Humanized or chimeric CD3 antibodies

Abstract

The present invention provides a humanized or chimeric antibody that binds to CD3, and uses thereof. The present invention provides a humanized or chimeric antibody, a bispecific antibody, that binds to CD3, comprising an Fc region modified to mediate Fc-mediated T cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody. The present invention also provides compositions, pharmaceutical compositions, uses of the antibody in the treatment of diseases, and methods of treatment that include the antibody.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to humanized or chimeric antibodies that bind to human CD3, compositions comprising the humanized or chimeric antibodies, and the use of the humanized or chimeric antibodies in the treatment of disease. [Background technology]

[0002] background Cluster of Differentiation 3 (CD3) has been known for a long time and has therefore been the subject of much interest. Specifically, antibodies raised against CD3 or against the T cell receptor complex containing CD3 are known. In vitro characterization of five humanized OKT3 effector function mutant antibodies has been described [1].

[0003] Treatment with the anti-CD3 monoclonal antibody hOKT3 gamma 1 (Ala-Ala) results in improved C-peptide responses and clinical parameters for at least two years after the onset of type 1 diabetes, in the absence of ongoing immunosuppressive medications [2].

[0004] A promising approach to improving targeted antibody therapy relies on the delivery of cytotoxic cells to antigen-expressing cancer cells. This concept of using T cells to efficiently kill tumor cells has been previously described [3]. However, early clinical studies were rather disappointing, primarily due to low efficacy, severe adverse effects (cytokine storm), and immunogenicity of bispecific antibodies [4]. Advances in the design and application of bispecific antibodies have partially overcome the initial obstacle of cytokine storm, improving clinical efficacy and eliminating dose-limiting toxicity [5].

[0005] For example, certain bispecific antibodies, which target an antigen on tumor cells with one arm and, for example, CD3 on T cells with the other, provide Fc receptor binding via the Fc region. Upon binding, complexes are formed between T cells, tumor cells, and effector cells that bind to the antibody's Fc region, leading to tumor cell killing. [4] Catumaxomab, a mouse IgG2a / rat IgG2b heterodimer, has been successfully administered intraperitoneally to treat cancer-associated ascites. [6] However, the mouse / rat hybrid is immunogenic. [7] Therefore, it cannot be used for long-term intravenous treatment in humans. Frequent treatment-related adverse events attributable to catumaxomab include cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) [8]-[9], which are related to the effector function of catumaxomab's Fc region. Another antibody, ertumaxomab (HER2×CD3), induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in phase II clinical development for metastatic breast cancer

[10] -

[11] .

[0006] Although CD3 antibodies that cross-react with cynomolgus and / or rhesus CD3 have been described

[12] -

[13] , such cross-reactive antibodies require further improvement. Summary of the Invention

[0007] It is an object of the present invention to provide humanized or chimeric CD3 antibodies. Accordingly, in one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, the antibody comprising a binding region comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NO:4, the sequence GTN, and the sequence shown in SEQ ID NO:5, respectively.

[0008] In another aspect, the present invention relates to a bispecific antibody comprising a first binding region of an antibody of the present invention and a second binding region that binds to a target different from the first antigen-binding region.

[0009] In another aspect, the present invention relates to nucleic acid constructs encoding one or more amino acid sequences of the present invention.

[0010] In another aspect, the present invention relates to an expression vector comprising (i) a nucleic acid sequence encoding the heavy chain sequence of a humanized or chimeric antibody of the present invention, (ii) a nucleic acid sequence encoding the light chain sequence of a humanized or chimeric antibody of the present invention, or (iii) both (i) and (ii).

[0011] In another aspect, the present invention relates to a host cell comprising the expression vector of the present invention.

[0012] In another aspect, the present invention relates to a composition comprising the antibody or bispecific antibody of the present invention.

[0013] In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0014] In another aspect, the present invention relates to the antibody or bispecific antibody, composition, or pharmaceutical composition of the present invention for use as a medicament.

[0015] In another aspect, the present invention relates to the antibody or bispecific antibody, composition, or pharmaceutical composition of the present invention for use in treating a disease.

[0016] In another aspect, the present invention relates to a method for treating a disease, comprising the step of administering to a subject in need thereof the antibody or bispecific antibody, composition, or pharmaceutical composition of the present invention.

[0017] In one aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, the method comprising the step of administering to a subject a humanized or chimeric antibody of the present invention, a composition of the present invention, or a pharmaceutical composition of the present invention, which may be labeled with a detectable agent.

