comprises a bispecific antibody; wherein (i) the first antigen is CD20 and the second antigen is CD3, specifically CD3 epsilon; (ii) the first Fab molecule of a) and the third Fab molecule of c) each comprise a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 4, a heavy chain CDR2 of SEQ ID NO: 5, a heavy chain CDR3 of SEQ ID NO: 6, a light chain CDR1 of SEQ ID NO: 7, a light chain CDR2 of SEQ ID NO: 8, and a light chain CDR3 of SEQ ID NO: 9, and the second Fab molecule of b) comprises a heavy chain CDR1 of SEQ ID NO: 32, a heavy chain CDR2 of SEQ ID NO: 33, a heavy chain CDR3 of SEQ ID NO: 34, a light chain CDR1 of SEQ ID NO: 35, a light chain CDR2 of SEQ ID NO: 36, and a light chain CDR3 of SEQ ID NO: 37; (iii) in the constant domain CL of the first Fab molecule of a) and the third Fab molecule of c), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), in the constant domain CH1 of the first Fab molecule of a) and the third Fab molecule of c), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat); and (iv) The first Fab molecule of a) is fused to the N-terminus of the Fab heavy chain of the second Fab molecule of b) at the C-terminus of the Fab heavy chain, and the second Fab molecule of b) and the third Fab molecule of c) are each fused to the N-terminus of one of the subunits of the Fc domain of d) at the C-terminus of the Fab heavy chain.

[0323] In one embodiment, the first Fab molecule of a) and the third Fab molecule of c) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 10 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 11.

[0324] In one embodiment, the first Fab molecule of a) and the third Fab molecule of c) each comprise the heavy chain variable region sequence of SEQ ID NO: 10 and the light chain variable region sequence of SEQ ID NO: 11.

[0325] In one embodiment, the second Fab molecule of b) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39.

[0326] In yet another embodiment, the second Fab molecule of b) comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

[0327] In certain embodiments, the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 44, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 45, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 46, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 47. In further particular embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 44, the polypeptide sequence of SEQ ID NO: 45, the polypeptide sequence of SEQ ID NO: 46, and the polypeptide sequence of SEQ ID NO: 47. (CD20XCD3 bsAB)

[0328] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD3 and comprises H1-HVR of SEQ ID NO: 120, H2-HVR of SEQ ID NO: 121, and H3-HVR of SEQ ID NO: 122; and a light chain variable region comprising L1-HVR of SEQ ID NO: 123, L2-HVR of SEQ ID NO: 124, and L3-HVR of SEQ ID NO: 125; and (ii) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD20 and comprises H1-HVR of SEQ ID NO: 128, H2-HVR of SEQ ID NO: 129, and H3-HVR of SEQ ID NO: 130; and a light chain variable region comprising L1-HVR of SEQ ID NO: 131, L2-HVR of SEQ ID NO: 132, and L3-HVR of SEQ ID NO: 133 comprising a bispecific antibody.

[0329] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region sequence that specifically binds to CD3; and a light chain variable region sequence of SEQ ID NO: 127; and (ii) an antigen-binding portion comprising a heavy chain variable region sequence that specifically binds to CD20; and a light chain variable region sequence of SEQ ID NO: 135 comprising a bispecific antibody.

[0330] In one embodiment, the bispecific antibody comprises an antigen-binding portion that specifically binds to CD19 and an antigen-binding portion that specifically binds to CD3. In one embodiment, the bispecific antibody comprises a first antigen-binding portion that specifically binds to CD3 and second and third antigen-binding portions that specifically bind to CD19. In one embodiment, the first antigen-binding portion is a crossover Fab molecule and the second and first antigen-binding portions are conventional Fab molecules, respectively. In one embodiment, the bispecific antibody further comprises an Fc domain. The bispecific antibody may comprise modifications to the Fc region and / or antigen-binding portions described herein.

[0331] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD3 and comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37; and (ii) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD19 and comprises heavy chain CDR (HCDR) 1 of SEQ ID NO: 48, HCDR2 of SEQ ID NO: 49, and HCDR3 of SEQ ID NO: 50; and a light chain variable region comprising light chain CDR (LCDR) 1 of SEQ ID NO: 51, LCDR2 of SEQ ID NO: 52, and LCDR3 of SEQ ID NO: 53 and comprises a bispecific antibody.

[0332] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region sequence of SEQ ID NO: 38 that specifically binds to CD3; and a light chain variable region sequence of SEQ ID NO: 39; and (ii) an antigen-binding portion comprising a heavy chain variable region sequence of SEQ ID NO: 54 that specifically binds to CD19; and a light chain variable region sequence of SEQ ID NO: 55 and comprises a bispecific antibody.

[0333] In certain embodiments, the therapeutic agent is a) A first Fab molecule that specifically binds to a first antigen; b) A second Fab molecule that specifically binds to a second antigen, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted with each other; c) A third Fab molecule that specifically binds to the first antigen; and d) An Fc domain consisting of first and second subunits capable of stable association comprising a bispecific antibody; wherein (i) The first antigen is CD19 and the second antigen is CD3, specifically CD3 epsilon; (ii) The first Fab molecule of a) and the third Fab molecule of c) each comprise a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 48, a heavy chain CDR2 of SEQ ID NO: 49, a heavy chain CDR3 of SEQ ID NO: 50, a light chain CDR1 of SEQ ID NO: 51, a light chain CDR2 of SEQ ID NO: 52, and a light chain CDR3 of SEQ ID NO: 53, and the second Fab molecule of b) comprises a heavy chain CDR1 of SEQ ID NO: 32, a heavy chain CDR2 of SEQ ID NO: 33, a heavy chain CDR3 of SEQ ID NO: 34, a light chain CDR1 of SEQ ID NO: 35, a light chain CDR2 of SEQ ID NO: 36, and a light chain CDR3 of SEQ ID NO: 37; (iii) In the constant domain CL of the first Fab molecule of a) and the third Fab molecule of c), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly arginine (R) (numbering according to Kabat). In the constant domain CH1 of the first Fab molecule of a) and the third Fab molecule of c), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat); and (iv) The first Fab molecule of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule of b), and the second Fab molecule of b) and the third Fab molecule of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d).

