Optimized anti-CD3 bispecific antibodies and their use

Abstract

The present invention provides antibodies that bind to CD3 with weak or no detectable binding affinity, and methods of using the same. [Solution] According to certain embodiments, antibodies of the present invention bind to human CD3 with low affinity and induce human T cell proliferation, thereby inducing T cell-mediated killing of tumor cells with high efficacy. According to certain embodiments, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds to human CD3 with weak or no detectable binding affinity in in vitro assays, and a second antigen-binding molecule that specifically binds to a human tumor-associated antigen. In certain embodiments, the bispecific antigen-binding molecules of the present invention can inhibit the growth of tumors expressing a target antigen, such as PSMA.

Description

Technical Field

[0001] Reference to Sequence Listing This application incorporates by reference a Sequence Listing submitted in computer-readable form as file 10151WO01_ST25.txt, created on September 22, 2016, and containing 264,418 bytes.

[0002] The present invention relates to bispecific antibodies that target effector antigens such as CD3 antigen and tumor-associated antigens, and methods of tumor killing. The present invention relates to methods of reducing or eliminating tumor tissue mass and controlling toxic side effects that may be associated with tumor immunotherapy. The present invention provides a bispecific antibody comprising an anti-CD3 antigen-binding arm that binds to CD3 with a weak affinity or that does not exhibit a detectable binding affinity, e.g., with a K greater than about 500 nM in an in vitro affinity binding assay. D and provides a bispecific antibody comprising an anti-CD3 antigen-binding arm that binds to CD3 with a weak affinity or that does not exhibit a detectable binding affinity, e.g., with a K greater than about 500 nM in an in vitro affinity binding assay.

Background Art

[0003] In particular, the future of therapeutic bispecific antibodies (bsAbs) in cancer immunotherapy aims to cross multiple antigen targets in order to elicit a more stable innate immune response against unwanted target-bearing cells or organisms.

[0004] Currently, it is well established that in order to mediate redirected lysis, bsAbs must directly cluster target cells onto inducible molecules on effector cells such as T cells. There are many factors that must be considered in bsAb design, for example size and composition affect biodistribution and stability in vivo (Non-Patent Document 1; Non-Patent Document 2). Distinguishable outcomes are difficult to predict depending on the subset of T cells that are induced to react and the state of the T cells that are stimulated. It is well known that bsAbs do not show consistent results (Non-Patent Document 3). For example, in the absence of appropriate cytokine production, CD3 crosslinking can induce an apoptotic response in T cells (Non-Patent Document 4). The subset of T cells and the differentiation state of such recruited T cells, such as naive T cells, are important for efficacy because naive T cells cannot lyse target cells without pre-activation (e.g., crosslinking with TCR in the presence of IL-2).

[0005] Certain bispecific therapies have been successful, but like many cancer therapies, they come with a cost. Toxicity is a major cause of failure among cancer therapies. It is well known that the toxicity of so-called chemotherapeutic agents is a major cause of side effects and secondary harms to patients. The "cell killing" action itself poses problems for patients. Excessive cytotoxic reactions can be induced by the activation of effector cells such as T cells and cancer target cells, but it is currently unclear which types of reactions are most beneficial in tumor immunotherapy. A method for identifying anti-CD3 antibodies for use in bispecific therapies with reduced side effects while maintaining efficacy and desired pharmacokinetic (PK) properties would be advantageous.

[0006] Technologies such as affinity maturation are described, which utilize mutagenesis to optimize antibodies to have increased and improved binding specificity or affinity for a target antigen compared to a starting antibody, based on structure / activity relationships (SAR) (see, for example, Patent Document 1 published on May 12, 2011). Modified OKT3 antibodies are described that can bind to and interact with CD3 to varying degrees and still exhibit moderate to high levels of T cell activation (Patent Document 2). However, methods for reducing the binding affinity of an antibody molecule to a level near or beyond the detection level of binding have not been described, nor has the efficacy required for tumor reduction or suppression been shown.

[0007] Therefore, there is a need for alternative bispecific antigen-binding molecules with controlled cytotoxicity and better PK properties. Such cancer therapies would be extremely useful at the site of treatment.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0010] In a first aspect, the present invention provides an antibody that binds to human CD3 and has a weak affinity for human and / or cynomolgus CD3 or no detectable affinity, and antigen-binding fragments thereof. Antibodies according to this aspect of the invention are useful, inter alia, for targeting T cells expressing CD3 in situations where, for example, T cell-mediated killing is beneficial or desirable, and for stimulating T cell activation. The anti-CD3 antibody of the present invention, or an antigen-binding portion thereof, may be included as part of a bispecific antibody that directs CD3-mediated T cell activation to a specific cell type such as a tumor cell or an infectious agent.

[0011] Exemplary anti-CD3 antibodies of the present invention are listed in Tables 2 and 3 herein. Table 2 lists the amino acid sequence identifiers of the heavy chain variable region (HCVR), as well as the heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3). Table 3 lists the sequence identifiers of nucleic acid molecules encoding the HCVR, HCDR1, HCDR2, and HCDR3 regions of the exemplary anti-CD3 antibodies. Tables 4 and 5 list the light chain variable region (LCVR) of the exemplary anti-CD3 antibodies, as well as the complementarity determining regions (LCDR1, LCDR2, and LCDR3).

[0012] The present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR comprising an amino acid sequence selected from the HCVR amino acid sequences listed in Table 2 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] The present invention also provides an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising any of the HCVR amino acid sequences listed in Table 2 that are paired with any of the LCVR amino acid sequences listed in Table 4, or a light chain obtained from the homologous light chain of an anti-TAA heavy chain, or a light chain that exhibits promiscuity or the ability to pair with a wide range of non-homologous heavy chains, i.e., a common or universal light chain, either known or obtained from a known light chain variable region. The present invention provides an antibody or an antigen-binding fragment thereof comprising the same. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-CD3 antibodies listed in Table 2 that are paired with the exemplary light chain variable regions listed in Table 4. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 10 / 162 (e.g., CD3-VH-G2); 18 / 162 (e.g., CD3-VH-G3); 26 / 162 (e.g., CD3-VH-G4); 34 / 162 (e.g., CD3-VH-G5); 42 / 162 (e.g., CD3-VH-G8); 50 / 162 (e.g., CD3-VH-G9); 58 / 162 (e.g., CD3-VH-G10); 66 / 162 (e.g., CD3-VH-G11); 74 / 162 (e.g., CD3-VH-G12); 82 / 162 (e.g., CD3-VH-G13); 90 / 162 (e.g., CD3-VH-G14); 98 / 162 (e.g., CD3-VH-G15); 106 / 162 (e.g., CD3-VH-G16); 114 / 162 (e.g., CD3-VH-G17); 122 / 162 (e.g., CD3-VH-G18); 130 / 162 (e.g., CD3-VH-G19); 138 / 162 (e.g., CD3-VH-G20); and 146 / 162 (e.g., CD3-VH-G21).

[0014] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR1 (HCDR1) selected from the amino acid sequences listed in Table 2, or any substantially similar sequence having at least 95%, at least 98%, or at least 99% sequence identity thereto. The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 178.

[0015] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR2 (HCDR2) selected from the amino acid sequences listed in Table 2, or any substantially similar sequence having at least 95%, at least 98%, or at least 99% sequence identity thereto. The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 179.

[0016] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR3 (HCDR3) selected from the amino acid sequences listed in Table 2, or any substantially similar sequence having at least 95%, at least 98%, or at least 99% sequence identity thereto. The present invention also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain CDR3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 180.

[0017] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light-chain CDR1 (LCDR1) selected from the amino acid sequences listed in Table 4, or any substantially similar sequence having at least 95%, at least 98%, or at least 99% sequence identity thereto. The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light-chain CDR1 (LCDR1) obtained from an allotypic light chain of an anti-TAA heavy chain, or a light chain obtained from a light chain that shows promiscuity or the ability to pair with a wide range of non-allotypic heavy chains, i.e., a universal or common light chain.

[0018] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain complementarity-determining region 2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 95%, at least 98%, or at least 99% sequence identity. The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) obtained from a homologous light chain of an anti-TAA heavy chain or a light chain obtained from a universal or common light chain, i.e., a light chain that exhibits promiscuity or the ability to pair with a wide range of non-homologous heavy chains.

[0019] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain complementarity-determining region 3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 4, or a substantially similar sequence having at least 95%, at least 98%, or at least 99% sequence identity. The present invention also provides an antibody or an antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) obtained from a homologous light chain of an anti-TAA heavy chain or a light chain obtained from a universal or common light chain, i.e., a light chain that exhibits promiscuity or the ability to pair with a wide range of non-homologous heavy chains.

[0020] The present invention also provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 2 that pair with any of the LCDR3 amino acid sequences listed in Table 4. According to certain embodiments, the present invention provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-CD3 antibodies listed in Table 2. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 16 / 168 (e.g., CD3-VH-G2); 24 / 168 (e.g., CD3-VH-G3); 32 / 168 (e.g., CD3-VH-G4); 40 / 168 (e.g., CD3-VH-G5); 48 / 168 (e.g., CD3-VH-G8); 56 / 168 (e.g., CD3-VH-G9); 64 / 168 (e.g., CD3-VH-G10); 72 / 168 (e.g., CD3-VH-G11); 80 / 168 (e.g., CD3-VH-G12); 88 / 168 (e.g., CD3-VH-G13); 96 / 168 (e.g., CD3-VH-G14); 104 / 168 (e.g., CD3-VH-G15); 112 / 168 (e.g., CD3-VH-G16); 120 / 168 (e.g., CD3-VH-G17); 128 / 168 (e.g., CD3-VH-G18); 136 / 168 (e.g., CD3-VH-G19); 144 / 168 (e.g., CD3-VH-G20); and 152 / 168 (e.g., CD3-VH-G21).

[0021] The present invention also provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-CD3 antibodies listed in Tables 2 and 4. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is SEQ ID NO: 12-14-16-164-166-168 (e.g., CD3-VH-G2); 20-22-24-164-166-168 (e.g., CD3-VH-G3); 28-30-32-164-166-168 (e.g., CD3-VH-G4); 36-38-40-164-166-168 (e.g., CD3-VH-G5); 44-46-48-164-166-168 (e.g., CD3-VH-G8); 52-54-56-164-166-168 (e.g., CD3-VH-G9); 60-62-64-164-166-168 (e.g., CD3-VH-G10); 68-70-72-164-166-168 (e.g., CD3-VH-G11); 76-78-80-164-166-168 (e.g., CD3-VH-G12); 84-86-88-164-166-168 (e.g., CD3-VH-G13); 92-94-96-164-166-168 (e.g., CD3-VH-G14); 100-102-104-164-166-168 (e.g., CD3-VH-G15); 108-110-112-164-166-168 (e.g., CD3-VH-G16); 116-118-120-164-166-168 (e.g., CD3-VH-G17); 124-126-128-164-166-168 (e.g., CD3-VH-G18); 132-134-136-164-166-168 (e.g., CD3-VH-G19); 140-142-144-164-166-168 (e.g., CD3-VH-G20); and 148-150-152-164-166-168 (e.g., CD3-VH-G21), and is selected from the group consisting of.

[0022] In related embodiments, the present invention relates to the exemplary anti-CD3 antibodies listed in Tables 2 and 4 Provided are antibodies or antigen-binding fragments thereof comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair as defined by any of them. For example, the present invention relates to antibodies or antigen-binding fragments thereof comprising an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained in an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO: 10 / 162 (e.g., CD3-VH-G2); 18 / 162 (e.g., CD3-VH-G3); 26 / 162 (e.g., CD3-VH-G4); 34 / 162 (e.g., CD3-VH-G5); 42 / 162 (e.g., CD3-VH-G8); 50 / 162 (e.g., CD3-VH-G9); 58 / 162 (e.g., CD3-VH-G10); 66 / 162 (e.g., CD3-VH-G11); 74 / 162 (e.g., CD3-VH-G12); 82 / 162 (e.g., CD3-VH-G13); 90 / 162 (e.g., CD3-VH-G14); 98 / 162 (e.g., CD3-VH-G15); 106 / 162 (e.g., CD3-VH-G16); 114 / 162 (e.g., CD3-VH-G17); 122 / 162 (e.g., CD3-VH-G18); 130 / 162 (e.g., CD3-VH-G19); 138 / 162 (e.g., CD3-VH-G20); and 146 / 162 (e.g., CD3-VH-G21).

[0023] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. For example, Kabat, "Sequences" See "of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0024] The present invention also provides a nucleic acid molecule encoding an anti-CD3 antibody or a portion thereof. For example, the present invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 3, and in certain embodiments, the nucleic acid molecule is selected from the HCVR nucleic acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and includes a polynucleotide sequence.

[0025] The present invention also provides a nucleic acid molecule encoding any of the LCVR amino acid sequences listed in Table 4; or a light chain obtained from a homologous light chain of an anti-TAA heavy chain, or a light chain showing promiscuity or the ability to pair with a wide range of non-homologous heavy chains, i.e., a light chain obtained from a universal or common light chain. In certain embodiments, the nucleic acid molecule is selected from the LCVR nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and includes a polynucleotide sequence.

[0026] The present invention also provides a nucleic acid molecule encoding any of the HCDR1 amino acid sequences listed in Table 2, and in certain embodiments, the nucleic acid molecule is selected from the HCDR1 nucleic acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and is selected from any of It includes a selected polynucleotide sequence.

[0027] The present invention also provides a nucleic acid molecule encoding any of the HCDR2 amino acid sequences listed in Table 2. In certain embodiments, the nucleic acid molecule is selected from the HCDR2 nucleic acid sequences listed in Table 3, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0028] The present invention also provides a nucleic acid molecule encoding any of the HCDR3 amino acid sequences listed in Table 2. In certain embodiments, the nucleic acid molecule is selected from the HCDR3 nucleic acid sequences listed in Table 3, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0029] The present invention also provides a nucleic acid molecule encoding the LCDR1 amino acid sequences listed in Table 4; or an LCDR1 obtained from the homologous light chain of an anti-TAA heavy chain, or from a light chain that exhibits promiscuity or the ability to pair with a wide range of non-homologous heavy chains, i.e., a universal or common light chain. In certain embodiments, the nucleic acid molecule is selected from the LCDR1 nucleic acid sequences listed in Table 5, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0030] The present invention also provides a nucleic acid molecule encoding any of the LCDR2 amino acid sequences listed in Table 4; or an LCDR2 obtained from an allotypic light chain of an anti-TAA heavy chain or from a light chain that exhibits promiscuity or the ability to pair with a wide range of non-allotypic heavy chains, i.e., a universal or common light chain. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0031] The present invention also provides a nucleic acid molecule encoding any of the LCDR3 amino acid sequences listed in Table 4; or an LCDR3 obtained from an allotypic light chain of an anti-TAA heavy chain or from a light chain that exhibits promiscuity or the ability to pair with a wide range of non-allotypic heavy chains, i.e., a universal or common light chain. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0032] The present invention also provides a nucleic acid molecule encoding an HCVR, which comprises a set of three CDRs (i.e., HCDR1 - HCDR2 - HCDR3), and the HCDR1 - HCDR2 - HCDR3 amino acid sequence set is as defined by any of the exemplary anti-CD3 antibodies listed in Table 2.

[0033] The present invention also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the set of LCDR1-LCDR2-LCDR3 amino acid sequences is as defined by any of the exemplary universal light chain antibodies described in Table 4; or, the LCDR1-LCDR2-LCDR3 is obtained from the homologous light chain of an anti-TAA heavy chain or from a light chain that shows promiscuity or the ability to pair with a wide range of non-homologous heavy chains, i.e., a universal or common light chain.