[0018] In another aspect, the present invention relates to a method for producing an antibody or bispecific antibody of the present invention, comprising the steps of a) culturing a host cell of the present invention, and b) purifying the antibody from the culture medium.

[0019] In another aspect, the present invention relates to a diagnostic composition comprising the antibody or bispecific antibody of the present invention.

[0020] In another aspect, the present invention relates to a method for detecting the presence of CD3 antigen or the presence of cells expressing CD3 in a sample, the method comprising the steps of a) contacting the sample with an antibody or bispecific antibody of the invention under conditions allowing the formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

[0021] In another aspect, the present invention relates to a kit for detecting the presence of CD3 antigen or the presence of cells expressing CD3 in a sample, comprising i) an antibody or bispecific antibody of the invention and ii) instructions for use of the kit.

[0022] In another aspect, the present invention relates to an anti-idiotype antibody that binds to an antibody of the present invention. [The present invention 1001] A humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:4, sequence GTN, and sequence shown in SEQ ID NO:5, respectively. [The present invention 1002] The VH region is (a) the VH sequence shown in SEQ ID NO:6; (b) the VH sequence shown in SEQ ID NO:8; (c) the VH sequence shown in SEQ ID NO:7, and (d) VH sequence shown in SEQ ID NO:9 1001. The antibody of the present invention, having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in a VH sequence selected from the group consisting of: [The present invention 1003] The VL region is (a) the VL sequence shown in SEQ ID NO: 10; (b) the VL sequence shown in SEQ ID NO:11, and (c) VL sequence shown in SEQ ID NO: 12 Any of the antibodies of the present invention, having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in a VL sequence selected from the group consisting of: [The present invention 1004] The VH region is (a) the VH sequence shown in SEQ ID NO:6; (b) the VH sequence shown in SEQ ID NO:8; (c) the VH sequence shown in SEQ ID NO:7, and (d) VH sequence shown in SEQ ID NO:9 Any of the antibodies of the present invention, selected from the group consisting of: [The present invention 1005] The VL region is (a) the VL sequence shown in SEQ ID NO: 10; (b) the VL sequence shown in SEQ ID NO:11, and (c) VL sequence shown in SEQ ID NO: 12 Any of the antibodies of the present invention, selected from the group consisting of: [The present invention 1006] The VH region and the VL region are (a) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; (b) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; (c) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10; (d) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:11; (e) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12; (f) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:10; (g) the VH sequence shown in SEQ ID NO: 7 and the VL sequence shown in SEQ ID NO: 11; (h) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:12; (i) a VH sequence shown in SEQ ID NO:8 and a VL sequence shown in SEQ ID NO:11; (j) a VH sequence shown in SEQ ID NO: 8 and a VL sequence shown in SEQ ID NO: 12; (k) the VH sequence shown in SEQ ID NO: 9 and the VL sequence shown in SEQ ID NO: 11; and (l) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12 Any of the antibodies of the present invention, selected from the group consisting of: [The present invention 1007] The binding region is (a) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; (b) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and (c) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10 Any of the antibodies of the present invention, comprising a VH sequence and a VL sequence selected from the group consisting of: [The present invention 1008] Any of the antibodies of the present invention, which is a humanized antibody. [The present invention 1009] The antibody of the present invention, which is a chimeric antibody. [The present invention 1010] Any of the antibodies of the present invention, which is a full-length antibody. [The present invention 1011] Any of the aforementioned antibodies of the invention, comprising an Fc region comprising a first and a second immunoglobulin heavy chain. [The present invention 1012] Any of the aforementioned antibodies of the present invention, wherein the first and second heavy chains are of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [The present invention 1013] comprising an Fc region modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type antibody; C1q binding determined by ELISA, Any of the antibodies of the present invention. [The present invention 1014] the antibody comprises an Fc region modified such that the antibody mediates Fc-mediated T cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody; The T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay. Any of the antibodies of the present invention. [The present invention 1015] the antibody comprises an Fc region that has been modified to reduce Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% when compared to a wild-type antibody; Fc-mediated CD69 expression is determined in a PBMC-based functional assay. Any of the antibodies of the present invention. [The present invention 1016] comprising a first and a second immunoglobulin heavy chain; in at least one of said first and said second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively; Any of the antibodies of the present