[0334] In one embodiment, the first Fab molecule of a) and the third Fab molecule of c) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55.

[0335] In one embodiment, the first Fab molecule of a) and the third Fab molecule of c) each comprise the heavy chain variable region sequence of SEQ ID NO: 54 and the light chain variable region sequence of SEQ ID NO: 55.

[0336] In one embodiment, the second Fab molecule of b) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39.

[0337] In yet another embodiment, the second Fab molecule of b) comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

[0338] In certain embodiments, the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 47, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 56, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 57, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 58. In further particular embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 47, the polypeptide sequence of SEQ ID NO: 56, the polypeptide sequence of SEQ ID NO: 57, and the polypeptide sequence of SEQ ID NO: 58.

[0339] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD3 and comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37; and (ii) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD19 and comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 59, HCDR2 of SEQ ID NO: 60, and HCDR3 of SEQ ID NO: 61; and a light chain variable region comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 62, LCDR2 of SEQ ID NO: 63, and LCDR3 of SEQ ID NO: 64 comprising a bispecific antibody.

[0340] In one embodiment, the therapeutic agent is (i) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD3 and comprises the heavy chain variable region sequence of SEQ ID NO: 38; and a light chain variable region sequence of SEQ ID NO: 39; and (ii) an antigen-binding portion comprising a heavy chain variable region that specifically binds to CD19 and comprises the heavy chain variable region sequence of SEQ ID NO: 65; and a light chain variable region sequence of SEQ ID NO: 66 comprising a bispecific antibody.

[0341] In certain embodiments, the therapeutic agent is a) a first Fab molecule that specifically binds to a first antigen; b) a second Fab molecule that specifically binds to a second antigen, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted with each other; c) a third Fab molecule that specifically binds to the first antigen; and d) an Fc domain consisting of first and second subunits capable of stable association comprising a bispecific antibody; wherein (i) the first antigen is CD19 and the second antigen is CD3, specifically CD3 epsilon; (ii) the first Fab molecule of a) and the third Fab molecule of c) each comprise a heavy chain complementarity determining region (CDR) 1 of SEQ ID NO: 59, a heavy chain CDR2 of SEQ ID NO: 60, a heavy chain CDR3 of SEQ ID NO: 61, a light chain CDR1 of SEQ ID NO: 62, a light chain CDR2 of SEQ ID NO: 63, and a light chain CDR3 of SEQ ID NO: 64, and the second Fab molecule of b) comprises a heavy chain CDR1 of SEQ ID NO: 32, a heavy chain CDR2 of SEQ ID NO: 33, a heavy chain CDR3 of SEQ ID NO: 34, a light chain CDR1 of SEQ ID NO: 35, a light chain CDR2 of SEQ ID NO: 36, and a light chain CDR3 of SEQ ID NO: 37; (iii) in the constant domain CL of the first Fab molecule of a) and the third Fab molecule of c), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), and the amino acid at position 123 is substituted by lysine (K) or arginine (R), particularly by arginine (R) (numbering according to Kabat), in the constant domain CH1 of the first Fab molecule of a) and the third Fab molecule of c), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat), and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat); and (iv) The first Fab molecule of a) is fused to the N-terminus of the Fab heavy chain of the second Fab molecule of b) at the C-terminus of the Fab heavy chain, and the second Fab molecule of b) and the third Fab molecule of c) are each fused to the N-terminus of one of the subunits of the Fc domain of d) at the C-terminus of the Fab heavy chain.

[0342] In one embodiment, the first Fab molecule of a) and the third Fab molecule of c) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 65 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 66.

[0343] In one embodiment, the first Fab molecule of a) and the third Fab molecule of c) each comprise the heavy chain variable region sequence of SEQ ID NO: 65 and the light chain variable region sequence of SEQ ID NO: 66.

[0344] In one embodiment, the second Fab molecule of b) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39.

[0345] In yet another embodiment, the second Fab molecule of b) comprises the heavy chain variable region sequence of SEQ ID NO: 38 and the light chain variable region sequence of SEQ ID NO: 39.

[0346] In certain embodiments, the bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 47, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 148, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 149, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 150. In a further particular embodiment, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 47, the polypeptide sequence of SEQ ID NO: 148, the polypeptide sequence of SEQ ID NO: 149, and the polypeptide sequence of SEQ ID NO: 150.

[0347] Antibody format The antibody components included in the therapeutic agent, specifically the multispecific antibodies, can be fused to each other in various configurations. Exemplary configurations are shown in FIG. 6.

[0348] In certain embodiments, the antigen-binding portion included in the antibody is a Fab molecule. In such embodiments, the first, second, third, etc. antigen-binding portions can be referred to as the first, second, third, etc. Fab molecules, respectively. Further, in certain embodiments, the antibody comprises an Fc domain consisting of first and second subunits capable of stable association.

[0349] In some embodiments, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0350] In one such embodiment, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In such a particular embodiment, the antibody consists essentially of the first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, where the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such configurations are schematically shown in FIGS. 6G and 6K. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may further be fused to each other.