[0034] The present invention also provides a nucleic acid molecule encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 2, and the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 4; or, the LCVR is obtained from the homologous light chain of an anti-TAA heavy chain or from a light chain that shows promiscuity or the ability to pair with a wide range of non-homologous heavy chains, i.e., a universal or common light chain. In certain embodiments, the nucleic acid molecule is selected from the polynucleotide sequences of any of the HCVR nucleic acid sequences listed in Table 2, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and the polynucleotide sequences of any of the LCVR nucleic acid sequences listed in Table 5, or substantially similar sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encoding both an HCVR and an LCVR is of fully human sequence or is obtained from human germline immunoglobulin sequences.

[0035] The present invention also provides a recombinant expression vector capable of expressing a polypeptide comprising a variable region of a heavy or light chain of an anti-CD3 antibody. For example, the present invention includes a recombinant expression vector comprising any of the above-described nucleic acid molecules, i.e., any nucleic acid molecule encoding an HCVR, LCVR, and / or CDR sequence as described in Table 2 or 4. Also included within the scope of the present invention are host cells into which such vectors have been introduced, and a method for producing an antibody or a portion thereof by culturing the host cells under conditions that permit the production of an antibody or antibody fragment and recovering the antibody and antibody fragment thus produced.

[0036] The present invention includes anti-CD3 antibodies and / or anti-TAA antibodies, as well as bispecific anti-CD3 / anti-TAA antibodies having a modified glycosylation pattern. In some embodiments, modifications for removing unwanted glycosylation sites, or fucose-depleted antibodies present on the oligosaccharide chain for increasing, for example, antibody-dependent cell cytotoxicity (ADCC) function may be useful (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation may be performed to modify complement-dependent cytotoxicity (CDC).

[0037] In one aspect, the present invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that i) cannot specifically bind to effector cells and ii) can specifically bind to target tumor cells, wherein the specific binding is measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay. In certain embodiments, the present invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that shows no detectable binding to effector cells and specifically binds to target tumor cells with a measurable binding affinity, wherein the binding affinity value is measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay.

[0038] In other embodiments, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule comprising: i) a first antigen-binding fragment (Fab1) that does not exhibit a detectable binding to CD3, and ii) a second antigen-binding fragment (Fab2) that specifically binds to target tumor cells with a measurable binding affinity, wherein the binding affinity value is measured in an in vitro FACS binding assay or an in vitro surface plasmon resonance binding assay. In some cases, the binding affinity is a monovalent binding affinity (e.g., in a bispecific antibody construct).

[0039] In another aspect, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that specifically binds to effector cells with a weak binding affinity, e.g., an EC 50 value of about 100 nM or greater than about 100 nM, and specifically binds to target tumor cells with a recognizable EC 50 value, or a high affinity EC 50 value such as less than 50 nM, wherein the EC binding affinity value is measured in an in vitro FACS binding assay. In certain embodiments, the invention provides a cytotoxic composition comprising a bispecific antigen-binding molecule that specifically binds to effector cells with an EC 50 value greater than about 500 nM, and specifically binds to target tumor cells with a recognizable EC 50 value, or a high affinity EC 50 value such as less than 50 nM, wherein the EC 50 binding affinity value is measured in an in vitro FACS binding assay. 50

[0040] In some examples, the bispecific antigen-binding molecule has an EC 50 ​It comprises Fab1 that specifically binds to human CD3 at a value. In some embodiments, the bispecific antigen-binding molecule binds with high affinity, for example, with an EC less than about 50 nM, less than about 40 nM, less than about 20 nM, less than about 10 nM, or less than about 6 nM (e.g., in relation to monovalent binding). 50 It comprises Fab2 obtained from a second antibody that specifically binds to target tumor cells at a value. In some cases, Fab1 has an EC greater than about 40 nM, or greater than about 100 nM, greater than about 200 nM, or greater than about 1 μM. 50 At a value, it specifically binds to each of human CD3 and cynomolgus CD3. In some cases, Fab1 specifically binds to each of human CD3 and cynomolgus CD3 with weak affinity or without showing a measurable affinity.

[0041] In some embodiments, the target tumor cells are human tumor cells. In some embodiments, Fab1 (or the bispecific antigen-binding molecule) has an EC less than about 1.3 nM as measured in an in vitro T cell-mediated tumor cell killing assay. 50 At a value, it induces T cell-mediated tumor cell killing.

[0042] In some uses, Fab1 or the bispecific antigen-binding molecule has a K greater than about 11 nM as measured in an in vitro surface plasmon resonance binding assay. D At a value, it specifically binds to human CD3. In other cases, Fab1 or the bispecific antigen-binding molecule has a K greater than about 15 nM, or greater than about 30 nM, greater than about 60 nM, greater than about 120 nM, or greater than about 300 nM as measured in an in vitro surface plasmon resonance binding assay. DIt specifically binds to human CD3 and cynomolgus CD3 by value. Further, in some applications, the Fab1 or bispecific antigen-binding molecule does not show detectable binding to human CD3 as measured in each of the in vitro surface plasmon resonance binding assay and the FACS binding assay, and also ii) attracts T cell-mediated tumor cell killing as measured in the in vitro T cell-mediated tumor cell killing assay.

[0043] In some applications, the bispecific antigen-binding molecule comprises a first heavy chain comprising an HCDR1 region comprising the amino acid sequence set forth in SEQ ID NO: 12 or 20. In some embodiments, the first heavy chain comprises an HCDR2 region comprising the amino acid sequence set forth in SEQ ID NO: 14 or 54. In other embodiments, the first heavy chain comprises an HCDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, or SEQ ID NO: 152. In other applications, the first heavy chain comprises an HCVR comprising an HCDR1-HCDR2-HCDR3 having the amino acid sequence of SEQ ID NO: 178-179-180. In other embodiments, the first heavy chain comprises a CDR1 comprising amino acid residues 1 to 7 of SEQ ID NO: 178, a CDR2 comprising amino acid residues 1 to 7 of SEQ ID NO: 179, and a CDR3 comprising amino acid residues 4 to 11 of SEQ ID NO: 180.

[0044] In a further embodiment, the first heavy chain comprises a variable domain framework region having an amino acid sequence selected from FR1 (SEQ ID NO: 174), FR2 (SEQ ID NO: 175), FR3 (SEQ ID NO: 176), and FR4 (SEQ ID NO: 177).

[0045] The present invention provides a bispecific antigen-binding molecule comprising a Fab1 HCVR and LCVR amino acid sequence pair (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 10 / 162; 18 / 162; 26 / 162; 34 / 162; 42 / 162; 50 / 162; 58 / 162; 66 / 162; 74 / 162; 82 / 162; 90 / 162; 98 / 162; 106 / 162; 114 / 162; 122 / 162; 130 / 162; 138 / 162; 146 / 162.

[0046] Antibodies, antigen-binding fragments, and bispecific antibodies thereof were prepared by stepwise replacement of parental amino acid residues based on the differences between germline sequences and parental antibody sequences. The present invention is a method for preparing a cytotoxic composition comprising: (a) identifying the amino acid sequence of a first heavy chain obtained from a first antibody that specifically binds to CD3 with high affinity, e.g., having a binding affinity EC 50 value of less than about 40 nM; (b) modifying selected amino acid residues within the heavy chain variable region of the first antibody to generate a modified antibody; (c) pairing the modified antibody with a second heavy chain obtained from a second antibody that specifically binds to a target tumor antigen to generate a bispecific antibody; (d) testing the bispecific antibody in a binding affinity assay; and if the binding affinity for CD3 has an EC 50 value greater than about 40 nM, or greater than 100 nM, or greater than 300 nM, or greater than 500 nM, or there is no detectable binding, (e) preparing a composition comprising the bispecific antibody and a pharmaceutically acceptable carrier or diluent. In addition to modifying the heavy chain variable region of a selected antibody to design an antigen-binding arm with weak or no affinity while specifically targeting effector cells, the present invention also provides herein a method for preparing and isolating a bispecific antibody by modifying the heavy chain constant region (e.g., C H 3 domain) of each binding arm.

[0047] An exemplary method is a method for generating a cytotoxic bispecific antibody, comprising: (a) identifying, from a plurality of species, a first human antibody or an antigen-binding fragment thereof that interacts with an effector cell antigen; (b) identifying germline amino acid residues of the heavy chain variable region (HCVR) of the human antibody; (c) comparing the amino acid sequence of the HCVR of the first human antibody with the amino acid sequence of the corresponding germline HCVR; (d) identifying amino acids within a modified region of the HCVR of the first antibody, wherein the modified region in the first antibody exhibits at least one amino acid modification by substitution, deletion, or addition of a single amino acid residue as compared to the same region in the germline HCVR; (e) generating a plurality of modified antibodies, each comprising at least one modified region of the HCVR; (f) screening each of the plurality of modified antibodies for monovalent affinity for the effector cell antigen; (g) selecting a modified antibody that exhibits a weaker binding affinity for the effector cell antigen or no detectable binding affinity as compared to the first antibody; and (h) pairing the selected first antibody with a second antibody that interacts with a tumor-associated antigen to generate a cytotoxic bispecific antibody.

[0048] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human bispecific antibody or a fragment thereof that specifically binds to CD3 and a pharmaceutically acceptable carrier. In a related aspect, the invention features a composition that is a combination of an anti-CD3 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with the anti-CD3 antibody. Exemplary agents that can be advantageously combined with the anti-CD3 antibody include, without limitation, other agents that bind to CD3 and / or activate CD3 signaling (including other antibodies or antigen-binding fragments thereof, etc.), and / or agents that activate immune cells or stimulate immune cell activation, even though they do not directly bind to CD3. Additional combination therapies and formulations comprising the anti-CD3 antibody of the invention are disclosed elsewhere herein.

[0049] In yet another aspect, the present invention provides a method of treatment for stimulating T cell activation using the anti-CD3 antibody or antigen-binding portion of the antibody of the present invention, the method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the bispecific antibody of the present invention, or an antigen-binding fragment thereof. The disorder to be treated is any disease or condition, such as cancer, that is ameliorated, remitted, inhibited or prevented by cytotoxic treatment targeted to a tumor-associated antigen.

[0050] According to another aspect, the present invention provides a bispecific antigen-binding molecule that binds to CD3 and a target antigen, particularly a tumor-associated antigen (TAA).

[0051] The present invention also includes the use of the anti-CD3 / anti-TAA bispecific antigen-binding molecule of the present invention in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by TAA expression. The present invention also provides the use of an anti-CD3 / anti-TAA bispecific antigen-binding molecule that exhibits a weak affinity for CD3-expressing effector cells and reduced clearance compared to an anti-CD3 / anti-TAA bispecific antigen-binding molecule that exhibits a high affinity for CD3-expressing effector cells in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by TAA expression.

[0052] Other embodiments will be apparent by considering the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053]

Figure 1-1

Figure 1-2

Figure 2A

Figure 2B

Figure 2C

DETAILED DESCRIPTION OF THE INVENTION

[0054] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, and thus the methods and conditions may vary. Also, since the scope of the present invention is limited only by the appended claims, it should be understood that the technical terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs. As used herein, the term "about," when used in reference to a specific recited numerical value, means that the value may vary within 1% of the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, as well as all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0056] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described herein.

[0057] Definitions The expression "CD3" refers to an antigen consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains, CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma, which are expressed on T cells as part of the multi-molecular T cell receptor (TCR). Human CD3-epsilon (hCD3ε) contains an amino acid sequence as set forth in SEQ ID NO: 169 (UniProtKB / Swiss-Prot: P07766.2). Human CD3-delta (hCD3δ) contains an amino acid sequence as set forth in SEQ ID NO: 170 (UniProtKB / Swiss-Prot: P04234.1). As used herein, all references to proteins, polypeptides, and protein fragments are intended to refer to the human version of each protein, polypeptide, or protein fragment, unless expressly specified to be from non-human species. Thus, the expression "CD3" means human CD3 unless specified to be from non-human species, such as "mouse CD3", "monkey CD3", etc.

[0058] The terms "antibody that binds to CD3" or "anti-CD3 antibody" include antibodies and antigen-binding fragments thereof that specifically recognize and associate with a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), and antibodies and antigen-binding fragments thereof that specifically recognize and associate with a dimeric complex of two CD3 subunits (e.g., epsilon / delta, epsilon / gamma, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3 and / or cell surface-expressed CD3. Soluble CD3 includes native CD3 protein, as well as recombinant CD3 protein variants that do not have a transmembrane domain or are otherwise not associated with the cell membrane, such as monomeric and dimeric CD3 constructs. The present invention provides antibodies that bind to and activate human and cynomolgus CD3 with weak binding affinity or without showing detectable binding affinity. "Binding to CD3 without showing detectable binding affinity" means that the interaction of the antibody and / or antigen-binding fragment with the CD3 target may not be measurable or detectable by known detection assays, such as FACS (cell-based) binding assays as described herein or surface plasmon resonance binding assays well known in the art as described herein. Other binding assays are well known in the art. The antibody and or antigen-binding fragment may recognize the CD3 target through very weak protein-protein biochemical interactions, but the interaction exceeds the detection limit of the assay, e.g., the measured value cannot be determined, so the determination of a specific KD or EC50 value cannot be measured. In another case, "not showing detectable binding affinity" is determined when the affinity of the antibody corresponding to the K D value is less than one-tenth of that for non-specific antigens such as BSA, casein, etc. "Binding to CD3 with weak binding affinity" includes interactions where the measured binding affinity value is at or slightly above the detection limit value of the assay, or equal to the binding affinity for non-specific antigens.

[0059] The expression "cell surface-expressed CD3" means one or more CD3 proteins that are expressed on the surface of a cell in vitro or in vivo such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. "Cell surface-expressed CD3" includes CD3 proteins that are contained in association with a functional T cell receptor within the membrane of the cell. The expression "cell surface-expressed CD3" includes CD3 proteins expressed as part of a homodimer or heterodimer (e.g., delta / epsilon, gamma / epsilon, and zeta / zeta CD3 dimers) on the surface of the cell. The expression "cell surface-expressed CD3" also includes CD3 chains (e.g., CD3-delta, CD3-epsilon or CD3-gamma) that are expressed alone on the surface of the cell without other CD3 chain types. "Cell surface-expressed CD3" may include or consist of CD3 proteins expressed on the surface of cells that normally express CD3 proteins. Alternatively, "cell surface-expressed CD3" may include or consist of CD3 expressed on the surface of a cell that does not normally express human CD3 on its surface but has been artificially modified to express CD3 on its surface.

[0060] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells may also include natural killer (NK) cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes). It is understood that the treatment can mediate an excessive cell-mediated immune response, or effector function, by Ig interaction with effector cell surface receptors, such as CD3 (T cell surface receptor), CD28 (T cell), Fcγ receptor (FcγR) (NK cells, activated macrophages, etc.). Subsequently, these interactions induce effector functions, such as cell killing, complement activation, phagocytosis and opsonization. Binding to effector cells and tumor target cells enables a valuable and effective immunotherapy design that propagates tumor cell death and recruits the endogenous immune function to fight tumors or cancer.

[0061] The expression "anti-CD3 antibody" includes both monovalent antibodies having a single specificity and bispecific antibodies comprising a first arm that binds to CD3 and a second arm that binds to a second (target) antigen, wherein the anti-CD3 arm comprises any of the HCVR / LCVR or CDR sequences as set forth in Tables 2, 3, 4, and / or 5 herein. Examples of anti-CD3 bispecific antibodies are described elsewhere herein. The term "antigen-binding molecule" includes antibodies, including, for example, bispecific antibodies, and antigen-binding fragments of antibodies.