invention. [The present invention 1017] 1016. The antibody of the invention, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D. [The present invention 1018] 1016. The antibody of the invention, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N. [The present invention 1019] 1016. The antibody of the invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively. [The present invention 1020] An antibody of any of the inventions 1016 and 1019, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively. [The present invention 1021] The antibody of the invention 1020, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively. [The present invention 1022] The antibody of the invention 1020, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively. [The present invention 1023] The antibody of any of claims 1001 to 1016, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively. [The present invention 1024] An antibody of the present invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively. [The present invention 1025] 1024. The antibody of the invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively. [The present invention 1026] The antibody of the present invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are A, A, and A, respectively. [The present invention 1027] 1016. An antibody of the invention, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively. [The present invention 1028] A bispecific antibody comprising a first binding domain of the antibody of any one of claims 1001 to 1012 and a second binding domain that binds to a target different from the first antigen-binding domain. [The present invention 1029] A bispecific antibody of the present invention 1028, comprising a first and a second heavy chain. [The present invention 1030] (a) the bispecific antibody comprises an Fc region modified according to any one of claims 1013 to 1015 of the present invention; or (b) at least one of the first and second heavy chains comprises one or more amino acids modified according to any one of claims 1016 to 1027 of the present invention; The bispecific antibody of the present invention. [The present invention 1031] A bispecific antibody according to any of claims 1028 to 1030, wherein the first and second heavy chains each comprise at least a hinge region, CH2 and CH3 regions, and wherein the first heavy chain has at least one amino acid substitution at a position corresponding to a position in a human IgG1 heavy chain selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409, and the second heavy chain has at least one amino acid substitution at a position corresponding to a position in a human IgG1 heavy chain selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409, and wherein the first and second heavy chains do not have substitutions at the same positions. [The present invention 1032] 1031. A bispecific antibody of the invention, wherein the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain, or vice versa. [The present invention 1033] The bispecific antibody of any of claims 1028 to 1032, wherein the first binding region is according to any of claims 1001 to 1007, and the second binding region binds to a target different from said first binding region. [The present invention 1034] A nucleic acid construct encoding one or more of the amino acid sequences set forth in Table 1. [This invention 1035] (i) a nucleic acid sequence encoding the heavy chain sequence of any one of the humanized or chimeric antibodies 1001 to 1033 of the present invention; (ii) a nucleic acid sequence encoding the light chain sequence of any one of the humanized or chimeric antibodies 1001 to 1033 of the present invention, or (iii) Both (i) and (ii) An expression vector comprising: [The present invention 1036] A host cell comprising an expression vector of the present invention. [This invention 1037] The host cell of the present invention 1036, which is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell. [The present invention 1038] A composition comprising any one of the antibodies of the present invention 1001 to 1027 or any one of the bispecific antibodies of the present invention 1028 to 1033. [This invention 1039] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1027 or any one of the bispecific antibodies of the present inventions 1028 to 1033, and a pharmaceutically acceptable carrier. [The present invention 1040] The antibody of any one of 1001 to 1027, the bispecific antibody of any one of 1028 to 1033, the composition of 1038, or the pharmaceutical composition of 1039, for use as a pharmaceutical. [The present invention 1041] The antibody of any of 1001 to 1027, the bispecific antibody of any of 1028 to 1033, the composition of 1038, or the pharmaceutical composition of 1039, for use in treating a disease. [The present invention 1042] A method for treating a disease, comprising the step of administering any one of the antibodies of the present inventions 1001 to 1027, any one of the bispecific antibodies of the present inventions 1028 to 1033, the composition of the present invention 1038, or the pharmaceutical composition of the present invention 1039 to a subject in need thereof. [This invention 1043] The use or method of any of claims 1040 to 1042, wherein the disease is cancer, an infectious disease, or an autoimmune disease. [This invention 1044] A method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising the step of administering to a subject any of the antibodies of the present inventions 1001 to 1027, any of the bispecific antibodies of the present inventions 1028 to 1033, the composition of the present invention 1038, or the pharmaceutical composition