[0351] In another such embodiment, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such a particular embodiment, the antibody consists essentially of the first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, where the first and second Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such configurations are schematically shown in FIGS. 6A and 6D. The first and second Fab molecules can be fused to the Fc domain either directly or via a peptide linker. In a particular embodiment, the first and second Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is the human IgG1 hinge region, particularly where the Fc domain is the IgG1 Fc domain.

[0352] In other embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0353] In one such embodiment, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. In such a particular embodiment, the antibody consists essentially of the first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, where the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. Such an arrangement is schematically shown in FIGS. 6H and 6L. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may further be fused to each other.

[0354] The Fab molecules can be fused to the Fc domain or to each other directly or via a peptide linker comprising one or more amino acids, typically about 2 - 20 amino acids. Peptide linkers are well known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or 4(SG4) n peptide linkers. "n" is usually a number from 1 to 10, typically 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment a length of 5 - 100, and in a further embodiment a length of 10 - 50 amino acids. In one embodiment, the peptide linker is (GxS) n or (GxS) n G mwhere G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5 and m = 0, 1, 2 or 3), in one embodiment, x = 4 and n = 2 or 3, and in a further embodiment, x = 4 and n = 2. In one embodiment, the peptide linker is (G4S)2. A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. Exemplary peptide linkers suitable for linking the Fab heavy chains of the first and second Fab molecules are sequences (D)-(G4S)2 (SEQ ID NOs: 118 and 119). Another suitable such linker comprises the sequence (G4S)4. Further, the linker may comprise (a part of) an immunoglobulin hinge region. In particular, when the Fab molecule is fused to the N-terminus of a subunit of the Fc domain, it can be fused via an immunoglobulin hinge region or a part thereof, with or without an additional peptide linker.

[0355] Antibodies having a single antigen-binding portion (such as a Fab molecule) that can specifically bind to a target cell antigen (e.g., shown in FIGS. 6A, D, G, H, K, L) are useful particularly when internalization of the target cell antigen is expected following binding of a high-affinity antigen-binding portion. In such cases, the presence of two or more antigen-binding portions specific for the target cell antigen may enhance internalization of the target cell antigen and thereby reduce its availability.

[0356] However, in many other cases, for example, to optimize targeting to a target site or to enable cross-linking of a target cell antigen, it would be advantageous to have an antibody comprising two or more antigen-binding portions (such as Fab molecules) specific for the target cell antigen (see the examples shown in FIGS. 6B, 6C, 6E, 6F, 6I, 6J, 6M or 6N).

[0357] Accordingly, in certain embodiments, the antibody further comprises a third Fab molecule that specifically binds to a first antigen. The first antigen is preferably a target cell antigen. In one embodiment, the third Fab molecule is a conventional Fab molecule. In one embodiment, the third Fab molecule is identical to the first Fab molecule (i.e., the first and third Fab molecules comprise the same heavy and light chain amino acid sequences and have the same arrangement of domains (i.e., conventional or crossover type)). In certain embodiments, the second Fab molecule specifically binds to an activated T cell antigen, specifically CD3, and the first and third Fab molecules specifically bind to a target cell antigen.

[0358] In another embodiment, the antibody further comprises a third Fab molecule that specifically binds to a second antigen. In these embodiments, the second antigen is preferably a target cell antigen. In one such embodiment, the third Fab molecule is a crossover Fab molecule (a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged / substituted with each other). In one such embodiment, the third Fab molecule is identical to the second Fab molecule (i.e., the second and third Fab molecules comprise the same heavy and light chain amino acid sequences and have the same arrangement of domains (i.e., conventional or crossover type)). In one such embodiment, the first Fab molecule specifically binds to an activated T cell antigen, specifically CD3, and the second and third Fab molecules specifically bind to a target cell antigen.

[0359] In one embodiment, the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0360] In certain embodiments, the second and third Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In such a particular embodiment, the antibody consists essentially of the first, second, and third Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such an arrangement is schematically shown in FIGS. 6B and 6E (in a particular embodiment, the third Fab molecule is a conventional Fab molecule, preferably identical to the first Fab molecule) and FIGS. 6I and 6M (in an alternative embodiment, the third Fab molecule is a crossover Fab molecule, preferably identical to the second Fab molecule). The second and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is the human IgG1 hinge region, particularly where the Fc domain is the IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may further be fused to each other.

[0361] In another embodiment, the first and third Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In such a particular embodiment, the antibody consists essentially of the first, second and third Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such an arrangement is schematically shown in FIGS. 6C and 6F (in a particular embodiment, the third Fab molecule is a conventional Fab molecule, preferably identical to the first Fab molecule) and FIGS. 6J and 6N (in an alternative embodiment, the third Fab molecule is a crossover Fab molecule, preferably identical to the second Fab molecule). The first and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In a particular embodiment, the first and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is the human IgG1 hinge region, particularly where the Fc domain is the IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may further be fused to each other.

[0362] In the conformation of an antibody in which the Fab molecules are fused at the C-terminus of the Fab heavy chains via the immunoglobulin hinge region to the N-terminus of each subunit of the Fc domain, the two Fab molecules, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In certain embodiments, the immunoglobulin molecule is an immunoglobulin of the IgG class. In more certain embodiments, the immunoglobulin is an immunoglobulin of the IgG1 subclass. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass. In more certain embodiments, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.

[0363] In some antibodies, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are optionally fused to each other via a peptide linker. Depending on the conformation of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces the mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required for the expression of some antibodies.