[0062] The term "antibody" includes any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CD3). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy-chain variable region (abbreviated herein as HCVR or V H and a heavy-chain constant region. The heavy-chain constant region comprises three domains, C H 1, C H 2 and C H 3. Each light chain comprises a light-chain variable region (abbreviated herein as LCVR or V L and a light-chain constant region. The light-chain constant region comprises one domain (C L 1). The V H and V L regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) that are interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt is composed of three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxy terminus. In different embodiments of the present invention, the FRs of the anti-CD3 antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be modified naturally or artificially. The amino acid consensus sequence can be determined based on the comparative analysis of two or more CDRs.

[0063] The term "antibody" also includes antigen-binding fragments of the complete antibody molecule. The terms "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody", etc., as used herein, refer to any naturally occurring, enzymatically obtained, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained, for example, from a complete antibody molecule using any suitable standard technique, such as proteolysis, or recombinant genetic modification techniques including the manipulation and expression of DNA encoding the antibody variable and optionally constant domains. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (including phage-antibody libraries), or may be synthesized. For example, the DNA may be sequenced or manipulated chemically or by molecular biological techniques to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, form cysteine residues, modify, add or delete amino acids, etc.

[0064] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or minimal recognition units consisting of amino acid residues mimicking a restricted FR3-CDR3-FR4 peptide. Other modified molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0065] An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in-frame with one or more framework sequences. The V L domain associated with the V H domain in an antigen-binding fragment having a V H and V L domain can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer, and can contain a V H -V H , V H -V L or V L -V L dimer. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0066] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within an antigen-binding fragment of an antibody of the present invention are: (i) V H -CH 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1 - C H 2; (v) V H -C H 1 - C H 2 - C H 3; (vi) V H -C H 2 - C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1 - C H 2; (xii) V L -C H 1 - C H 2 - C H 3; (xiii) V L -C H 2 - C H 3; and (xiv) V L -C L including. In any configuration of variable and constant domains including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Further, the antigen-binding fragments of the antibodies of the present invention may non-covalently associate with each other and / or with one or more monomeric V H or V L domains (e.g., by disulfide bond(s)) and may include homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above.

[0067] Similar to full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, each of which can specifically bind to a distinct antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use related to the antigen-binding fragments of the antibodies of the present invention using conventional techniques available in the art.

[0068] The antibodies of the present invention can function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors recognize the bound antibody on the target cell, thereby resulting in the lysis of the target cell. CDC and ADCC can be measured using assays well-known and available in the art. (See, for example, U.S. Patent No. 5,500,362 and U.S. Patent No. 5,821,337, as well as Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of the antibody is important in the ability of the antibody to mediate cell-dependent cytotoxicity by engaging complement. Thus, the isotype of the antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0069] In certain embodiments of the invention, the anti-CD3 antibodies (monospecific or bispecific) of the invention are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions obtained from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences obtained from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0070] The term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, formed, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (further described below), antibodies isolated from a recombinant combinatorial human antibody library (further described below), antibodies isolated from an animal (e.g., a mouse) into which human immunoglobulin genes have been introduced (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, formed, or isolated by any other means that includes splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions obtained from human germline immunoglobulin sequences. However, in certain embodiments such recombinant human antibodies are subjected to in vitro mutagenesis (or, if an animal into which human Ig sequences have been introduced is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the V H and V L regions of the recombinant antibody may differ from the amino acid sequences of human germline V H and V LSequences obtained from an array and related thereto that are not necessarily naturally present in the human antibody germline repertoire in vivo.

[0071] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of about 150-160 kDa, and the dimer is held together by interchain heavy chain disulfide bonds. In the second form, the dimer does not link by interchain disulfide bonds, and a molecule of about 75-80 kDa composed of covalently linked light and heavy chains is formed (half antibody). These forms were extremely difficult to separate even after affinity purification.

[0072] The frequency of appearance of the second form in various intact IgG isotypes is due, inter alia, to structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form to levels typically observed using the human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention encompasses antibodies having one or more mutations in the hinge C H 2 or C H 3 region, which may be desirable, for example, to improve the yield of the desired antibody form in production.

[0073] The antibody of the present invention may be an isolated antibody. As used herein, "isolated antibody" means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an "isolated antibody" for the purposes of the present invention. Isolated antibodies also include in situ antibodies in recombinant cells. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, the isolated antibody may be substantially free of other cellular materials and / or chemical substances.

[0074] The present invention also includes single-arm antibodies that bind to CD3. The term "single-arm antibody" means an antigen-binding molecule that includes a single antibody heavy chain and a single antibody light chain. The single-arm antibodies of the present invention may include any of the HCVR / LCVR or CDR amino acid sequences as described in Table 2 herein.

[0075] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have two or more epitopes. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain situations, an epitope may include a saccharide, phosphoryl group, or sulfonyl group moiety on an antigen.

[0076] The terms "substantially identical" or "substantially the same", when referring to a nucleic acid or a fragment thereof, mean that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity of at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm for sequence identity such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially the same amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially the same amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0077] When applied to polypeptides, the terms "substantial similarity" or "substantially the same" mean that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity when optimally aligned using an initial gap weight, for example, by programs such as GAP or BESTFIT. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the degree of percent sequence identity or similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains cysteine and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, conservative replacements are any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science is any change having a positive value in the PAM250 log-likelihood matrix disclosed in 256:1443-1445. A "moderately conservative" replacement is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0078] The sequence similarity of polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that can be used with initial parameters to determine sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein. For example, see GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with initial or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignment of the best overlapping regions and percent sequence identity between a query and a search sequence (Pearson (2000), supra). Another preferred algorithm when comparing the sequences of the present invention to a database containing numerous sequences from different organisms is BLAST, particularly BLASTP or TBLASTN, a computer program using initial parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0079] Germline mutation The anti-CD3 antibodies disclosed herein contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy chain variable domain compared to the corresponding germline sequence from which the antibody was obtained.

[0080] The invention also includes antibodies and antigen-binding fragments thereof obtained from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was obtained, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations") and have weak binding to the CD3 antigen or no detectable binding. Some such exemplary antibodies that recognize CD3 are described in Table 2 herein.

[0081] Furthermore, the antibodies of the invention may contain any combination of two or more germline mutations within the framework and / or CDR regions. For example, a particular individual residue may be mutated to the corresponding residue of a particular germline sequence, while a particular other residue that is different from the original germline sequence is either maintained or mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be tested for one or more desired properties, such as improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, and the like. Antibodies and antigen-binding fragments obtained in this general manner in consideration of the guidance of the present disclosure are encompassed by the invention.

[0082] The present invention also includes anti-CD3 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences described in Table 2 herein. The antibodies and bispecific antigen-binding molecules of the present invention include one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains, compared to the corresponding germline sequences from which the individual antigen-binding domains were obtained, while maintaining or improving undetectable binding from the desired weak binding to the CD3 antigen. "Conservative amino acid substitutions" are those in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein, i.e., the amino acid substitution maintains or improves undetectable binding affinity from the desired weak binding affinity in the case of an anti-CD3 binding molecule. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains cysteine and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, conservative replacements are described in Gonnet et al. (1992) Science Any change having a positive value in the PAM250 log-likelihood matrix disclosed at 256:1443-1445. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0083] The invention also includes an antigen-binding domain having an HCVR and / or CDR amino acid sequence that is substantially identical to any of the HCVR and / or CDR amino acid sequences disclosed herein, while including an antigen-binding molecule that maintains or improves the desired weak affinity for the CD3 antigen. When referring to amino acid sequences, the terms "substantial identity" or "substantially identical" mean that two amino acid sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity, when optimally aligned using, for example, the GAP or BESTFIT programs with initial gap weights. Preferably, the non-identical residue positions differ by conservative amino acid substitutions. When two or more amino acid sequences differ from each other by conservative substitutions, the degree of percent sequence identity or similarity can be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331. The sequence similarity of a polypeptide, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that can be used with initial parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein. For example, GCG Version

[0084] ​See also 6.1. The polypeptide sequences can also be compared using FASTA with the initial or recommended parameters which are programs in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the best overlapping regions and the percent sequence identity between the query and the search sequences (Pearson (2000), supra). Another preferred algorithm when comparing the sequences of the present invention to a database containing numerous sequences from different organisms is BLAST, a computer program using the initial parameters, particularly BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

[0085] Once obtained, antigen-binding domains containing one or more germline mutations were tested for reduced binding affinity using one or more in vitro assays. Antibodies that recognize a particular antigen are typically screened for that purpose by testing for high (i.e., strong) binding affinity to the antigen, whereas the antibodies of the present invention exhibit weak binding or no detectable binding. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are also encompassed by the present invention and have been found to be advantageous as affinity-driven tumor therapy.

[0086] Unexpected benefits, such as improved pharmacokinetic properties and low toxicity to patients, can be realized from the methods described herein.

[0087] Binding properties of antibodies As used herein, the term "binding" in connection with the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to, for example, any of a given antigen, e.g., a cell surface protein or fragment thereof, typically refers to the interaction or association between at least two entities or molecular structures, e.g., an antibody-antigen interaction.

[0088] For example, when the binding affinity is typically determined using surface plasmon resonance (SPR) technology, such as on a BIAcore 3000 instrument, with an antigen as a ligand and an antibody, Ig, antibody binding fragment, or Fc-containing protein as a sample (or anti-ligand), it is about 10 -7 M or less, for example about 10 -8 M or less, for example about 10 -9 M or less of K D value. Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also routinely used, and FACS data correlates well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0089] Thus, the antibodies or antigen-binding proteins of the present invention can bind to a predetermined antigen or cell surface molecule (receptor such as CD3) having an affinity corresponding to a K D value that is at least one-tenth of the affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, when the affinity of an antibody corresponding to the K D value is one-tenth or less of that of a non-specific antigen, this may be considered undetectable binding, but such an antibody may be paired with a second antigen-binding arm for the production of the bispecific antibodies of the present invention.

[0090] The term "K D " (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody binding fragment that binds to an antigen. There is an inverse relationship between K D and the binding affinity, and thus K DThe smaller the value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" relate to a higher ability to form an interaction and thus a smaller K D value, and conversely, the terms "lower affinity" or "weaker affinity" relate to a lower ability to form an interaction and thus a larger K D value. In some situations, a higher binding affinity (or K D ) of a molecule (e.g., an antibody) for an interaction partner molecule (e.g., antigen X) compared to its binding affinity for another interaction partner molecule (e.g., antigen Y) of the same molecule (e.g., an antibody) can be expressed as a binding ratio determined by dividing the larger K D value (lower, or weaker affinity) by the smaller K D (higher, or stronger affinity), and can be expressed, for example, in some cases as a 5 - fold or 10 - fold higher binding affinity. For example, "low affinity" refers to a weaker binding interaction. In some embodiments, a low binding affinity corresponds to a K D greater than about 1 nM, greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or greater than about 40 nM, and such K D binding affinity values are measured in an in vitro surface plasmon resonance binding assay, or an equivalent biomolecular interaction detection assay. In some embodiments, a low binding affinity corresponds to an EC D greater than about 10 nM, an EC 50 greater than about 15 nM, an EC 50 of 20 nM, an EC 50 greater than about 25 nM, an EC 50 of 30 nM, an EC 50 greater than about 35 nM, or an EC 50 greater than about 40 nM, and such EC 50 corresponds to an EC 50The binding affinity value is measured in an in vitro FACS binding assay, or an equivalent cell-based binding assay. "Weak affinity" refers to a weak binding interaction. In some embodiments, the weak binding affinity is a K D or EC50 greater than about 100 nM, greater than about 200, 300, or greater than about 500 nM K D or EC50, and such K D The binding affinity value is measured in an in vitro surface plasmon resonance binding assay, or an equivalent biomolecular interaction detection assay, and such EC50 binding affinity value is measured in an in vitro FACS binding assay, or an equivalent cell-based interaction detection assay for detecting monovalent binding. Failing to show detectable binding means that the affinity between two biomolecules, for example, particularly between a monovalent antibody binding arm and its target antigen, exceeds the detection limit of the assay being used.

[0091] The term "k d " (sec-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody binding fragment. The said value is also called the k off value.

[0092] The term "k a " (M-1×sec-1 or 1 / M) refers to the association rate constant of a specific antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0093] The term "k A " (M-1 or 1 / M) refers to the association equilibrium constant of a specific antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody binding fragment. The association equilibrium constant is obtained by dividing k a by k d .

[0094] The term "EC50" or "EC 50 " refers to the maximum half-effective concentration, including the concentration of an antibody that induces a reaction halfway between the baseline and the maximum value after a specific exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximum effect is observed. In certain embodiments, the EC 50 value is equal to the concentration of the antibody of the invention that exhibits half-maximal binding to cells expressing CD3 or a tumor-associated antigen, as determined, for example, by a FACS binding assay. Thus, at increased EC 50 values or half-maximal effective concentration values, reduced or weaker binding is observed. Thus, an EC 50 of 500 nM exhibits a weaker binding affinity than an EC 50 of 50 nM.

[0095] In one embodiment, the reduced binding can be defined as an increased EC 50 antibody concentration, which enables binding to half-maximum of the target cells.

[0096] In other experimental measurements, the EC 50 value represents the concentration of the antibody of the invention that elicits half-maximal depletion of target cells by the cytotoxic activity of T cells. Thus, increased cytotoxic activity (e.g., T cell-mediated tumor cell death) is observed at a reduced EC 50 or half-maximal effective concentration value.

[0097] Bispecific antigen-binding molecules The antibodies of the invention may be bispecific or multispecific. Multispecific antibodies may be specific for one effector molecule, such as CD3, in combination with different epitopes of one target polypeptide, or may contain antigen-binding domains specific for two or more target polypeptides. For example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends See Biotechnol. 22:238-244. The anti-CD3 antibodies of the present invention may be bound to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof may be functionally bound (e.g., by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second binding specificity.

[0098] The use of the expression "anti-CD3 antibody" herein is intended to include both monospecific anti-CD3 antibodies and bispecific antibodies comprising a CD3-binding arm and a second arm that binds to a target antigen. Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds to human CD3 and the other arm of the immunoglobulin is specific for a target antigen. The target antigen to which the other arm of the CD3 bispecific antibody binds may be any antigen expressed on or near a cell, tissue, organ, microorganism or virus for which a targeted immune response is desired. The CD3-binding arm may comprise any of the HCVR or CDR amino acid sequences as described in Table 2 herein. In certain embodiments, the CD3-binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm binds weakly to human CD3 and induces tumor-associated antigen-expressing cell death in the context of a bispecific or multispecific antibody. In other embodiments, the CD3-binding arm binds or associates weakly with human and cynomolgus monkey (monkey) CD3, but the binding interaction is not detectable by in vitro assays known in the art. In some embodiments of the present invention, the CD3-binding arm does not bind or associate with human and cynomolgus monkey (monkey) CD3, but the bispecific molecule still causes tumor associated cell death.

[0099] In connection with the bispecific antibodies of the invention in which one arm of the antibody binds to CD3 and the other arm binds to a target antigen, the target antigen may be a tumor-associated antigen (TAA). Non-limiting examples of certain tumor-associated antigens are, for example, AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0100] The inventors contemplate that the invention includes numerous examples of bispecific antibodies having a weak anti-CD3 binding arm made according to the invention.

[0101] According to certain exemplary embodiments, the invention includes bispecific antigen-binding molecules that specifically bind to CD3 and PSMA. Such molecules may be referred to herein as "anti-CD3 / anti-PSMA", or "anti-CD3×PSMA", or "CD3×PSMA" bispecific molecules, etc. The term "PSMA", as used herein, refers to the human PSMA protein unless specified to be from a non-human species (e.g., "mouse PSMA", "monkey PSMA", etc.).