of the present invention 1039, optionally wherein the antibody or the bispecific antibody is labeled with a detectable agent. [This invention 1045] (a) culturing a host cell of any one of 1036 to 1037 of the present invention; and (b) Purifying the antibody from the culture medium A method for producing any one of the antibodies of the present inventions 1001 to 1027 or any one of the bispecific antibodies of the present inventions 1028 to 1033, comprising: [The present invention 1046] A diagnostic composition comprising any one of the antibodies of the present invention 1001 to 1027 or any one of the bispecific antibodies of the present invention 1028 to 1033. [This invention 1047] (a) contacting a sample with any one of the antibodies of the present invention 1001 to 1027 or any one of the bispecific antibodies of the present invention 1028 to 1033 under conditions that allow the formation of a complex between the antibody or bispecific antibody and CD3; and (b) analyzing whether a complex is formed; A method for detecting the presence of CD3 antigen or cells expressing CD3 in a sample, comprising: (i) any one of the antibodies 1001 to 1027 of the present invention or any one of the bispecific antibodies 1028 to 1033 of the present invention, and (ii) Instructions for use of the kit 1. A kit for detecting the presence of CD3 antigen or the presence of cells expressing CD3 in a sample, comprising: [This invention 1049] An anti-idiotype antibody that binds to any one of antibodies 1001 to 1027 of the present invention. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1A shows the binding curves of the monospecific antibody variant IgG1-huCD3 and (Figure 1B) the bispecific antibody variant bsIgG1 huCD3xHER2 to the human T cell line Jurkat. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as described in Example 2. The table shows the antibody concentrations (μg / mL) that resulted in half-maximal binding (EC50). [Figure 2]Figure 2A shows the binding curves of the monospecific antibody variant IgG1-huCD3 and (Figure 2B) the bispecific antibody variant bsIgG1 huCD3xHER2 to the cynomolgus monkey T cell line HSC-F. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as described in Example 2. [Figure 3] T cell activation by IgG1-huCD3 antibody variants. CD69 expression on T cells from humans (FIG. 3A) and cynomolgus monkeys (FIG. 3B) in PBMC cultures was measured by FACS analysis as described in Example 3. These experiments were performed in duplicate. Representative results from one experiment are shown. [Figure 4] IgG1-huCD3 antibody variants induce T cell proliferation. Human (FIG. 4A) or cynomolgus monkey (FIG. 4B) PBMCs were incubated with IgG1-huCD3 antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 5] Induction of human (FIG. 5A) and cynomolgus monkey (FIG. 5B) T cell-mediated cytotoxicity by huCD3 antibody variants carrying a non-activating LFLEDA mutation was determined as described in Example 5. Representative results from two independent experiments performed in duplicate are shown. [Figure 6A] Figure 1 shows the binding curves of non-activating monospecific antibody variants of IgG1-huCD3 to the human T cell line Jurkat. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as described in Example 2. The table shows the antibody concentrations (μg / mL) that result in 50% maximal binding (EC50). [Figure 6B] Figure 1 shows the binding curve of the non-activating bispecific antibody variant bsIgG1-huCD3xHER2 of IgG1-huCD3 to the human T cell line Jurkat. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that resulted in 50% maximal binding (EC50). [Figure 7A]Figure 1 shows the binding curves of non-activating monospecific antibody variants of IgG1-huCD3 to the cynomolgus monkey T cell line HSC-F. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as described in Example 2. The table shows the antibody concentrations (μg / mL) that resulted in 50% maximal binding (EC50). [Figure 7B] Figure 1 shows the binding curve of the non-activating bispecific antibody variant bsIgG1-huCD3xHER2 of IgG1-huCD3 to the cynomolgus monkey T cell line HSC-F. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that resulted in 50% maximal binding (EC50). [Figure 8A] T cell activation with non-activating monospecific IgG1-huCD3. CD69 expression on human-derived T cells in PBMC cultures was measured by FACS analysis as described in Example 3. These experiments were performed in duplicate. Representative results from one experiment are shown. [Figure 8B] T cell activation with non-activating monospecific IgG1-huCD3. CD69 expression on T cells from cynomolgus monkeys in PBMC cultures was measured by FACS analysis as described in Example 3. These experiments were performed twice. Representative results from one experiment are shown. [Figure 8C] T cell activation with non-activating bispecific bsIgG1-huCD3xHER2 antibody variants. CD69 expression on human-derived T cells in PBMC cultures was measured by FACS analysis as described in Example 3. These experiments were performed in duplicate. Representative results from one experiment are shown. [Figure 8D] T cell activation with non-activating bispecific bsIgG1-huCD3xHER2 antibody variants. CD69 expression on T cells from cynomolgus monkeys in PBMC cultures was measured by FACS analysis as described in Example 3. These experiments were performed in duplicate. Representative results from one experiment are shown. [Figure 9A]T cell proliferation induced by non-activating monospecific IgG1-huCD3. T cell proliferation was measured in human PBMCs. These PBMCs were incubated with the various antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 