[0364] In certain embodiments, the antibody is a polypeptide (VL (2) -CH1 (2) -CH2-CH3(-CH4)) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit, and a polypeptide (VH (1) -CH1 (1)-CH2-CH3(-CH4)), and in some embodiments, the antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ), and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0365] In certain embodiments, the antibody comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit, and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (2) -CL (2) -CH2-CH3(-CH4)), and a polypeptide in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (1) -CH1 (1) -CH2-CH3(-CH4)). In some embodiments, the antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ), and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0366] In some embodiments, the antibody is a polypeptide in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (2) -CH1 (2) -VH (1) -CH1 (1) -CH2-CH3(-CH4)). In other embodiments, the antibody is a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VL (2) -CH1 (2) -CH2-CH3(-CH4)).

[0367] In some of these embodiments, the antibody further comprises a crossover Fab light chain polypeptide of the second Fab molecule (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and a Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In other of these embodiments, the antibody further comprises a polypeptide (VH (2) -CL (2) -VL (1)-CL (1) ) or the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (polypeptide (VL (1) -CL (1) -VH (2) -CL (2) ) is included as needed.

[0368] Antibodies according to these embodiments further include (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a subunit of the Fc domain (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0369] In some embodiments, the antibody has a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -VH (1) -CH1 (1)-CH2-CH3(-CH4)). In other embodiments, the antibody is such that the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (1) -CH1 (1) -VH (2) -CL (2) -CH2-CH3(-CH4)).

[0370] In some of these embodiments, the antibody further comprises a crossover Fab light chain polypeptide (VL (2) -CH1 (2) ) of the second Fab molecule, in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In other of these embodiments, the antibody further comprises a polypeptide (VL (2) -CH1 (2) -VL (1) -CL (1) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule, or a polypeptide (VL (1) -CL (1) -VH (2) -CL (2) ) in which the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, as needed.

[0371] Antibodies according to these embodiments further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with a subunit of the Fc domain (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide (VL (3) -CL (3) ). In certain embodiments, the polypeptides are covalently linked, for example, by disulfide bonds.

[0372] In some embodiments, the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the antibody does not contain an Fc domain. In certain embodiments, the antibody consists essentially of the first and second Fab molecules, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in FIGS. 6O and 6S.

[0373] In other embodiments, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the antibody does not contain an Fc domain. In certain embodiments, the antibody consists essentially of the first and second Fab molecules, and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in FIGS. 6P and 6T.

[0374] In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the antibody further comprises a third Fab molecule, wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In certain such embodiments, the third Fab molecule is a conventional Fab molecule. In other such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged / substituted with each other. In certain such embodiments, the antibody consists essentially of the first, second, and third Fab molecules, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such configurations are schematically shown in FIGS. 6Q and 6U. (In certain embodiments, the third Fab molecule is a conventional Fab molecule, preferably the same as the first Fab molecule).

[0375] In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the antibody further comprises a third Fab molecule, wherein the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. In certain such embodiments, the third Fab molecule is the crossover Fab molecule described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged / substituted with each other. In other such embodiments, the third Fab molecule is a conventional Fab molecule. In certain such embodiments, the antibody consists essentially of the first, second, and third Fab molecules, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. Such configurations are schematically shown in FIGS. 6W and 6Y. (In certain embodiments, the third Fab molecule is a crossover Fab molecule, preferably the same as the second Fab molecule).

[0376] In some embodiments, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the antibody further comprises a third Fab molecule, wherein the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain. In certain such embodiments, the third Fab molecule is a conventional Fab molecule. In other such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged / substituted with each other. In certain such embodiments, the antibody consists essentially of the first, second, and third Fab molecules, and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain. Such configurations are schematically shown in FIGS. 6R and 6V. (In certain embodiments, the third Fab molecule is a conventional Fab molecule, preferably the same as the first Fab molecule).

[0377] In some embodiments, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the antibody further comprises a third Fab molecule, wherein the third Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. In certain such embodiments, the third Fab molecule is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged / substituted with each other. In other such embodiments, the third Fab molecule is a conventional Fab molecule. In certain such embodiments, the antibody consists essentially of the first, second, and third Fab molecules, and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain. Such configurations are schematically shown in FIGS. 6X and 6Z. (In certain embodiments, the third Fab molecule is a crossover Fab molecule, preferably the same as the first Fab molecule).

[0378] In certain embodiments, the antibody comprises a polypeptide (VH (1) -CH1 (1) -VL (2) -CH1 (2) ) in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region). (2) -CL (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide (VH

[0379] In certain embodiments, the antibody comprises a polypeptide (VL (2) -CH1 (2) -VH (1) -CH1 (1) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule.

[0380] In certain embodiments, the antibody comprises a polypeptide (VH (2) -CL (2) -VH (1) -CH1 (1) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide (VL (2) -CH1 (2) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule.

[0381] In certain embodiments, the antibody is a polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) ) in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region). In some embodiments, the antibody further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further comprises the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0382] In certain embodiments, the antibody is a polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VH (2) -CL (2) ) in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region). In some embodiments, the antibody further comprises a polypeptide (VL(2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody is the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0383] In certain embodiments, the antibody is such that the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule, a polypeptide (VL (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) ). In some embodiments, the antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ), and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further comprises the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0384] In certain embodiments, the antibody is a polypeptide (VH (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) ) in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule includes a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. In some embodiments, the antibody further includes a polypeptide (VL (2) -CH1 (2) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further includes the Fab light chain polypeptide (VL (3) -CL (3) ) of the third Fab molecule.

[0385] In certain embodiments, the antibody is a polypeptide (VH (1) -CH1 (1) -VL (2) -CH1 (2) -VL (3) -CH1(3) ) includes. In some embodiments, the antibody further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide (VH (3) -CL (3) ) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule.