[0102] The term "PSMA" refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1) (UniProtKB / Swiss-Prot.No.Q04609; SEQ ID NO: 171). PSMA is an integral non-cleavable membrane glycoprotein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer.

[0103] According to other exemplary embodiments, the invention includes bispecific antigen-binding molecules that specifically bind to CD3 and EGFRvIII. Such molecules may be referred to herein as "anti-CD3 / anti-EGFRvIII", or "anti-CD3×EGFRvIII", or "CD3×EGFRvIII" bispecific molecules, etc. The term "EGFRvIII", as used herein, refers to the human EGFRvIII protein unless specified to be from a non-human species (e.g., "mouse EGFRvIII", "monkey EGFRvIII", etc.).

[0104] The term "EGFRvIII" refers to class III variant of the epidermal growth factor receptor (EGFRvIII; SEQ ID NO: 172), which is the most frequently seen EGFR variant in glioblastoma (Bigner et al., 1990, Cancer Res 50:8017-8022; Humphrey et al., 1990, Proc Natl Acad Sci USA 87:4207-4211; Yamazaki et al., 1990, Jap J Cancer Res 81:773-779; Ekstrand et al., 1992, Proc Natl Acad Sci USA 89:4309-4313; Wikstrand et al., 1995, Cancer Res 55:3140-3148; and Frederick et al., 2000, Cancer Res 60:1383-1387). EGFRvIII is characterized by a deletion of exons 2-7 of the EGFR gene, resulting in an in-frame deletion of 801 base pairs in the coding region, i.e., a deletion of amino acid residues 6-273 (based on the number of residues in mature EGFR; see UniProtKB / Swiss-Prot.No. P00533), and the generation of a new glycine at the fusion junction (Humphrey et al., 1988, Cancer Res 48:2231-2238; Yamazaki et al., 1990, supra). EGFRvIII has been shown to have non-ligand-dependent, weak but constitutively active kinase activity, and enhanced tumorigenicity (Nishikawa et al., 1994, Proc Natl Acad Sci USA 91:7727-7731; and Batra et al., 1995, Cell Growth and Differentiation 6:1251-1259). In addition to glioblastoma, EGFRvIII has been detected in ductal and intraductal carcinoma of the breast (Wikstrand et al., 1995, Cancer Res 55:3140-3148), non-small cell lung cancer (Garcia de Palazzo et al., 1993, Cancer Res 53:3217-3220), ovarian cancer (Moscatello et al., 1995, Cancer Res 55:5536-5539), prostate cancer (Olapade-Olaopa et al., 2000, British J Cancer 82:186-194), as well as squamous cell carcinoma of the head and neck (Tinhofer et al., 2011, Clin Cancer Res 17(15):5197-5204).

[0105] In yet other exemplary embodiments, the invention includes bispecific antigen-binding molecules that specifically bind to CD3 and MUC16. Such molecules may be referred to herein as "anti-CD3 / anti-MUC16", or "anti-CD3×MUC16", or "CD3×MUC16" bispecific molecules, etc. The term "MUC16" refers to the human MUC16 protein unless specified to be from a non-human species (e.g., "mouse MUC16", "monkey MUC16", etc.).

[0106] Mucin 16 (MUC16; NCBI reference sequence: NP_078966.2, SEQ ID NO: 173), also known as cancer antigen 125 (CA-125), is a mucin encoded by the MUC16 gene in humans. The family of mucin proteins is known to protect the body from infection by binding of pathogens to oligosaccharides in the extracellular domain and to prevent the morphological form of pathogens from reaching the cell surface. For years, the overexpression of MUC16 / CA125 has been used as a prognostic and diagnostic marker for ovarian cancer (Yin and Lloyd, 2001, J. Biol. Chem. 276(29), 27371-27375; O´Brien, TJ, et al, 2001, Tumour Biol. 22(6), 348-366; Leggieri, C. et al., 2014, Eur. J. Gynaecol. Oncol. 35(4), 438-441). MUC16 has been shown to protect tumor cells from the immune system by its highly glycosylated tandem repeat domain that can bind galectin-1 (an immunosuppressive protein) (Seelenmeyer, C., et al., 2003, J. Cell. Sci. 116(Pt 7):1305-18; O´Brien, TJ, et al., 2002, Tumour Biol. 23(3), 154-169). Natural killer cells and monocytes are unable to attack tumor cells that express high levels of MUC16. In its normal physiological role, the MUC16-galectin interaction functions as a barrier against bacterial and viral infections, but MUC16 is immunoprotective in relation to tumor cells, thereby lysing cancer cells is thought to prevent (Felder, M. et al., 2014, Molecular Cancer, 13:129). Thus, MUC16 is a desirable target for immunotherapeutic bispecific antibody molecules administered for treating ovarian cancer by activating immune effector cells.

[0107] In yet another exemplary embodiment, the invention includes a bispecific antigen-binding molecule that specifically binds to CD3 and STEAP2. Such molecules may be referred to herein as "anti-CD3 / anti-STEAP2", or "anti-CD3×STEAP2", or "CD3×STEAP2" bispecific molecules, etc. The term "STEAP2" refers to the human STEAP2 protein unless specified to be from a non-human species (e.g., "mouse STEAP2", "monkey STEAP2", etc.). Six-transmembrane epithelial antigen of the prostate 2 (STEAP2; UniProtKB / Swiss-Prot: Q8NFT2.3) is a 490-amino acid protein encoded by the STEAP2 gene located on human chromosome region 7q21.

[0108] The above-described bispecific antigen-binding molecule that specifically binds to a tumor-associated antigen binds to CD3 with a weak binding affinity or shows no detectable binding affinity, e.g., a K greater than about 100 nM, 300 nM or 500 nM as measured by an in vitro affinity binding assay. D including an anti-CD3 antigen-binding molecule showing.

[0109] As used herein, the expression "antigen-binding molecule" means a protein, polypeptide or molecular complex that includes, or consists of, at least one complementarity-determining region (CDR) that specifically binds to a particular antigen, alone or in combination with one or more additional CDRs and / or framework regions (FRs). In certain embodiments, the antigen-binding molecule is an antibody or a fragment of an antibody, the terms of which are defined elsewhere in this specification.

[0110] As used herein, the expression "bispecific antigen-binding molecule" means a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding domain specifically binds to a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds to a second, different antigen (e.g., PSMA, MUC16, EGFRvIII or STEAP2).

[0111] In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In relation to a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be prefixed with "A1", and the CDRs of the second antigen-binding domain may be prefixed with "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0112] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain brings the two antigens Promote the association between binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, a multimerization domain is an immunoglobulin C H The polypeptide may contain 3 domains. Non-limiting examples of multimerization components are immunoglobulin (C H 2-C H The Fc portion containing 3 domains), for example, isotypes IgG1, IgG2, IgG3, and IgG4, and the Fc domain of IgG selected from any allotype within each isotype group.

[0113] The bispecific binding molecule of the present invention typically comprises two multimerization domains, for example, two Fc domains, each of which is part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0114] In certain embodiments, the multimerization domain is an Fc fragment containing at least one cysteine residue, or an amino acid sequence having a length of 1 to about 200 amino acids. In other embodiments, the multimerization domain is a cysteine residue, or a short-chain cysteine-containing peptide. Other multimerization domains include peptides or polypeptides containing or consisting of leucine zippers, helix-loop motifs, or coiled-coil motifs.

[0115] In order to produce the bispecific antigen-binding molecules of the present invention, any bispecific antibody format or technology can be used. For example, an antibody or a fragment thereof having a first antigen-binding specificity is functionally bound (e.g., by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, e.g., another antibody or antibody fragment having a second antigen-binding specificity, to generate a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in connection with the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain having knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) bodies, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 including bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein for consideration of the above formats).

[0116] In connection with the bispecific antigen-binding molecules of the present invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions or substitutions) compared to the wild-type native version of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules that contain one or more modifications to the Fc domain that result in a modified Fc domain having a modified binding interaction (e.g., improved or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule is C H 2 or C HThe modification is included in the 3 domains, and the modification increases the affinity of the Fc domain for FcRn in an acidic environment (for example, in an endosome where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are, for example, modifications at position 250 (for example, E or Q); positions 250 and 428 (for example, L or F); positions 252 (for example, L / Y / F / W or T), 254 (for example, S or T) and 256 (for example, S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (for example, L / R / S / P / Q or K) and / or position 434 (for example, H / F or Y); or modifications at positions 250 and / or at position 428; or modifications at positions 307 or 308 (for example, 308F, V308F) and position 434. In one embodiment, the modification is 428L (for example, M428L) and 434S (for example, N434S) modifications; 428L, 259I (for example, V259I) and 308F (for example, V308F) modifications; 433K (for example, H433K) and 434 (for example, 434Y) modifications; 252, 254, and 256 (for example, 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (for example, T250Q and M428L); and / or 307 and / or 308 modifications (for example, 308F or 308P).

[0117] The present invention also provides a first C H 3 domain and a second Ig C H 3 domain-containing bispecific antigen-binding molecule, wherein the first and second Ig C H 3 domains differ from each other by at least one amino acid, and at least one amino acid difference reduces the binding of the bispecific antibody to protein A as compared to a bispecific antibody having no amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A, and the second Ig C H 3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R according to the EU numbering). The second C H3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). A further modification found within the second C H 3 may include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); for IgG2 antibodies, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I according to EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU).

[0118] In certain embodiments, the Fc domain may be a chimeric combined Fc sequence obtained from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may be from a portion or all of the C H 2 region obtained from human IgG1, human IgG2 or human IgG4 C H 2 sequence, as well as a portion or all of the C H 3 sequence. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence obtained from the human IgG1, human IgG2 or human IgG4 hinge region combined with a "lower hinge" sequence obtained from the human IgG1, human IgG2 or human IgG4 hinge region. Specific examples of chimeric Fc domains that may be included in any of the antigen-binding molecules described herein include, from N-terminus to C-terminus, [IgG4 C H 1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein includes, from N-terminus to C-terminus, [IgG1 C HIt comprises [IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These, and other examples of the chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention, are described in PCT International Publication No. WO2014 / 121087A1 published on August 7, 2014, which is hereby incorporated by reference in its entirety. Chimeric Fc domains having these general structural arrangements and variants thereof can have altered Fc receptor binding, which on the one hand affects Fc effector functions.

[0119] In certain embodiments, the present invention provides an antibody heavy chain comprising an amino acid sequence in which the heavy chain constant region (CH) region is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190 or SEQ ID NO: 191. In some embodiments, the heavy chain constant region (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190 and SEQ ID NO: 191.

[0120] In other embodiments, the present invention provides an antibody heavy chain comprising an amino acid sequence in which the Fc domain is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200 or SEQ ID NO: 201. In some embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200 and SEQ ID NO: 201.

[0121] Other Fc variants According to certain embodiments of the present invention, provided are anti-CD3 antibodies and anti-CD3 / anti-TAA bispecific antigen-binding molecules comprising an Fc domain comprising one or more mutations that improve or decrease antibody binding to the FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present invention relates to the C H 2 or C H 3 regions of the Fc domain, and the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); positions 250 and 428 (e.g., L or F); positions 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and modifications at 307 and / or 308 (e.g., 308F or 308P).

[0122] For example, the invention includes anti-CD3 antibodies and anti-CD3 / anti-TAA bispecific antigen binding molecules comprising an Fc domain that comprises one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the above Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated within the scope of the invention.

[0123] Biological characteristics of antibodies and bispecific antigen-binding molecules The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that can simultaneously bind to human CD3 and human TAA. According to certain embodiments, the bispecific antigen-binding molecules of the present invention specifically interact with cells expressing CD3 and / or a TAA, such as PSMA, EGFRvIII, or MUC16. The binding arms may have weak to undetectable binding as measured in a suitable in vitro binding assay. The extent to which a bispecific antigen-binding molecule binds to cells expressing CD3 and / or TAAs may be assessed by fluorescence-activated cell sorting (FACS), as shown in Example 4 herein.

[0124] For example, the invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that specifically bind to human T cell lines (e.g., Jurkat) that express CD3 but not TAAs, primate T cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]), and / or TAA-expressing cells. The invention provides antibodies, antigen-binding fragments, and bispecific antibodies thereof that specifically bind to human T cell lines (e.g., Jurkat) that express CD3 but not TAAs, primate T cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]), and / or TAA-expressing cells, with a binding capacity of about 1.8×10 as determined using a FACS binding assay as described in Example 4 or a substantially similar assay. -8 (18 nM) to approximately 2.1 × 10 -7 EC50 > 210nM 50A bispecific antigen-binding molecule that binds to either of the above-mentioned T cells and T cell lines with a value (i.e., a weaker affinity), and also an EC 50 A bispecific antibody that is undetectable. In certain embodiments, the antibodies, antigen-binding fragments, and bispecific antibodies of the invention have an EC 50 Greater than about 30 nM, greater than about 40 nM, greater than about 45 nM, greater than about 50 nM, greater than about 55 nM, greater than about 60 nM, greater than about 65 nM, greater than about 70 nM, greater than about 75 nM, at least 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 500 nM, greater than about 1 μM, greater than about 2 μM, or greater than about 3 μM, as measured by FACS binding using an assay format such as that defined in Example 4 herein, or by a substantially similar assay, and binds to CD3 without showing a detectable binding affinity.

[0125] The present invention also provides antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to TAA-expressing cells and cell lines such as PSMA, EGFRvIII, STEAP2, and MUC16-expressing cell lines with an EC -9 Less than about 100 nM, or at an even lower concentration (i.e., a stronger affinity) required for binding, e.g., less than 5.6 nM (5.6×10 50 ), as determined using a FACS binding assay as described in Example 4 herein, or a substantially similar cell-based assay. The present invention includes bispecific antigen-binding molecules that bind to any of the above-mentioned tumor cell lines with an EC 50 Less than about 50 nM, less than about 45 nM, less than about 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 6 nM, less than about 5 nM, or less than about 1 nM, as measured using, for example, the above-mentioned assay.

[0126] The present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to human CD3 with low affinity, weak affinity, or no further detectable affinity. According to certain embodiments, the present invention has a K greater than about 11 nM (at about 37 °C), as measured by surface plasmon resonance using an assay format such as defined in Example 5 herein. D It includes antibodies and antigen-binding fragments of antibodies that bind to human CD3 with a K greater than about 100 nM or greater than 500 nM. D It includes antibodies that bind to CD3 with a K greater than about 100 nM or greater than 500 nM, and also includes antibodies that do not have a detectable binding affinity. In certain embodiments, the antibodies or antigen-binding fragments of the present invention have a K greater than about 15 nM, greater than about 20 nM, greater than about 25 nM, greater than about 30 nM, greater than about 35 nM, greater than about 40 nM, greater than about 45 nM, greater than about 50 nM, greater than about 55 nM, greater than about 60 nM, greater than about 65 nM, greater than about 70 nM, greater than about 75 nM, at least 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 1 μM, greater than about 2 μM, or greater than about 3 μM, as measured by surface plasmon resonance using an assay format (e.g., mAb capture or antigen capture format) such as defined in Example 5 herein, or by a substantially similar assay. D Or it binds to CD3 without showing a detectable affinity. Bind.