[0386] In certain embodiments, the antibody comprises a polypeptide (VH (1) -CH1 (1) -VH (2) -CL (2) -VH (3) -CL (3) ) in which the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region). In some embodiments, the antibody further comprises a polypeptide (VL (2) -CH1 (2) ) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1)) is included. In some embodiments, the antibody further comprises a polypeptide (VL (3) -CH1 (3) ) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule.

[0387] In certain embodiments, the antibody comprises a polypeptide (VL (3) -CH1 (3) -VL (2) -CH1 (2) -VH (1) -CH1 (1) ) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule. In some embodiments, the antibody further comprises a polypeptide (VH (3) -CL (3) ) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule.

[0388] In certain embodiments, the antibody is a polypeptide in which the Fab heavy chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VH (3) -CL (3) -VH (2) -CL (2) -VH (1) -CH1 (1) ). In some embodiments, the antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ), and the Fab light chain polypeptide of the first Fab molecule (VL (1) -CL (1) ). In some embodiments, the antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ).

[0389] In any of the above embodiments, the components of the antibody (e.g., Fab molecules, Fc domains) are fused either directly or via various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids. Such linkers are described herein or are known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4(SG4) nA peptide linker is included, where n is usually an integer from 1 to 10, typically from 2 to 4.

[0390] Fc domain An antibody, e.g., a bispecific antibody or an immunoconjugate included in a therapeutic agent, may comprise an Fc domain consisting of a pair of polypeptide chains comprising the heavy chain domains of the antibody molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each of its subunits comprises CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other.

[0391] In one embodiment, the Fc domain is an IgG Fc domain. In certain embodiments, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84 - 91 (2010)). In an even more specific embodiment, the Fc domain is human. An exemplary sequence of the human IgG1 Fc region is provided in SEQ ID NO: 30.

[0392] (i) Modifications of the Fc domain that promote heterodimerization Antibodies contained in a therapeutic agent, specifically bispecific antibodies or immunoconjugates, can contain different components (e.g., antigen-binding domains, cytokines) fused to one or the other of the two subunits of the Fc domain, and thus the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. The recombinant co-expression of these polypeptides and subsequent dimer formation results in multiple possible combinations of the two polypeptides. To improve the yield and purity of such antibodies in recombinant production, it would be advantageous to introduce modifications into the Fc domain of the antibody that promote the association of the desired polypeptides.

[0393] Accordingly, in certain embodiments, the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Accordingly, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0394] To force heterodimerization, there are several approaches for modification in the CH3 domain of the Fc domain, for example, as described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both modified in a complementary manner, and each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself, but is forced to heterodimerize with the other complementarily modified CH3 domain (so that the first and second CH3 domains heterodimerize and no homodimer is formed between two first or two second CH3 domains). These different approaches for improved heavy chain heterodimerization, which reduce light chain mispairing and Bence Jones type by-products, are considered as different options in combination with heavy chain light chain modifications (such as exchange / substitution of variable or constant regions in the Fab arm, or introduction of substitutions of charged amino acids with opposite charges at the CH1 / CL interface).

[0395] In a particular embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0396] The knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by substituting small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created at the contact surface of the second polypeptide by substituting large amino acid side chains with smaller ones (e.g., alanine or threonine).

[0397] Thus, in certain embodiments, in the CH3 domain of the first subunit of the Fc domain, an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby generating a protrusion within the CH3 domain of the first subunit that can be positioned within the cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit that can accommodate the protrusion within the CH3 domain of the first subunit.

[0398] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0399] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0400] Protrusions and cavities can be created by modifying a nucleic acid encoding a polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0401] In certain embodiments, in the CH3 domain of the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, further, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0402] In yet another embodiment, in the first subunit of the Fc domain, further, the serine residue at position 354 is replaced with a cysteine residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain, further, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0403] In certain embodiments, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0404] In certain embodiments, the mutant IL-2 polypeptide in the immunoconjugates described herein or the CD3 antigen-binding portion in the bispecific antibodies described herein is fused to the first subunit of the Fc domain (including the "knob" modification). Without wishing to be bound by theory, fusing the IL-2 polypeptide or the CD3 antigen-binding portion to the knob-containing subunit of the Fc domain may (further) minimize the production of each of the immunoconjugates containing two IL-2 polypeptides or the bispecific antibodies containing two CD3 antigen-binding portions (steric collisions of two knob-containing polypeptides).

[0405] Other techniques for forcing heterodimerization by CH3 modification are contemplated as alternatives according to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0406] In one embodiment, the heterodimerization approach described in EP 1870459 (A1) is alternatively used. This approach is based on the introduction of charged amino acids having opposite charges at specific amino acid positions at the CH3 / CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment is the amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and the amino acid mutations D399K; E357K in the other of the CH3 domains of the Fc domain (numbering according to the Kabat EU index).

[0407] In another embodiment, the antibody comprises the amino acid mutations T366W in the CH3 domain of the first subunit of the Fc domain, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further the amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutations D399K;E357K (numbering according to Kabat's EU index) in the CH3 domain of the second subunit of the Fc domain.

[0408] In another embodiment, the antibody comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain, and Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain, and S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further the amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutations D399K;E357K (all numbering is according to Kabat's EU index) in the CH3 domain of the second subunit of the Fc domain.

[0409] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K, and the second CH3 domain comprises the amino acid mutation L351D (numbering according to Kabat's EU index). In a further embodiment, the first CH3 domain further comprises the additional amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation (preferably L368E) selected from Y349E, Y349D and L368E (numbering according to Kabat's EU index).

[0410] In one embodiment, the heterodimerization approach described in International Publication No. WO 2012 / 058768 is alternatively used. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises additional amino acid mutations selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to Kabat's EU index) at positions T411, D399, S400, F405, N390, or K392. In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutation Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to Kabat's EU index).