[0127] The present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to cynomolgus monkey (i.e., cynomolgus macaque) CD3 with low affinity, weak affinity, or no further detectable affinity. According to certain embodiments, the present invention has a K greater than about 10 nM (at about 37 °C), as measured by surface plasmon resonance using an assay format such as defined in Example 5 herein. D It includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to human CD3 with a K greater than about 100 nM or greater than 500 nM. DIt includes an antibody that binds to CD3 and also includes an antibody that does not have a detectable binding affinity. In certain embodiments, the antibody or antigen-binding fragment of the invention has a K greater than about 15 nM, greater than about 20 nM, greater than about 25 nM, greater than about 30 nM, greater than about 35 nM, greater than about 40 nM, greater than about 45 nM, greater than about 50 nM, greater than about 55 nM, greater than about 60 nM, greater than about 65 nM, greater than about 70 nM, greater than about 75 nM, at least 80 nM, greater than about 90 nM, greater than about 100 nM, greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 1 μM, greater than about 2 μM, or greater than about 3 μM, as measured by surface plasmon resonance using an assay format (e.g., mAb capture or antigen capture format) as defined, for example, in Example 5 herein, or by a substantially similar assay. D and binds to CD3 without showing a detectable affinity.

[0128] The present invention includes antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind to human CD3 and induce T cell activation. For example, the present invention has an EC less than about 113 pM, as measured by an in vitro T cell activation assay using an assay format as defined, for example, in Example 6 herein [e.g., evaluation of percent activated (CD69+) cells from total T cells (CD2+) in the presence of an anti-CD3 antibody], or by a substantially similar assay that evaluates T cells in their activated state. 50It includes anti-CD3 antibodies that induce human T cell activation at a value. In certain embodiments, the antibody or antigen-binding fragment of the present invention induces in vitro T cell activation assays using an assay format as defined in Example 6 herein, or substantially similar assays, at an EC of less than about 100 pM, less than about 50 pM, less than about 20 pM, less than about 19 pM, less than about 18 pM, less than about 17 pM, less than about 16 pM, less than about 15 pM, less than about 14 pM, less than about 13 pM, less than about 12 pM, less than about 11 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM. 50 50 has a weak binding to CD3 or no detectable binding, and induces human T cell activation [e.g., percent activated (CD69+) T cells] at a value. Anti-CD3 antibodies with weak binding affinity to CD3 or no detectable binding affinity, as exemplified in Example 6 herein, have the ability to induce T cell activation with high potency (i.e., in the pM range).

[0129] The present invention also includes antibodies, antigen-binding fragments, and bispecific antibodies that bind to human CD3 and induce T cell-mediated killing of tumor antigen-expressing cells. For example, the present invention uses an in vitro T cell-mediated tumor cell killing assay (e.g., evaluation of tumor antigen-expressing cell death, such as PSMA-expressing, EGFRvIII-expressing, or MUC16-expressing cells, by human PBMC in the presence of an anti-CD3 antibody) using an assay format as defined in Example 6 herein, or substantially similar assays, at an EC of less than about 1.3 nM to induce T cell-mediated killing of tumor cells. 50 It includes anti-CD3 antibodies that induce T cell-mediated killing of tumor cells at a value. In certain embodiments, the antibody or antigen-binding fragment of the present invention induces in vitro T cell-mediated tumor cell killing assays using an assay format as defined in Example 6 herein, or substantially similar assays, at an EC of less than about 1 nM, less than about 400 pM, less than about 250 pM, less than about 100 pM, less than about 50 pM, less than about 40 pM. EC at levels of full, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM 50 induces T cell-mediated tumor cell killing (e.g., PBMC-mediated killing of OVCAR3 cells) at the value.

[0130] The present invention also includes antibodies, antigen-binding fragments, and bispecific antibodies that bind to CD3 with a dissociation half-life (t1 / 2) of less than about 10 minutes, as measured by surface plasmon resonance at 25° C. or 37° C. using an assay format such as that defined in Example 5 herein, or by an assay substantially similar thereto. In certain embodiments, the antibody or antigen-binding fragment of the present invention binds to CD3 with a t1 / 2 of less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, less than about 1.9 minutes, or less than about 1.8 minutes, as measured by surface plasmon resonance at 25° C. or 37° C. using an assay format (e.g., mAb capture or antigen capture format) such as that defined in Example 5 herein, or by an assay substantially similar thereto, or exhibits very weak binding or no detectable binding.

[0131] The anti-CD3 / anti-TAA bispecific antigen-binding molecules of the present invention can additionally exhibit one or more characteristics selected from the group consisting of: (a) inducing PBMC proliferation in vitro; (b) activating T cells by inducing IFN-gamma release and CD25 upregulation in human whole blood; and (c) inducing T cell-mediated cytotoxicity against anti-TAA-resistant cell lines.

[0132] The present invention includes anti-CD3 / anti-TAA bispecific antigen-binding molecules capable of depleting tumor antigen-expressing cells in a subject (see, for example, Example 7). For example, according to certain embodiments, a single administration of 1 μg, or 10 μg, or 100 μg of the bispecific antigen-binding molecule to a subject (e.g., a dose of about 0.1 mg / kg, about 0.08 mg / kg, about 0.06 mg / kg, about 0.04 mg / kg, about 0.04 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, or less) results in a reduction to below a detectable level in the number of tumor antigen-expressing cells in the subject (e.g., tumor growth in the subject is suppressed or inhibited), and anti-CD3 / anti-PSMA, anti-CD3 / anti-MUC16, or anti-CD3 / anti-STEAP2 bispecific antigen-binding molecules are provided. In certain embodiments, a single administration of the anti-CD3 / anti-PSMA bispecific antigen-binding molecule at a dose of about 0.4 mg / kg results in a reduction to below a detectable level in tumor growth in the subject by about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day after administration of the bispecific antigen-binding molecule to the subject. According to certain embodiments, a single administration of the anti-CD3 / anti-PSMA bispecific antigen-binding molecule of the present invention at a dose of at least about 0.01 mg / kg maintains the number of PSMA-expressing tumor cells below a detectable level for at least about 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, or more after administration. As used herein, the expression "below a detectable level" means that tumor cells growing subcutaneously in a subject cannot be detected directly or indirectly, for example, using standard caliper measurement methods as described in Example 7 herein. In certain embodiments, a single administration of the anti-CD3 / anti-MUC16 bispecific antigen-binding molecule at a dose of about 10 μg results in suppression of tumor growth in the subject on about the 6th day after administration of the bispecific antigen-binding molecule to the subject and maintains tumor suppression until at least the 26th day. In patients receiving a single administration of the anti-CD3 / anti-MUC16 bispecific antigen-binding molecule at a dose of about 10 μg at least 7 days after tumor implantation, the bispecific antigen-binding molecule is effective in suppressing further growth of established tumors in the subject on about the 26th day after tumor implantation in the subject.According to certain embodiments, a single administration of the anti-CD3 / anti-MUC16 bispecific antigen-binding molecule of the invention at a dose of at least about 0.1 mg / kg inhibits the growth of MUC16-expressing tumor cells for at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more after administration of the bispecific molecule. See, for example, Example 8. 7, 18, 19, 20 days or more after administration of the bispecific molecule. See, for example, Example 8.

[0133] In certain embodiments, a single administration of the anti-CD3 / anti-STEAP2 bispecific antigen-binding molecule at a dose of about 0.1 mg / kg or 0.01 mg / kg maintains inhibition of tumor growth until at least day 46 after administration of the bispecific antigen-binding molecule and the tumor to the subject. According to certain embodiments, a single administration of the anti-CD3 / anti-STEAP2 bispecific antigen-binding molecule of the invention at a dose of at least about 0.1 mg / kg, about 0.08 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, about 0.01 mg / kg, or less inhibits the growth of STEAP2-expressing tumor cells for at least about 20, 30, 35, 40, 45 days or more after administration of the bispecific molecule. See, for example, Example 10.

[0134] In other embodiments, an anti-CD3 / anti-TAA bispecific antigen-binding molecule having a CD3-targeting binding arm with a weak binding affinity to effector cells exhibits a reduced drug disappearance rate compared to a bispecific antibody containing the same anti-TAA binding arm and a strong CD3 binding arm administered in an in vivo pharmacokinetic study. The results suggest that bispecific molecules containing a CD3-targeting arm with weaker binding can exhibit beneficial drug exposure levels (AUC last ) and drug disappearance profiles (antibody clearance). See, for example, Example 9.

[0135] The present invention provides anti-CD3 / anti-PSMA, anti-CD3 / anti-MUC16 and anti-CD3 / anti-STEAP2 bispecific antigen-binding molecules (i.e., anti-CD3 / anti-TAA bispecific antigen-binding molecules) that exhibit one or more characteristics selected from the group consisting of: (a) inhibition of tumor formation in immunocompromised mice bearing human prostate cancer xenografts; (b) inhibition of tumor growth in immunocompetent mice bearing human prostate cancer xenografts; (c) suppression of tumor growth of established tumors in immunocompromised mice bearing human prostate cancer xenografts; and (d) reduction of tumor growth of established tumors in immunocompetent mice bearing human prostate cancer xenografts (see, e.g., Examples 7, 8 and 10). The present invention also provides anti-CD3 / anti-PSMA, anti-CD3 / anti-MUC16 and anti-CD3 / anti-STEAP2 bispecific antibodies (i.e., anti-CD3 / anti-TAA bispecific antibodies) comprising: i) a first heavy chain directed to an effector T cell (i.e., CD3), and ii) a second heavy chain directed to a target tumor cell, wherein the bispecific antibody exhibits weak binding to effector cells, or no detectable binding, and exhibits tumor growth inhibition and reduced antibody clearance (i.e., disappearance) from the body as compared to a bispecific antibody that exhibits strong binding to effector cells.

[0136] Epitope mapping and related techniques The epitope on CD3 to which the antigen-binding molecule of the present invention binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD3 protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of CD3. The antibodies of the present invention may interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or with amino acids on two or more different CD3 chains. The term "epitope" as used herein refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be conformational or linear. Conformational epitopes are generated by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. Linear epi topes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include a saccharide, phosphoryl group, or sulfonyl group moiety on an antigen.

[0137] To determine whether an antigen-binding domain of an antibody "interacts with one or more amino acids" within a polypeptide or protein, various techniques known to those of skill in the art may be used. Exemplary techniques include, for example, Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Conventional cross - blocking assays, alanine - scanning mutagenesis analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443 - 463), and peptide cleavage analysis, such as those described in Spring Harb., NY), are included. Additionally, epitope cleavage, epitope extraction, and chemical modification of the antigen may be used (Tomer, 2000, Protein Science 9:487 - 496). Another method that can be used to identify amino acids within a polypeptide with which the antigen - binding domain of an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest followed by binding of the antibody to the deuterium - labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen - deuterium exchange to occur at all residues except those protected by the antibody (where deuterium labeling is maintained). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectral analysis, thereby revealing deuterium - labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252 - 259; Engen and Smith (2001) Anal.Chem.73:256A - 265A. X - ray crystallography of the antigen / antibody complex can also be used for epitope mapping purposes.

[0138] The present invention further includes anti - PSMA antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences as described in Table 6 herein). Similarly, the present invention also includes anti - PSMA antibodies that compete with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences as described in Table 6 herein) with respect to binding to PSMA. The anti - PSMA antibodies disclosed in U.S. Patent Application Publication No. 15 / 223,434 are incorporated herein by reference into this application.

[0139] The present invention also includes a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human CD3 and / or cynomolgus CD3 with low or detectable binding affinity, and a second antigen-binding domain that specifically binds to a human tumor-associated antigen (TAA), wherein the first antigen-binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen-binding domains described herein.

[0140] Similarly, the present invention also includes a bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to human CD3 and / or cynomolgus CD3 with low or detectable binding affinity, and a second antigen-binding domain that specifically binds to a human tumor-associated antigen (TAA), wherein the first antigen-binding domain competes with any of the specific exemplary CD3-specific antigen-binding domains described herein with respect to binding to CD3.

[0141] Whether a particular antigen-binding molecule (e.g., an antibody) or its antigen-binding domain binds to the same epitope as the reference antigen-binding molecule of the present invention or competes with it for binding can be readily determined by using conventional methods known in the art. For example, to determine whether a test antibody binds to the same epitope on CD3 (or TAA) as the reference bispecific antigen-binding molecule of the present invention, the reference bispecific molecule First, bind the child to the CD3 protein (or TAA protein). Next, evaluate the ability of the test antibody to bind to the CD3 molecule. If the test antibody can bind to CD3 (or TAA) after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to an epitope of CD3 (or TAA) different from that of the reference bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CD3 (or TAA) molecule after saturation binding with the reference bispecific antigen-binding molecule, the test antibody can bind to the same epitope of CD3 (or TAA) as the epitope bound by the reference bispecific antigen-binding molecule of the present invention. Then, to confirm whether the observed loss of binding of the test antibody is actually due to binding to the same epitope as the reference bispecific antigen-binding molecule, or whether steric hindrance blocks (or another phenomenon) causes the observed loss of binding, additional conventional experiments (such as peptide mutagenesis and binding analysis) can be performed. If the reference antibody has no measurable binding as exemplified herein, the reference antibody can be mutated to the germline sequence to determine binding to CD3 for the purpose of comparing epitope interactions or comparing its binding characteristics with those of test antibodies as described herein. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody-binding assay available in the art. According to certain embodiments of the present invention, for example, if one antigen-binding protein at 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess inhibits the binding of the other by at least 50% but preferably 75%, 90%, or even 99% as measured in a competitive binding assay, the two antigen-binding proteins bind to the same (or overlapping) epitope (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if essentially all amino acid mutations within the antigen that reduce or abolish the binding of one antigen-binding protein also reduce or abolish the binding of the other, the two antigen-binding proteins are considered to bind to the same epitope.If only a subset of the amino acid mutations that reduce or abolish the binding of one antigen-binding protein also reduce or abolish the binding of the other, the two antigen-binding proteins are considered to have "overlapping epitopes."

[0142] To determine whether an antibody or its antigen-binding domain competes with respect to binding to a reference antigen-binding molecule, the binding methods described above are performed in two directions. In the first direction, the reference antigen-binding molecule is bound to the CD3 protein (or TAA protein) under saturation conditions, followed by evaluation of the binding of the test antibody to the CD3 (or TAA) molecule. In the second direction, the test antibody is bound to the CD3 (or TAA) molecule under saturation conditions, followed by evaluation of the binding of the reference antigen-binding molecule to the CD3 (or TAA) molecule. If only the first (saturating) antigen-binding molecule can bind to the CD3 (or TAA) molecule in both directions, it is concluded that the test antibody and the reference antigen-binding molecule compete with respect to binding to CD3 (or TAA). As will be understood by those skilled in the art, antibodies that compete with respect to binding to a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically hinder the binding of the reference antibody by binding to overlapping or adjacent epitopes. If the reference antibody does not have a measurable binding as exemplified herein, the reference antibody can be mutated to the germline sequence for the purpose of determining binding to CD3 for the purpose of comparing epitope interactions or comparing its binding properties or blocking interactions with test antibodies as described herein.

[0143] Preparation of Antigen-Binding Domains and Construction of Bispecific Molecules Antigen-binding domains specific for a particular antigen can be prepared by any antibody generation technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD3 and TAA) can be used to prepare the bispecifics of the invention using conventional methods. They can be appropriately arranged relative to each other to generate a specific antigen-binding molecule. (A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present invention is described elsewhere in this specification.) In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the present invention are obtained from chimeric, humanized, or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules of the present invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody production technology), high-affinity chimeric antibodies against specific antigens (e.g., CD3 or TAA) having human variable regions and mouse constant regions are first isolated. The antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. To generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules of the present invention, the mouse constant regions are replaced with the desired human constant regions.

[0144] To generate a human bispecific antigen-binding molecule, a genetically modified animal can be used. For example, a genetically engineered mouse that cannot rearrange and express an endogenous mouse immunoglobulin light chain variable sequence may be used, and the mouse expresses only one or two human light chain variable domains encoded by a human immunoglobulin sequence operably linked to a mouse kappa (κ) constant gene at the endogenous mouse kappa (κ) locus. Such a genetically engineered mouse can be used to generate a fully human bispecific antigen-binding molecule that comprises two different heavy chains that associate with the same light chain that comprises a variable domain obtained from one of two different human light chain variable region gene fragments. (For a detailed discussion of such modified mice and their use for generating bispecific antigen-binding molecules, see, for example, US2011 / 0195454.) The antibodies of the present invention may comprise immunoglobulin heavy chains associated with a common light chain. The common light chain may be obtained from the isotype light chain of an anti-TAA heavy chain, or from a light chain that exhibits promiscuity or the ability to pair with a wide range of non-isotype heavy chains, i.e., a universal or common light chain, i.e., a known or a light chain variable domain obtained from a known light chain variable domain. The antibodies of the present invention may comprise immunoglobulin heavy chains associated with a single rearranged light chain. In some embodiments, the light chain comprises a variable domain obtained from a human Vκ1-39 gene fragment or a Vκ3-20 gene fragment. In other embodiments, the light chain comprises a variable domain obtained from a human Vκ1-39 gene fragment rearranged with a human Jκ5 or a human Jκ1 gene fragment, or a Vκ3-20 gene fragment rearranged with a human Jκ1 gene fragment, or a Vκ1-39 gene fragment rearranged with a human Jκ1 gene fragment.

[0145] Biological equivalents The present invention encompasses antigen-binding molecules that have an amino acid sequence different from those of the exemplary molecules disclosed herein but that maintain the ability to bind or interact with CD3 and / or TAA. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids compared to the parental sequence, but exhibit a biological activity that is essentially equivalent to that of the bispecific antigen-binding molecules described.

[0146] The present invention includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered to be biologically equivalent, for example, if they show no significant difference when administered at the same molar dose, whether as a single dose or multiple doses, under experimental conditions where the rate and extent of absorption are similar, and are pharmaceutical equivalents or pharmaceutical alternatives. Some antigen-binding proteins are considered to be equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in the rate of absorption, provided that such differences in absorption rate are intentional and reflected in the labeling and are not essential, for example, for achieving an effective drug concentration in the body during chronic use and are not considered medically significant for the particular formulation being studied, and thus can be considered to be biologically equivalent. This can be done.

[0147] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, and potency.

[0148] In one embodiment, two antigen-binding proteins are biologically equivalent if a patient can be switched between a reference product and a biological product one or more times without a clinically significant change in immunogenicity or a predicted increase in the risk of side effects, including decreased efficacy, compared to continued treatment without switching between the reference product and the biological product.

[0149] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by a common mechanism of action (or mechanisms) on the conditions of use (if any), as long as such a mechanism is known.

[0150] Biological equivalence can be demonstrated by in vivo and in vitro methods. Means of biological equivalence include, for example: (a) in vivo tests in humans or other animals in which the concentration of an antibody or its metabolite in blood, plasma, serum, or other biological fluids is measured as a function of time; (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other animals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, bioavailability, or biological equivalence of the antigen-binding protein.

[0151] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity may be deleted or substituted with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges upon regeneration. In other situations, biologically equivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that modify the glycosylation properties of the molecule, such as mutations that abolish or remove glycosylation.

[0152] Species selectivity and cross-reactivity According to certain embodiments of the invention, antigen-binding molecules are provided that show weak or no interaction with human CD3 and weak or no interaction with CD3 from other species such as cynomolgus CD3. Also provided are antigen-binding molecules that bind to human TAA but not to TAA from other species. The invention also includes antigen-binding molecules that bind to human CD3 and CD3 from one or more non-human species; and / or antigen-binding molecules that bind to human TAA and TAA from one or more non-human species.

[0153] According to certain exemplary embodiments of the present invention, provided are antigen-binding molecules that may or may not bind weakly to human CD3 and / or TAA, and in some cases, may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey or chimpanzee CD3 and / or TAA. For example, in certain exemplary embodiments, provided are bispecific antigen-binding molecules of the present invention comprising a first antigen-binding domain that binds weakly to human CD3 and cynomolgus monkey CD3, and a second antigen-binding domain that specifically binds to human PSMA, MUC16, EGFRvIII or STEAP2.

[0154] Immunoconjugate The present invention encompasses antigen-binding molecules conjugated to a therapeutic moiety (an "immunoconjugate"), such as a cytotoxin, chemotherapeutic agent, immunosuppressive agent or radioisotope. Cytotoxic agents include any agent that is harmful to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art (see, for example, WO05 / 103081).

[0155] Therapeutic Formulations and Administration The present invention provides a pharmaceutical composition comprising an antigen-binding molecule of the present invention. The pharmaceutical composition of the present invention is formulated with a suitable carrier, excipient, and other agents that provide improved movement, delivery, resistance, etc. A number of suitable formulations can be found in the formulary known to all pharmacists: Remington´s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) - containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA complexes, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al., "Compendium of excipients for parenteral formulations", PDA (1998) J Pharm Sci Technol 52:238-311.

[0156] The dosage of the antigen-binding molecule administered to a patient can vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred dosages are typically calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in adult patients, it may be advantageous to administer the bispecific antigen-binding molecule of the present invention intravenously in a single dose of usually about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule for administering the bispecific antigen-binding molecule can be determined experimentally, for example, the patient's progress is monitored by regular evaluation and the dosage is adjusted accordingly. Furthermore, interspecies increases or decreases in dosage may be made using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut.Res. 8:1351).

[0157] A variety of delivery systems are known, such as encapsulation within liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432), and can be used to administer the pharmaceutical compositions of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by injection or bolus injection, or by absorption through epithelial or skin mucosal linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and can also be administered together with other biologically active agents. Administration can be systemic or local.

[0158] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. Further, with respect to subcutaneous delivery, pen delivery devices are readily applicable for the delivery of the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused obtained. Disposable pen delivery devices do not have replaceable cartridges. Rather, disposable pen delivery devices are filled with the pharmaceutical composition held within a container within the device. Once the pharmaceutical composition has been depleted from the container, the entire device is discarded.

[0159] A number of reusable pens and autoinjector delivery devices are applicable in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Some examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Some examples of disposable pen delivery devices applicable in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ Pen (Abbott Labs, Abbott Park IL).

[0160] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; see also Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be placed in proximity to the target of the composition and thus may require only a very small fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0161] Injectable formulations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable formulations can be prepared by known methods. For example, an injectable formulation can be prepared by dissolving, suspending, or emulsifying, for example, the above-described antibody or a salt thereof in a sterile aqueous medium or an oily medium conventionally used for injection. Examples of aqueous media for injection include isotonic solutions containing physiological saline, glucose, and other adjuvants, which can be used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, which can be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The injectable thus prepared is preferably filled into an appropriate ampoule.

[0162] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a unit dosage form adapted to suit the dosage of the active ingredient. Such dosage forms in unit dosage include, for example, tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the antibody described above contained is generally about 5 to about 500 mg per unit dosage form; particularly in the form of injection, it is preferably contained at about 5 to about 100 mg of the antibody described above, and in other dosage forms, it is preferably contained at about 10 to about 250 mg.

[0163] Therapeutic Use of Antigen-Binding Molecules The present invention includes a method comprising administering to a subject in need thereof a therapeutic composition comprising an anti-tumor antibody or an antigen-binding fragment thereof, or a bispecific antigen-binding molecule that binds weakly or has no detectable binding to CD3 and binds to a tumor-associated antigen. The therapeutic composition may comprise either an antibody or a bispecific antigen-binding molecule as disclosed herein, and a pharmaceutically acceptable carrier or diluent. As used herein, the expression "a subject in need thereof" refers to a subject exhibiting one or more symptoms or signs of cancer (e.g., a subject expressing a tumor or suffering from any of the cancers mentioned hereinafter herein), or alternatively, a human or non-human animal that would benefit from inhibition or reduction of tumor activity, or depletion of tumor cells (e.g., PSMA++ prostate cancer cells).

[0164] The antibodies and bispecific antigen-binding molecules of the present invention (as well as therapeutic compositions containing the same) are useful, inter alia, for the treatment of any disease or disorder in which the stimulation, activation and / or targeting of an immune response would be beneficial. In particular, the bispecific antigen-binding molecules of the present invention can be used for the treatment, prevention and / or remission of any disease or disorder associated with or mediated by the growth of cells expressing a TAA, such as cells expressing or active in PSMA or PSMA+ cells. The mechanism of action by which the therapeutic methods of the present invention are achieved involves the killing of cells expressing tumor-associated antigens in the presence of effector cells, for example by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms. Cells expressing tumor-associated antigens such as PSMA, MUC16, STEAP2 or EGFRvIII that can be inhibited or killed using the bispecific antigen-binding molecules of the present invention include, for example, prostate tumor cells.

[0165] The antigen-binding molecules of the present invention can be used, for example, to treat primary and / or metastatic tumors that occur in the brain and meninges, head and neck, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive organs, muscle, bone, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, kidney, bladder and / or special sense organs such as the eye. In certain embodiments, the bispecific antigen-binding molecules of the present invention are used for the treatment of one or more of the following cancers: pancreatic cancer, head and neck cancer, prostate cancer, glioblastoma multiforme, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer due to MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma glioma, breast cancer (e.g., ductal or intraductal carcinoma, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobe renal carcinoma, (renal) oncocytoma, (renal) transitional cell carcinoma, urothelial carcinoma, (bladder) adenocarcinoma, or (bladder) small cell carcinoma), but are not limited thereto. According to certain embodiments of the present invention, the bispecific antibody is useful for the treatment of patients suffering from refractory or treatment-resistant cancers, such as castration-resistant prostate cancer. According to an exemplary embodiment of the present invention, a method is provided that includes administering to a patient suffering from castration-resistant prostate cancer an anti-CD3 / anti-PSMA bispecific antigen-binding molecule as disclosed herein. To determine whether a patient has castration-resistant tumors, analytical / diagnostic methods known in the art, such as tumor screening, can be used.

[0166] The present invention also includes a method for treating residual cancer in a subject. As used herein, the term "residual cancer" means the presence or persistence of one or more cancer cells in a subject after treatment with anti-cancer therapy, such as primary treatment or standard therapy.

[0167] According to certain embodiments, the invention provides a method for treating cancer associated with TAA expression (e.g., prostate cancer associated with PSMA expression or STEAP2 expression, glioblastoma associated with EGFRvIII expression, or ovarian cancer associated with MUC16 expression), the method comprising administering to a subject one or more of the bispecific antigen-binding molecules described elsewhere herein after the subject has been determined to have cancer. For example, the invention provides a method for treating prostate cancer, the method comprising administering an anti-CD3 / anti-TAA bispecific antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or later after the subject has received prior treatment.

[0168] Combination Therapies and Formulations The present invention provides a method comprising administering a pharmaceutical composition comprising any of the exemplary antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents.Exemplary additional therapeutic agents that can be combined with or administered in combination with the antigen-binding molecules of the present invention include, for example, anti-programmed cell death 1 antibodies (e.g., anti-PD1 antibodies such as those described in US Patent Application Publication No. US2015 / 0203579A1), anti-programmed cell death ligand-1 (e.g., anti-PD-L1 antibodies such as those described in US Patent Application Publication No. US2015 / 0203580A1), EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), antagonists of another EGFR family member such as Her2 / ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2, anti-ErbB3 or anti-ErbB4 antibodies or small molecule inhibitors of ErbB2, ErbB3 or ErbB4 activity), antagonists of EGFRvIII (e.g., antibodies that specifically bind to EGFRvIII), cMET antagonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies), VEGF antagonists (e.g., VEGF-Trap, see, e.g., US7,087,411 (also referred to herein as "VEGF-inhibitory fusion protein"), anti-VEGF antibodies (e.g., bevacizumab), small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib or pazopanib)), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US2009 / 0142354, e.g., REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US2011 / 0027286, e.g., H1H685P), FOLH1 (PSMA) antagonists, PRLR antagonists (e.g., anti-PRLR antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., anti-CA9 antibodies), uroplakin antagonists (e.g., anti-uroplakin antibodies), and the like.Other agents that can be beneficially administered in combination with the antigen-binding molecules of the present invention include small molecule cytokine inhibitors, and cytokine inhibitors including antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors. The pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising anti-CD3 / anti-PSMA bispecific antigen-binding molecules as disclosed herein) also include "ICE": ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); "DHAP": dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platino. l®-AQ); and "ESHAP": etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ), and may be administered as part of a treatment regimen comprising one or more therapeutic combinations selected from.

[0169] The present invention also includes a pharmaceutical composition comprising any of the antigen-binding molecules referred to herein and one or more inhibitors of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the above-mentioned cytokines, wherein the inhibitor is an aptamer, an antisense molecule, a ribozyme, siRNA, a peptibody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab´)2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other modified molecules, such as diabody, triabody, tetrabody, minibody and minimal recognition unit). The antigen-binding molecule of the present invention may also be administered and / or formulated in combination with an antiviral agent, an antibiotic, an analgesic, a corticosteroid and / or an NSAID. The antigen-binding molecule of the present invention may also be administered as part of a treatment regimen that also includes radiation treatment and / or conventional chemotherapy.

[0170] The additional therapeutic active ingredient(s) may be administered immediately before, simultaneously with, or immediately after administration of the antigen-binding molecule of the present invention (for the purposes of this disclosure, such an administration schedule is considered an administration of the antigen-binding molecule "in combination with" the additional therapeutic active ingredient).

[0171] The present invention includes a pharmaceutical composition in which the antigen-binding molecule of the present invention is formulated in combination with one or more of the additional therapeutic active ingredient(s) as described elsewhere herein.

[0172] Administration schedule According to certain embodiments of the present invention, multiple doses of a bispecific antigen-binding molecule (e.g., an anti-TAA bispecific antigen-binding molecule) may be administered to a subject over a defined period of time. The method according to this aspect of the present invention includes sequentially administering multiple doses of the antigen-binding molecule of the present invention to a subject. As used herein, "sequentially administering" means that each dose of the antigen-binding molecule is administered to the subject at different times, e.g., on different days separated by a predetermined interval (e.g., time, day, week, or month). The present invention includes methods that include sequentially administering to a patient a single initial dose of an antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and optionally followed by one or more tertiary doses of the antigen-binding molecule.

[0173] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the chronological order of administration of the antigen-binding molecule of the present invention. Thus, an "initial dose" is a dose administered at the start of a treatment plan (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of the antigen-binding molecule, but generally may differ from each other with respect to the frequency of administration. However, in certain embodiments, the amounts of the antigen-binding molecule contained in the initial, secondary, and / or tertiary doses differ from each other during the course of treatment (e.g., are adjusted up or down as appropriate). In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the start of a treatment plan, followed by subsequent doses being administered less frequently (e.g., a "maintenance dose").

[0174] In one exemplary embodiment of the present invention, the secondary and / or tertiary doses are each from 1 to 26 (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, It is administered after 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, 13, 13 and 1 / 2, 14, 14 and 1 / 2, 15, 15 and 1 / 2, 16, 16 and 1 / 2, 17, 17 and 1 / 2, 18, 18 and 1 / 2, 19, 19 and 1 / 2, 20, 20 and 1 / 2, 21, 21 and 1 / 2, 22, 22 and 1 / 2, 23, 23 and 1 / 2, 24, 24 and 1 / 2, 25, 25 and 1 / 2, 26, 26 and 1 / 2, or more weeks. The phrase "immediately preceding dose", as used herein, in the context of multiple administrations, means the dose of the antigen-binding molecule that is administered to the patient immediately prior to the administration of the very next dose in the sequence, without intervening doses.