[0411] In one embodiment, the heterodimerization approach described in International Publication No. WO 2011 / 143545 having amino acid modifications at positions (numbering according to Kabat's EU index) selected from the group consisting of, for example, 368 and 409 is alternatively used.

[0412] In one embodiment, an alternative use is made of the heterodimerization approach described in WO 2011 / 090762, which uses the knob-into-hole technology described above. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the EU index of Kabat).

[0413] In one embodiment, the antibody or its Fc domain is of the IgG2 subclass and an alternative use is made of the heterodimerization approach described in WO 2010 / 129304.

[0414] In another embodiment, the modification that promotes the association of the first and second subunits of the Fc domain includes, for example, the modification that mediates the electrostatic steering effect described in International Publication No. WO 2009 / 089004 of the PCT publication. Generally, this method involves substituting one or more amino acid residues with charged amino acid residues at the interface of the two Fc domain subunits such that homodimer formation is electrostatically unfavorable while heterodimer is electrostatically favorable. In one such embodiment, the first CH3 domain includes an amino acid substitution of a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) for K392 or N392, preferably K392D or N392D, and the second CH3 domain includes an amino acid substitution of a positively charged amino acid (e.g., lysine (K) or arginine (R)) for D399, E356, D356 or E357, preferably D399K, E356K, D356K, or E357K, more preferably D399K and E356K. In a further embodiment, the first CH3 domain further includes an amino acid substitution of a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) for K409 or R409, preferably K409D or R409D. In a further embodiment, the first CH3 domain further includes an amino acid substitution of a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) for K439 and / or K370 (all numbers are according to Kabat's EU index).

[0415] In yet another embodiment, the heterodimerization approach described in International Publication No. WO 2007 / 147901 is alternatively used. In one embodiment, the first CH3 domain includes the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain includes the amino acid mutations D239K, E240K, and K292D (numbering according to Kabat's EU index).

[0416] In yet another embodiment, the heterodimerization approach described in International Publication No. WO 2007 / 110205 is alternatively used.

[0417] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0418] (ii) Modifications of the Fc domain that reduce binding to Fc receptors and / or effector functions The Fc domain confers desirable pharmacokinetic properties to antibodies, such as bispecific antibodies or immunoconjugates, and such pharmacokinetic properties include a long serum half-life that contributes to good accumulation in target tissues and a desirable tissue-blood distribution ratio. However, at the same time, it can lead to undesirable targeting of the antibody to cells that express Fc receptors rather than to preferably antigen-bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway can cause cytokine release, which, in combination with other immune-activating properties that the antibody may have and the long half-life of the antibody, leads to over-activation of cytokine receptors and causes severe side effects when administered systemically.

[0419] Accordingly, in certain embodiments, the Fc domain of an antibody, specifically a bispecific antibody or an immunoconjugate, contained in a therapeutic agent exhibits a reduced binding affinity for Fc receptors and / or a reduced effector function when compared to the native IgG1 Fc domain. In such an embodiment, the Fc domain (or a molecule containing said Fc domain, such as an antibody) exhibits a binding affinity for Fc receptors that is less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native IgG1 Fc domain (or the corresponding molecule containing the native IgG1 Fc domain), and / or exhibits an effector function that is less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to the native IgG1 Fc domain (or the corresponding molecule containing the native IgG1 Fc domain). In one embodiment, the Fc domain (or a molecule containing said Fc domain, such as an antibody) does not substantially bind to Fc receptors and / or does not induce an effector function. In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits a binding affinity for the neonatal Fc receptor (FcRn) that is substantially similar to that of the native IgG1 Fc domain. A substantially similar binding affinity for FcRn is achieved when the Fc domain (or a molecule containing said Fc domain, such as an antibody) exhibits a binding affinity for FcRn that is greater than about 70%, specifically greater than about 80%, more specifically greater than about 90% of the native IgG1 Fc domain (or the corresponding molecule containing the native IgG1 Fc domain).

[0420] In certain embodiments, the Fc domain is engineered to have a reduced binding affinity for Fc receptors and / or a reduced effector function as compared to an unengineered Fc domain. In certain embodiments, the Fc domain comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain for Fc receptors. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain for Fc receptors. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain for Fc receptors to at least one-half, at least one-fifth, or at least one-tenth. In one embodiment in which there are multiple amino acid mutations that reduce the binding affinity of the Fc domain for Fc receptors, the combination of these amino acid mutations reduces the binding affinity of the Fc domain for Fc receptors to at least one-tenth, at least one-twentieth, or at least one-fiftieth. In one embodiment, an antibody comprising the engineered Fc domain exhibits a binding affinity for Fc receptors that is less than 20%, specifically less than 10%, more specifically less than 5% compared to the corresponding molecule comprising the unengineered Fc domain. In one particular embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In one particular embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity for complement components, specifically for C1q, is also reduced. In one embodiment, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain for said receptor, is achieved when the Fc domain (or a molecule comprising said Fc domain, such as an antibody) exhibits a binding affinity for FcRn that exceeds about 70% of the unmanipulated form of the Fc domain (or the corresponding molecule comprising the unmanipulated form of the Fc domain). The Fc domain, or a molecule comprising said Fc domain (e.g., an antibody), can exhibit an affinity exceeding about 80%, and even exceeding about 90%, of such affinity. In certain embodiments, the Fc domain is engineered to have reduced effector function compared to the unmanipulated Fc domain. Reduction of effector function can include, but is not limited to, reduction of complement-dependent cytotoxicity (CDC), reduction of antibody-dependent cell-mediated cytotoxicity (ADCC), reduction of antibody-dependent cell phagocytosis (ADCP), reduction of cytokine secretion, reduction of immune complex-mediated antigen uptake by antigen-presenting cells, reduction of binding to NK cells, reduction of binding to macrophages, reduction of binding to monocytes, reduction of binding to polymorphonuclear cells, reduction of direct signaling to induce apoptosis, reduction of cross-linking of target-binding antibodies, reduction of dendritic cell maturation, or reduction of T cell priming. In one embodiment, the reduction of effector function is one or more selected from the group consisting of reduction of CDC, reduction of ADCC, reduction of ADCP, and reduction of cytokine secretion. In certain embodiments, the reduction of effector function is reduction of ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by the unmanipulated Fc domain (or the corresponding molecule comprising the unmanipulated Fc domain).