[0175] The method according to this aspect of the invention may include administering to the patient any number of secondary and / or tertiary doses of the antigen-binding molecule (e.g., an anti-TAA bispecific antigen-binding molecule). For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0176] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency at which secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The dosing frequency may also be adjusted by the physician during the course of treatment, depending on the needs of the individual patient after clinical trials.

Examples

[0177] The following examples are presented to provide a complete disclosure and description to those skilled in the art of how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0178] Example 1: Generation of anti-CD3 antibodies The following procedure was aimed at identifying antibodies that specifically recognize CD3 (T cell co-receptor) as an antigen.

[0179] A pool of anti-CD3 antibodies was obtained by immunizing genetically engineered mice. Briefly, mice genetically modified to express a reverse chimeric (human variable, mouse constant) and immunoglobulin heavy chain associated with a single rearranged light chain (e.g., Vκ1-39 / J or Vκ3-20 / J) were immunized with the CD3 antigen to generate B cells containing diverse human VH rearrangements to express a diverse repertoire of high-affinity antigen-specific antibodies. Certain exemplified antibodies described in this application are made recombinantly and express the same light chain sequence of Vκ1-39JK5 (LCVR described in SEQ ID NO: 162), while other antibodies made recombinantly express one allotypic light chain of the heavy chain arm (e.g., the tumor target arm).

[0180] The generated antibodies were tested for affinity for human and cynomolgus CD3 antigens in an in vitro binding assay, and for example, one CD3 antibody designated CD3-V H-P (HCVR described in SEQ ID NO: 154) was identified, which binds to both human and cynomolgus CD3 and has an affinity (+++) EC between 1 and 40 nM as determined in FACS titrations of Jurkat cells and cynomolgus T cells, respectively. 50It has been found to have. See, for example, the FACS binding experiment outlined in Example 4 below in this specification.

[0181] Subsequently, the germline amino acid residues of CD3-VH-P were identified, and the antibody symbolized as "CD3-VH-G" was modified to contain only the germline framework. Other antibody derivatives were modified by well-known molecular cloning techniques, and amino acid residues were replaced stepwise based on the differences between the germline sequence and the CD3-VH-P sequence. Each antibody derivative is assigned a numerical symbol designation of "CD3-VH-G". See Table 1 and Figure 1.

[0182] A bispecific antibody was prepared that includes a first binding arm obtained from the modified anti-CD3 antibody with its symbol designation and description shown in Table 1, and a second binding arm obtained from an anti-TAA antibody, and was tested for monovalent affinity for CD3-bearing cells in a FACS assay (as described in Example 4). The results of the monovalent binding affinity of these bispecific antibodies are shown in the two rightmost columns of Table 1. In a specific example, the bispecific antibodies having TAA-binding arms and CD3-binding arms symbolized as "CD3-VH-G", "CD3-VH-G5", and "CD3-VH-G20", respectively, had EC 50 , no detectable binding, and an EC of 5.5E-07 50 and bound to Jurkat cells.

[0183]

Table 1

[0184] CD3-VH-G and several other modified antibodies maintained their binding affinity as seen in the FACS assay, but several anti-CD3 antibodies bound to human or cynomolgus CD3 with weak (+ / -) to undetectable (-) affinity in vitro. Subsequently, bispecific The binding affinity, binding reaction rate, and other biological properties were investigated for the neutralizing antibody to elucidate cytotoxicity and pharmacokinetic (pK) profiles, which will be described in detail in the examples below.

[0185] Example 2: Heavy and light chain variable regions (amino acid and nucleic acid sequences of CDRs) The amino acid and nucleic acid sequences were determined for each antibody heavy chain sequence. Each antibody heavy chain as a derivative of the germline sequence IGHV3-9*01 / D5-12*01 / J6*02 (SEQ ID NO: 181) was assigned a "G" numerical symbol designation for consistent naming. Table 2 describes the amino acid sequence identifiers of the heavy chain variable region and CDRs of the modified anti-CD3 antibodies of the present invention. The corresponding nucleic acid sequence identifiers are described in Table 3. Also, the amino acid and nucleic acid sequence identifiers of the light chain variable region and CDRs for constructing each recombinant antibody are specified in Tables 4 and 5 below, respectively.

[0186] [Table 2]

[0187] [Table 3]

[0188] [Table 4]

[0189] [Table 5]

[0190] The control 1 antibody designated "CD3-L2K" was constructed based on a known anti-CD3 antibody (i.e., the anti-CD3 antibody "L2K" as described in WO2004 / 106380).

[0191] The isotype control antibodies referred to in the following examples of this specification are isotype-matched (engineered IgG4) antibodies that interact with an irrelevant antigen, namely the FelD1 antigen.

[0192] Example 3: Generation of ULC Bispecific Antibodies That Bind to CD3 and Tumor-Associated Antigen (TAA) Bispecific antibodies comprising anti-CD3 specific binding domains and anti-TAA specific binding domains such as PSMA, EGFRvIII, MUC16, or STEAP2 were constructed using standard molecular biology methods utilizing the heavy chains from the anti-CD3 antibodies described herein, the heavy chains from the anti-TAA antibodies, and a common or universal light chain (ULC). The anti-TAA antibodies used to construct the bispecific antibodies of the invention were obtained by immunizing transgenic mice.

[0193] An overview of the components of the antigen-binding domains of the various bispecific antibodies made according to this example is set forth in Tables 6, 7, and 8 below. All bispecific antibodies having an engineered (chimeric) IgG4 Fc domain were produced as described in U.S. Patent Application Publication No. US20140243504A1, published Aug. 28, 2014. Exemplary EGFRvIII×CD3 bispecific antibodies can be prepared using any of the heavy chain and light chain variable regions (or CDRs) of the EGFRvIII antibodies discussed in U.S. Patent Application Publication No. US20150259423, which is hereby incorporated by reference in its entirety in combination with any of the variable regions or CDRs of the anti-CD3 antibodies discussed herein.

[0194] [Table 6]

[0195] [Table 7]

[0196] [Table 8]

[0197]

Table 9

[0198] Each of the exemplary bispecific antibodies was tested in various bioassays as described below in this specification.

[0199] Example 4: Binding Affinity of Exemplary Bispecific Antibodies as Measured by FACS Analysis In this example, the ability of CD3×TAA bispecific antibodies to bind to human and cynomolgus monkey CD3-expressing cell lines was determined by FACS. Furthermore, the ability of these bispecific antibodies to bind to target-specificity (TAA-specificity) cell lines was also confirmed. As described above, the various bispecific antibodies of the present invention utilized a single TAA-specific binding arm (PSMA, EGFRvIII, MUC16, or STEAP2; see Examples 3, Tables 6, 7, and 8) paired with one of a panel of anti-CD3 binding arms (see Examples 1 and 2 above), as well as a common light chain. Also, as shown in Example 5, the CD3×TAA bispecific antibodies showed a range of affinities for the human soluble heterodimer hCD3ε / δ.mFc protein by surface plasmon resonance.

[0200] Briefly, 2×10 5 Cells / well of human CD3-expressing Jurkat, cynomolgus monkey T, or TAA-specific expressing cells were incubated with serial dilutions of the bispecific antibody for 30 minutes at 4°C. After incubation, the cells were washed and goat F(ab´)2 anti-human FcγPE-labeled secondary antibody (Jackson Immunolabs) was added to the cells for an additional 30 minutes. Next, the cells were washed and resuspended in cold PBS + 1% BSA and analyzed by flow cytometry on a BD FACS Canto II.

[0201] For FACS analysis, cells were gated by forward scatter height versus forward scatter area for single event selection, followed by side and forward scatter. EC 50 was determined using PRISM™ software (GraphPad Software, Inc., La Jolla, Calif.). Values ​​were calculated using four-parameter nonlinear regression analysis.

[0202] [Table 10] [Table 11]

[0203] As shown in Table 10A, the CD3 binding arm of each CD3 x PSMA bispecific antibody exhibited a range of cell binding affinities to human CD3 expressing Jurkat cells (EC of 15-300 nM). 50 range). Importantly, CD3 arms showing weak to no binding to human CD3 heterodimeric protein by surface plasmon resonance (see Table 11 herein below) also correlated with weak to unobservable binding to Jurkat cells (i.e., CD3-VH-G2, CD3-VH-G3, CD3-VH-G5). Undetectable or no detectable binding in a FACS assay or equivalent assay means that the affinity between the antibody and its target antigen exceeds the detection limit of the assay (e.g., >1 μM). Some CD3 binding arms also showed cross-reactivity to cynomolgus T cells. All tested bispecific antibodies showed similar cell binding to each PSMA, EGFRvIII and MUC16 expressing cell line, confirming that bispecific pairing with individual CD3 arms does not affect or reduce TAA-specific binding (TAA-specific binding was 5.6 nM or less (high affinity) in all cases tested).

[0204] Antibodies showing weak to undetectable binding to human CD3 and weak to no binding to cynomolgus CD3 were deemed favorable for affinity-driven bispecific pairing according to the present invention and were further tested for cytotoxicity in in vitro and in vivo assays.

[0205] Example 5: Binding Affinities of Exemplary Antibodies as Measured by Surface Plasmon Resonance Binding Assay The binding affinity and kinetic constants of anti-TAA x anti-CD3 bispecific antibodies to the soluble heterodimeric hCD3ε / δ.mFc protein (hCD3ε=UniProtKB / Swiss-Prot: P07766.2; SEQ ID NO: 169; hCD3δ=UniProtKB / Swiss-Prot: P04234.1, SEQ ID NO: 170) were determined by surface plasmon resonance using either an antigen capture format (Table 11) or an antibody capture format (data not shown). The CD3 binding arm was determined at 37°C. In this example, BSPSMA / CD3 bispecific antibodies were utilized because these pairings represented the use of a broader panel of antibodies with CD3 binding arms. Measurements were performed on a Sierra Sensors MASS-1 instrument.

[0206] In the antigen capture format, a MASS-1 high density amine sensor surface was derivatized with a goat anti-mouse IgG2a polyclonal antibody (Southern Biotech). Soluble heterodimeric CD3 protein was captured and each antibody was injected onto the captured antigen.

[0207] Data were processed using MASS-1 Analyser R2 curve fitting software and fitted to a 1:1 binding model to determine the kinetics of association (k a ) and dissociation (k d The binding / dissociation equilibrium constant (K D ) and dissociation half-life (t 1 / 2 ) was calculated from the kinetic rate constants as follows: K D (M)=k d / k a; and t 1 / 2 (min) = (ln2 / (60 * k d ).

[0208]

Table 12

[0209] As shown in Table 11, the anti-CD3 × anti-PSMA bispecific antibody maintained a very weak binding to soluble CD3 in the surface plasmon resonance binding assay, for example, with K D values ranging from greater than 11 nM to a maximum of 334 nM, which were weaker than those of the bispecific anti-CD3 arm, CD3-VH-G, obtained from the germline framework.

[0210] Some bispecific antibodies showed K D values greater than 50 nM, and some showed K -7 values greater than 100 nM (> 1 × 10 D ), i.e., BSPSMA / CD3-900, BSPSMA / CD3-1000, BSPSMA / CD3-1900), K -7 values greater than 300 nM (> 3 × 10 D ), i.e., BSPSMA / CD3-005), and some even exceeded the detection limit of the assay (> 500 nM; > 5 × 10 -7 ), i.e., did not show detectable binding to soluble human CD3 (i.e., BSPSMA / CD3-200, BSPSMA / CD3-300, BSPSMA / CD3-400, BSPSMA / CD3-004, and BSPSMA / CD3-1800).

[0211] Example 6: T cell activation and tumor-specific cytotoxicity shown by the bispecific antibodies of the present invention as measured in vitro In this example, the specific killing of PSMA-, EGFRvIII- or MUC16-expressing TAA target cells in the presence of a CD3-based bispecific antibody was monitored by flow cytometry. As previously reported, the bispecific antibodies showed a range of affinities for the CD3 protein and CD3-expressing cell lines (i.e., weak, moderate and strong binding). The same panel of this bispecific antibody was tested for its ability to re-induce killing of target-expressing cells in naive human T cells.

[0212] Briefly, PSMA-expressing (C4-2, 22Rv1 and TRAMPC2_PSMA), EGFRvIII-expressing (U87 / EGFRvIII) or MUC16-expressing (OVCAR3) cell lines were labeled with 1 μM of the fluorescent tracking dye Violet Cell Tracker. After labeling, the cells were seeded at 37 °C overnight. Separately, human PBMCs were seeded at 1 × 10 6 cells / mL in supplemented RPMI medium and incubated overnight at 37 °C to enrich lymphocytes by depleting adherent macrophages, dendritic cells, and some monocytes. The next day, the target cells were co-incubated with adherent cell-depleted naive PBMCs (effector / target cell ratio 4:1) and serial dilutions of the relevant bispecific antibody or isotype control (concentration range: 66.7 nM to 0.25 pM) at 37 °C for 48 h. The cells were removed from the cell culture plates using enzyme-free cell dissociation buffer and analyzed by FACS.

[0213] For FACS analysis, the cells were stained with a dead / live far-red cell tracker (Invitrogen). Immediately before FACS analysis, 5 × 10 5 counting beads were added to each well. 1 × 10 4 beads were collected for each sample. To assess the specificity of killing, the cells were gated on the live Violet-labeled population. The percentage of the live population was recorded and used to calculate the normalized survival rate.

[0214] T cell activation was evaluated by incubating cells with a direct conjugate antibody to CD2 and CD69 and reporting the percentage of activated (CD69+) T cells among total T cells (CD2+).

[0215] As shown by the results in Tables 12A - 12C, depletion of TAA-expressing cells was observed with anti-PSMA, EGFRvIII or MUC16×CD3 bispecific antibodies. Most of the bispecific antibodies tested activated human T cells and induced depletion of target cells in the picomolar range. Furthermore, the observed target cell lysis (depletion) was related to upregulation of CD69 cells relative to CD2+ T cells and was in the picomolar (pM) EC 50 range. 50

[0216] Importantly, the results of this example demonstrate that several bispecific antibodies utilizing a CD3-binding arm (i.e., CD3-VH-G5) that shows weak to undetectable binding to the CD3 protein or CD3-expressing cells still retain the ability to activate T cells and show potent cytotoxicity against tumor antigen-expressing cells.

[0217] [Table 13] [Table 14]

[0218] Example 7: Anti-PSMA / anti-CD3 bispecific antibody shows potent antitumor effect in vivo To determine the in vivo efficacy of exemplary anti-PSMA / anti-CD3 bispecific antibodies that were identified as having weak binding affinity or no detectable binding affinity for human and cynomolgus monkey CD3, studies were conducted in immune-compromised mice bearing human prostate cancer xenografts. Additional studies were also conducted in immune-competent mice bearing mouse prostate cancer xenografts engineered to express human PSMA.​

[0219] Efficacy of Anti-PSMA / Anti-CD3 Bispecific Antibodies in a Human Tumor Xenograft Model To evaluate the in vivo efficacy of anti-PSMA / anti-CD3 bispecific antibodies in a human tumor xenograft assay, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Maine) were co-transplanted with 22Rv1 or C4-2 human prostate tumor cells that endogenously express PSMA and human peripheral blood mononuclear cells (PBMC).

[0220] Briefly, 4×10 6 of 22Rv1 or 5×10 6 of C4-2 cells (MD Anderson, TX) were co-transplanted s.c. into the right flank of male NSG mice with 1×10 of human PBMC (ReachBio, LLC., Seattle, WA) in a 50:50 mix of Matrigel matrix (BD 6 Biosciences). In the C4-2 study, mice were treated i.p. with 0.1 mg / kg of BSPSMA / CD3-003 or BSPSMA / CD3-005 on days 0, 4, and 7 post-tumor implantation.