[0421] In one embodiment, the amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain for Fc receptors are amino acid substitutions. In one embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to the EU index of Kabat). In a more specific embodiment, the Fc domain comprises amino acid substitutions at positions selected from the group of L234, L235 and P329 (numbering according to the EU index of Kabat). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the EU index of Kabat). In such an embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at the position of P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the EU index of Kabat). In one embodiment, the Fc domain comprises an amino acid substitution at the position of P329 and further comprises amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering according to the EU index of Kabat). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D or P331S. In certain embodiments, the Fc domain comprises amino acid substitutions at the positions of P329, L234 and L235 (numbering according to the EU index of Kabat). In a more specific embodiment, the Fc domain comprises the amino acid substitutions L234A, L235A and P329G ("P329G LALA"). In such an embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The combination of amino acid substitutions "P329G LALA" almost completely abrogates the binding of the human IgG1 Fc domain to Fcγ receptors (as well as complement), as described in WO 2012 / 130831, which is hereby incorporated by reference in its entirety.WO 2012 / 130831 also describes methods for preparing such variant Fc domains and methods for determining their properties such as Fc receptor binding or effector function.

[0422] IgG4 antibodies exhibit reduced binding affinity for Fc receptors and reduced effector function compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). In order to further reduce its binding affinity for Fc receptors and / or its effector function, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). In certain embodiments, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically the amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is hereby incorporated by reference in its entirety.

[0423] In certain embodiments, the Fc domain that exhibits reduced binding affinity for Fc receptors and / or reduced effector function compared to the native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbering according to the Kabat EU index).

[0424] In certain embodiments, N-glycosylation of the Fc domain is excluded. In such an embodiment, the Fc domain comprises an amino acid substitution at position N297, specifically an amino acid substitution that replaces asparagine with alanine (N297A) or aspartic acid (N297D) or glycine (N297G).

[0425] In addition to the Fc domains described herein and in International Publication No. WO 2012 / 130831, Fc domains having reduced Fc receptor binding and / or effector function also include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbering according to Kabat's EU index). Such Fc variants include Fc variants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant having substitutions of alanine at residues 265 and 297 (U.S. Patent No. 7,332,581).

[0426] Variant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, and the like. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0427] Binding to the Fc receptor can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard measurement means such as a BIAcore instrument (GE Healthcare), and such Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of the Fc domain, or a molecule containing the Fc domain, for the Fc receptor may be evaluated using cell lines known to express a particular Fc receptor, such as NK cells that express the FcγIIIa receptor.

[0428] The effector functions of the Fc domain, or of the molecules (e.g., antibodies) of the present invention that contain the Fc domain, can be measured by methods well known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for evaluating the ADCC activity of the molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assays (see CellTechnology, Inc., Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0429] In some embodiments, binding of the Fc domain to complement components, particularly binding to C1q, is reduced. Thus, in some embodiments where the Fc domain is modified such that effector function is reduced, the reduction of said effector function includes reduction of CDC. A C1q binding assay can be performed to determine whether an Fc domain, or a molecule comprising the Fc domain (e.g., an antibody), can bind to C1q and thereby have CDC activity. See, for example, the C1q and C3c binding ELISAs of WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, et al., Blood 101:1045-1052 (2003); and Cragg, and Glennie, Blood 103:2738-2743 (2004)).

[0430] antigen-binding portion The antibody included in the therapeutic agent may be bispecific, i.e., it comprises at least two antigen-binding portions capable of specifically binding to two different antigenic determinants. According to certain embodiments, the antigen-binding portion is a Fab molecule (i.e., an antigen-binding domain consisting of a heavy chain and a light chain each comprising a variable and a constant domain). In one embodiment, the Fab molecule is human. In another embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule comprises human heavy and light chain constant domains.

[0431] In some embodiments, at least one of the antigen-binding portions is a crossover Fab molecule. Such modifications reduce the mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the antibody in recombinant production. In certain crossover Fab molecules useful for antibodies, the variable domains of the Fab light chain and Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of the antibody may include certain by-products due to so-called Bence Jones-type interactions between mispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). To further reduce the mispairing of heavy and light chains from different Fab molecules and thus increase the purity and yield of the desired antibody according to the present invention, charged amino acids having opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of either a Fab molecule that specifically binds to a target cell antigen or a Fab molecule that specifically binds to an activated T cell antigen. The charge modification is performed in either a conventional Fab molecule contained in the antibody (e.g., such as those shown in FIGS. 1A-C, G-J), or a crossover Fab molecule contained in the antibody (e.g., such as those shown in FIGS. 1D-F, K-N) (but not both). In certain embodiments, the charge modification is performed in a conventional Fab molecule contained in the antibody (which, in certain embodiments, specifically binds to a target cell antigen).

[0432] In certain embodiments according to the present invention, the antibody can simultaneously bind to a target cell antigen, specifically a tumor cell antigen, and an activated T cell antigen, specifically CD3. In one embodiment, the antibody can bridge T cells and target cells by simultaneously binding to the target cell antigen and the activated T cell antigen. In a more specific embodiment, such simultaneous binding causes lysis of the target cells, particularly tumor cells. In one embodiment, such simultaneous binding causes activation of T cells. In other embodiments, such simultaneous binding causes a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activation, and expression of activation markers. In one embodiment, binding of the antibody to the activated T cell antigen, specifically CD3, without concomitant binding to the target cell antigen does not cause activation of T cells.

[0433] In one embodiment, the antibody can re-direct the cytotoxic activity of T cells to target cells. In a particular embodiment, said re-direction is independent of MHC-mediated peptide antigen presentation by the target cells and / or the specificity of the T cells.

[0434] In particular, the T cells according to any of the embodiments of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, particularly CD8 + T cells.

[0435] (i) Activated T cell antigen-binding portion In some embodiments, the antibody contained in the therapeutic agent, specifically the bispecific antibody, comprises at least one antigen-binding portion that specifically binds to an activated T cell antigen, particularly a Fab molecule (also referred to herein as the "activated T cell antigen-binding portion, or activated T cell antigen-binding Fab molecule"). In a particular embodiment, the antibody comprises at most one antigen-binding portion that can specifically bind to the activated T cell antigen. In one embodiment, the antibody provides a monovalent binding to the activated T cell antigen.

[0436] In certain embodiments, the antigen-binding portion that specifically binds to an activated T cell antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged / replaced with each other. In such embodiments, the antigen-binding portion that specifically bin...

Claims

1. A medicament comprising a T cell activation therapeutic agent for use in a method of treating a disease in a subject, wherein the method comprises: (i) administering to the subject a type II anti-CD20 antibody, and, after a certain period, consecutively (ii) administering to the subject a T cell activation therapeutic agent including a treatment regimen, the type II anti-CD20 antibody being obinutuzumab, the T cell activation therapeutic agent being (a) (i) a heavy chain variable region comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 32, an HCDR2 of SEQ ID NO: 33, and an HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 35, an LCDR2 of SEQ ID NO: 36, and an LCDR3 of SEQ ID NO: 37; or (ii) an antigen-binding portion comprising a heavy chain variable region sequence of SEQ ID NO: 38 and a light chain variable region sequence of SEQ ID NO: 39, which specifically binds to CD3; and (b) (i) a heavy chain variable region comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 4, an HCDR2 of SEQ ID NO: 5, and an HCDR3 of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 7, an LCDR2 of SEQ ID NO: 8, and an LCDR3 of SEQ ID NO: 9; or (ii) an antigen-binding portion comprising a heavy chain variable region sequence of SEQ ID NO: 10 and a light chain variable region sequence of SEQ ID NO: 11, which specifically binds to CD20 including a bispecific antibody, the disease being a B cell proliferative disorder, and the period between the administration of the type II anti-CD20 antibody and the administration of the therapeutic agent being sufficient to reduce the number of B cells in the subject in response to the administration of the type II anti-CD20 antibody, the medicament.

2. The medicament according to claim 1, wherein the treatment regimen effectively reduces cytokine release associated with the administration of the T cell activation therapeutic agent in the subject as compared to a corresponding treatment regimen not involving the administration of the type II anti-CD20 antibody.

3. The period between the administration of the type II anti-CD20 antibody and the administration of the T cell activation therapeutic agent is (i) from 3 days to 21 days, from 5 days to 20 days, from 7 days to 21 days, from 7 days to 14 days, from 5 days to 15 days, from 7 days to 15 days, from 8 days to 15 days, from 10 days to 20 days, from 10 days to 15 days, from 11 days to 14 days, or from 12 days to 13 days; or (ii) from 5 days to 10 days. The medicament according to claim 1 or 2.

4. The period between the administration of the type II anti-CD20 antibody and the administration of the T cell activation therapeutic agent is 7 days. The medicament according to any one of claims 1 to 3.

5. The medicament according to any one of claims 1 to 4, wherein the administration of the type II anti-CD20 antibody is (i) a single administration, or (ii) two or more separate administrations.

6. The medicament according to any one of claims 1 to 5, wherein the administration of the type II anti-CD20 antibody is at a dose of about 1000 mg or about 2 g of the type II anti-CD20 antibody.

7. The medicament according to any one of claims 1 to 6, wherein the administration of the type II anti-CD20 antibody is a single administration at a dose of about 1000 mg of the type II anti-CD20 antibody, and the period between the administration of the type II anti-CD20 antibody and the administration of the therapeutic agent is 7 days.

8. The medicament according to any one of claims 1 to 7, wherein the T cell activation therapeutic agent is administered parenterally, specifically intravenously.

9. The bispecific antibody comprises a polypeptide that is at least 95% identical to the sequence of SEQ ID NO: 44, a polypeptide that is at least 95% identical to the sequence of SEQ ID NO: 45, a polypeptide that is at least 95% identical to the sequence of SEQ ID NO: 46, and a polypeptide that is at least 95% identical to the sequence of SEQ ID NO:

47. The medicament according to any one of claims 1 to 8.

10. The bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 44, the polypeptide sequence of SEQ ID NO: 45, the polypeptide sequence of SEQ ID NO: 46, and the polypeptide sequence of SEQ ID NO:

47. The medicament according to any one of claims 1 to 8.

11. The disease is (i) a CD20-positive B cell disorder, and / or (ii) a disease selected from the group consisting of non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin lymphoma (HL). The medicament according to any one of claims 1 to 10.

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