[0221] In an additional xenograft model, anti-PSMA / anti-CD3 bispecific antibodies were tested in mice engrafted with human hematopoietic CD34+ stem cells. Briefly, neonatal SIR Pα BALB / c-Rag2-IL2rγ- (BRG) were engrafted with hCD34+ fetal liver cells. Three to six months later, SIRPα BRG mice engrafted with hCD34 were transplanted with C4-2 cells (5×10 6 s.c. in Matrigel). Eight days later, mice were treated with 10 μg of BSPSMA / CD3-004 or an isotype control antibody, and subsequent twice-weekly dosing was performed throughout the study.

[0222] In all tests, tumor size was measured twice a week using calipers, and tumor volume was calculated as volume = (length × width 2 ) / 2.

[0223] As shown by the results in Table 13, all of the bispecific antibodies tested in the above xenograft model were effective in suppressing tumor growth compared to treatment with isotype controls.

[0224]

Table 15

[0225] Efficacy of anti-PSMA / anti-CD3 bispecific antibody in immunocompetent tumor model Furthermore, the anti-PSMA / anti-CD3 bispecific antibody was evaluated for antitumor activity in an immunocompetent model (U.S. Provisional Patent Application No. 62 / 083,653, filed November 24, 2014). Mice humanized for the three chains (δγε) of CD3 were also humanized for PSMA and transplanted with the mutant mouse prostate cancer cell line TRAMP-C2 transfected with human PSMA.

[0226] Prior to the start of the test, the tumorigenic cell line mutant TRAMP-C2_hPSMAv#1 was generated. Briefly, 7.5 × 10 6 TRAMP-C2_hPSMA cells were transplanted s.c. into the right flank of male mice humanized for CD3 and PSMA. The tumors were excised, cut into 3 mm fragments, and then transplanted into the right flank of new male humanized mice. Then, the tumors arising from the transplanted tumor fragments were harvested and dispersed into a single cell suspension. These cells (TRAMP-C2_hPSMAv#1) were then cultured in vitro under G418 selection. Then, for the efficacy test of the bispecific antibody, 4 × 10 6 cells of this mutant cell line were transplanted into the right flank of male PSMA / CD3 humanized mice.

[0227] Humanized PSMA / CD3 mice engrafted with TRAMPC2_hPSMAv#1 were treated twice a week starting on the day of tumor engraftment with 100 μg or 10 μg of the anti-PSMA / anti-CD3 bispecific antibody BSPSMA / CD3-004 or isotype controls. Serum cytokine levels 4 hours after injection were also tested, along with splenic T cell levels. The study was terminated on day 27.

[0228] As shown by the results in Table 14, the tested anti-PSMA / anti-CD3 bispecific molecule, BSPSMA / CD3-004, demonstrated efficacy in a significant delay of tumor growth across treatment groups. Minimal cytokine release was observed following administration of BSPSMA / CD3-004, presumably due to weak binding of anti-CD3. Both antibodies tested demonstrated anti-tumor effects without depleting T cells within the spleen.

[0229]

Table 16

[0230] In summary, the anti-PSMA / anti-CD3 bispecific antibodies of the invention demonstrate potent anti-tumor effects in both immunocompromised and immunocompetent tumor models, despite having low to undetectable binding to the CD3 antigen.

[0231] Example 8: Anti-MUC16 / anti-CD3 bispecific antibodies demonstrate potent anti-tumor effects in vivo To determine the in vivo efficacy of exemplary anti-MUC16 / anti-CD3 bispecific antibodies that were identified as having weak binding affinity or no detectable binding affinity for human and cynomolgus CD3, studies were conducted in immunocompromised mice bearing human prostate cancer xenografts. The efficacy of the selected bispecific antibodies was tested in both immediate treatment and therapeutic treatment dosing models.

[0232] Efficacy of anti-MUC16 / anti-CD3 bispecific antibodies in human tumor xenograft models To evaluate the in vivo efficacy of an anti-MUC16 / anti-CD3 bispecific antibody in a human tumor xenograft assay, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Maine) were pre-transplanted with human peripheral blood mononuclear cells (PBMC; ReachBio LLC., Seattle, WA), and then ascites cells from the luciferase-transduced human ovarian cancer cell line OVCAR-3 (American Type Tissue Culture, Manassas, VA) (OVCAR-3 / Luc) were given. OVCAR-3 cells endogenously express MUC-16.

[0233] Briefly, NSG mice were injected intraperitoneally (i.p.) with 5.0 × 10 6 human PBMC. Eight days later, 1.5 × 10 6 ascites cells from the OVCAR-3 / Luc cell line pre-passaged in vivo were administered i.p. to NSG mice engrafted with PBMC. In the immediate treatment group, on the day of OVCAR-3 / Luc cell transplantation, the mice were treated i.p. with the MUC16 / CD3 bispecific antibodies BSMUC16 / CD3-001 or BSMUC16 / CD3-005, or an isotype control, at a dose of 10 μg / mouse (N = 5 mice / treatment group). In the therapeutic dosing model, seven days after tumor transplantation, the mice were treated i.p. with the above-described MUC16 / CD3 bispecific or control antibodies at a dose of 10 μg / mouse (N = 5 / treatment group).

[0234] In all studies, tumor growth was monitored by bioluminescence imaging (BLI). The mice were injected i.p. with the luciferase substrate D-luciferin (150 mg / kg) suspended in PBS, and imaged 10 minutes later under isoflurane anesthesia. BLI was performed using a Xenogen IVIS system (Perkin Elmer, Hopkinton, MA), and the BLI signal was extracted using Living Image software (Xenogen / Perkin Elmer). Regions of interest around each cell population were delineated, and the photon intensity was measured in photons (p) / second (s) / cm 2Recorded as steradians (sr). In the immediate treatment group, the data are shown as the BLI level 26 days after tumor transplantation (Table 15). In the treatment group, the data are shown as the fold change in BLI between day 6 (1 day before treatment) and the end of the study (26 days after tumor transplantation; Table 16).

[0235] As the results show, when BLI was measured on day 26 in the immediate dosing model, both BSMUC16 / CD3-001 and BSMUC16 / CD3-005 showed similar efficacy in suppressing tumor growth compared to the isotype control. Also, both anti-MUC16 / anti-CD3 bispecific antibodies suppressed the growth of established tumors when administered 7 days after tumor transplantation compared to the control. In summary, the bispecific anti-MUC16 / anti-CD3 antibodies of the present invention show a potent anti-tumor effect in several models.

[0236]

Table 17

[0237]

Table 18

[0238] Example 9: Pharmacokinetic Evaluation of Anti-MUC16×CD3 Bispecific Antibodies The pharmacokinetics of the anti-MUC16×CD3 bispecific antibodies BSMUC16 / CD3-001 and BSMUC16 / CD3-005, and the isotype control were evaluated in humanized MUC16×CD3 mice (homozygous mice for human MUC16 and CD3 expression, MUC16 hu / hu ×CD3 hu / hu ), CD3 humanized mice (homozygous mice for human CD3 expression, CD3 hu / hu) And were performed in congenic (75% C57BL, 25% 129Sv) wild-type (WT) mice. Each cohort contained 4-5 mice per test antibody and per mouse strain. All mice received a single intraperitoneal (i.p.) dose of 0.4 mg / kg. Blood samples were collected 3 and 6 hours, 1, 3, 7, 14, and 28 days after dosing. Blood was processed into serum and frozen at -80 °C until analysis.

[0239] Circulating antibody concentrations were determined by total human IgG antibody analysis using GyroLab xPlore™ (Gyros, Uppsala, Sweden). Briefly, to capture human IgG present in serum, biotinylated goat anti-human IgG polyclonal -nal antibody (Jackson ImmunoResearch, West Grove, PA) was captured on streptavidin-coated beads on a Gyrolab Bioaffy 200 CD (Gyros). After affinity column capture, bound human IgG antibodies in the sample were detected with Alexa-647-labeled goat anti-human IgG (Jackson ImmunoResearch). The fluorescence signal on the column enabled detection of bound IgG, and the instrument read out the reaction units (RU). Sample concentrations were determined by interpolation from a standard curve fitted using 5-parameter logarithmic curve fitting with the Gyrolab Evaluator Software.

[0240] PK parameters were determined by non-compartmental analysis (NCA) using Phoenix® WinNonlin® software version 6.3 (Certara, L.P., Princeton, NJ) and an extravascular dosing model. For each antibody, using each mean concentration value, all PK parameters including the maximum concentration observed in serum (C max ), the observed estimated half-life (t 1 / 2 ), and the area under the concentration curve (AUC last ) to the last measurable concentration were determined using linear interpolation and the linear trapezoidal rule with uniform weighting.

[0241] After i.p. administration in WT mice, the total IgG concentration-time profiles of BSMUC16 / CD3-001, BSMUC16 / CD3-005 and the isotype control were all similar, characterized by an initial short drug distribution phase followed by a single drug elimination phase throughout the remainder of the study. The maximum serum concentrations (C max ) and calculated drug exposures (AUC last ) of the three antibodies were equivalent (within 1.3-fold of each other). CD3 hu / hu After i.p. administration of the antibodies in CD3 max mice, BSMUC16 / CD3-001, BSMUC16 / CD3-005 and the isotype control had equivalent C max concentrations (4.6, 3.6 and 4.1 μg / mL, respectively). BSMUC16 / CD3-005 and the isotype control showed similar drug elimination curves, while BSMUC16 / CD3-001 showed more rapid drug elimination than both, suggesting that human CD3 target binding drives clearance. The final antibody concentration of BSMUC16 / CD3-001 was 0.03 μg / mL, which is approximately 1 / 28 of the final antibody concentration (0.85 μg / mL) determined for the isotype control and 1 / 22 of the BSMUC16 / CD3-005 (0.66 μg / mL) serum concentration.

[0242] MUC16 hu / hu ×CD3 hu / hu In Muc16×CD3 dual humanized mice, the Muc16×CD3 bispecific and isotype control antibodies had equivalent C max concentrations (C max range: 4.5 - 6.9 μg / mL). Both bispecific antibodies showed more rapid drug elimination than the isotype control, suggesting a target-mediated effect. The final antibody concentrations for BSMUC16 / CD3-001 and BSMUC16 / CD3-005 were approximately 1 / 29 and 1 / 2.9 of the final antibody concentration (0.86 μg / mL) determined for the isotype control, respectively.

[0243] Summary of data on total anti-MUC16×CD3 bispecific antibody and isotype control antibody concentrations are summarized in Table 17. Mean PK parameters are described in Tables 18A and 18B. Mean total antibody concentrations over time are shown in Figures 2A, 2B, and 2C. As a conclusion, the MUC16×CD3 bispecific antibody showed a similar C max and drug elimination curve in WT mice, but BSMUC16 / CD3-001 showed a steeper elimination rate than BSMUC16 / CD3-005 and the isotype control in CD3 single humanized mice and MUC16 / CD3 double humanized mice. Since the bispecific antibodies administered in this PK study were composed of the same anti-MUC16 binding arm, the results suggest that the binding strength of the CD3 targeting arm may play a role in drug exposure levels (AUC last ) and drug elimination rate. Neither BSMUC16 / CD3-001 nor BSMUC16 / CD3-005 binds to mouse MUC16 or mouse CD3.

[0244]

Table 19

[0245]

Table 20

[0246] Example 10: Anti-STEAP2 / anti-CD3 bispecific antibody shows potent antitumor effect in vivo To determine the in vivo efficacy of exemplary anti-STEAP2 / anti-CD3 bispecific antibodies that have been identified as having weak binding affinity or no detectable binding affinity for human and cynomolgus monkey CD3, studies were conducted in immunocompromised mice bearing human prostate cancer xenografts.

[0247] To evaluate the in vivo efficacy of an anti-STEAP2 / anti-CD3 bispecific antibody in human tumor xenograft assays, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Maine) were co-transplanted with human prostate cancer C4-2 cells (MD Anderson Cancer Center, Houston, TX) that endogenously express STEAP2, along with human peripheral blood mononuclear cells (PBMC; ReachBio LLC., Seattle, WA).

[0248] Briefly, 5.0×10 6 C4-2 cells were co-transplanted subcutaneously (s.c.) into the right flank of male NSG mice, along with 1.25×10 6 human PBMC in a 50:50 mix of Matrigel matrix (BD Biosciences, San Jose, CA). On the day of transplantation (immediate treatment model), the mice were treated intraperitoneally (i.p.) with the anti-STEAP2 / anti-CD3 bispecific antibody BSSTEAP2 / CD3-001, BSSTEAP2 / CD3-002 or BSSTEAP2 / CD3-003, or an isotype control (which does not bind to C4-2 tumor cells) at a dose of 0.1 or 0.01 mg / kg (N = 5 mice / group).

[0249] Tumor size was measured twice / week using calipers, and tumor volume was calculated as volume = (length × width 2 ) / 2. Data are shown as tumor size (mm 3 ) at the end point of the study, 46 days after tumor implantation (Table 19).

[0250] As shown by the results in Table 19, BSSTEAP2 / CD3-001, BSSTEAP2 / CD3-002 and BSSTEAP2 / CD3-003 significantly inhibited tumor growth compared to the isotype control when tumor size was measured at the end point of the study. Importantly, the anti-STEAP2 / anti-CD3 bispecific antibody was effective in inhibiting C4-2 tumor growth even at the lowest dose of 0.1 mg / kg.

[0251]

Table 21

[0252] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising a cytotoxic bispecific antibody for use in a method of treating cancer in a subject, wherein the bispecific antibody comprises a first antigen-binding arm that exhibits weak to undetectable binding to human CD3 and cynomolgus CD3 and a second antigen-binding arm that binds to a tumor-associated antigen, the first antigen-binding arm comprising a heavy chain and a light chain comprising a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 138, the light chain being common to both the first antigen-binding arm and the second antigen-binding arm, and the light chain being a cognate light chain of the second antigen-binding arm that binds to the tumor-associated antigen.

2. The pharmaceutical composition according to claim 1, wherein the bispecific antibody exhibits T cell activation in vitro.

3. The pharmaceutical composition according to claim 1 or 2, wherein the tumor-associated antigen is expressed on human tumor cells.

4. The bispecific antibody according to any one of claims 1 to 3, which induces T cell-mediated tumor cell killing at an EC 50 value of less than 1.3 nM as measured in an in vitro T cell-mediated tumor cell killing assay.

5. The tumor-associated antigen is AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE protein, GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE protein, MART-1, mesothelin, ML-IAP, Mu The pharmaceutical composition according to any one of claims 1 to 4, which is selected from the group consisting of c1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

6. The pharmaceutical composition according to claim 5, wherein the tumor-associated antigen is EGFRvIII.

7. The pharmaceutical composition according to claim 5, wherein the tumor-associated antigen is PSMA.

8. The pharmaceutical composition according to claim 5, wherein the tumor-associated antigen is MUC16.

9. The pharmaceutical composition according to claim 5, wherein the tumor-associated antigen is STEAP2.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the bispecific antibody comprises an HCVR comprising the amino acid sequence of SEQ ID NO:

34.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the bispecific antibody comprises an HCVR comprising the amino acid sequence of SEQ ID NO:

138.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the cancer is selected from the group consisting of pancreatic cancer, melanoma, glioblastoma, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer, malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma glioma, breast cancer, squamous cell carcinoma, esophageal cancer, clear cell renal cell carcinoma, chromophobic renal carcinoma, renal oncocytoma, renal transitional cell carcinoma, urothelial carcinoma, adenocarcinoma, small cell carcinoma or primary or metastatic tumors of the immune system.

13. The pharmaceutical composition according to any one of claims 6 to 11, wherein the cancer is glioblastoma, prostate cancer, or ovarian cancer.

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