9B] T cell proliferation induced by non-activating monospecific IgG1-huCD3. T cell proliferation was measured in cynomolgus monkey PBMCs. These PBMCs were incubated with various antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 9C] T cell proliferation induced by non-activating bispecific bsIgG1-huCD3xHER2 antibody variants. T cell proliferation was measured in human PBMCs. These PBMCs were incubated with the various antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 9D] Figure 1. T cell proliferation induced by non-activating bispecific bsIgG1-huCD3xHER2 antibody variants. T cell proliferation was measured in cynomolgus monkey PBMCs. These PBMCs were incubated with the various antibody variants for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 10] Induction of human (FIG. 10A) and cynomolgus monkey (FIG. 10B) T cell-mediated cytotoxicity by huCD3 antibody variants with a non-activating LFLEDA mutation was determined as described in Example 5. Representative results from two independent experiments performed in duplicate are shown. [Figure 11] Rhesus T cell activation by IgG1-huCD3 antibody variants. CD69 expression on T cells from rhesus macaques in PBMC cultures was measured by FACS analysis as described in Example 6. [Figure 12]T cell activation by non-activating mutants of the huCLB-T3 / 4 antibody. IgG1-huCLB-T3 / 4 mutants were titrated against PBMCs. CD69 expression on T cells in PBMC cultures was measured by FACS analysis as described in Example 7. A representative example of three experiments is shown. [Figure 13] T cell proliferation with non-activating mutants of the huCLB-T3 / 4 antibody. PBMCs were incubated with the antibodies for 3 days, after which proliferation was measured by cell proliferation ELISA as described in Example 8. Representative results from two independent experiments are shown. [Figure 14A] In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 14B] In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 14C] In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 14D] In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 14E]In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 14F] In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 14G] In vitro T cell-mediated cytotoxicity induced by non-activating antibody mutants of CD3 antibodies. The induction of T cell-mediated cytotoxicity by antibody mutants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figures 14A-G]) was determined as described in Example 9. Averages from two experiments performed in duplicate are shown. [Figure 15] In vitro T cell-mediated cytotoxicity induced by non-activating huCLB-T3 / 4 mutants. Induction of T cell-mediated cytotoxicity by antibody mutants (LFLEDA LAL [Figures 15A-C]) was determined as described in Example 9. Averages from one experiment performed in duplicate are shown. [Figure 16A] Assessment of C1q binding to non-activating huCLB-T3 / 4 antibody variants. C1q binding to monospecific IgG1 huCLB-T3 / 4 (FIGS. 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (FIGS. 16B-D) and their non-activating antibody variants was assessed by ELISA as described in Example 10. Results in the graphs are representative of n=2 experiments. [Figure 16B]Assessment of C1q binding to non-activating huCLB-T3 / 4 antibody variants. C1q binding to monospecific IgG1 huCLB-T3 / 4 (FIGS. 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (FIGS. 16B-D) and their non-activating antibody variants was assessed by ELISA as described in Example 10. Results in the graphs are representative of n=2 experiments. [Figure 16C] Assessment of C1q binding to non-activating huCLB-T3 / 4 antibody variants. C1q binding to monospecific IgG1 huCLB-T3 / 4 (FIGS. 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (FIGS. 16B-D) and their non-activating antibody variants was assessed by ELISA as described in Example 10. Results in the graphs are representative of n=2 experiments. [Figure 16D] Assessment of C1q binding to non-activating huCLB-T3 / 4 antibody variants. C1q binding to monospecific IgG1 huCLB-T3 / 4 (FIGS. 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (FIGS. 16B-D) and their non-activating antibody variants was assessed by ELISA as described in Example 10. Results in the graphs are representative of n=2 experiments. [Figure 17] Pharmacokinetic (PK) analysis of the inactive huCLB-T3 / 4 antibody mutant was compared with that of the wild-type IgG1-huCLB-T3 / 4 antibody, as described in Example 11. Plasma concentrations of human IgG1 were plotted against time (FIG. 17A). Plasma clearance rates were calculated as described in FIG. 11 (FIG. 17B). The horizontal dotted line represents the mean clearance rate (10 mL / day / kg) of human IgG1 antibodies in SCID mice. [Figure 18] Frequency of positive T cell responses in HLA-typed healthy donors. A SI index of ≥ 1.9 in both proliferation and IL-2 secretion assays was considered a positive response. Humanized A33 was used as a clinical benchmark control antibody, which has shown high levels of immunogenicity in clinical settings and consistently elicits 20–30% T cell responses in the EpiScreen assay. KLH responses were included to check the quality of PBMCs (after thawing). [Figure 19] Sequence alignment of the heavy chain (VH) and light chain (VL) of the humanized CD3 antibody of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 comprising the sequences shown in SEQ ID NO:4, the sequence GTN, and the sequence shown in SEQ ID NO:5, respectively.

[0025] As used herein, the term "antibody" refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either that has the ability to specifically bind to an antigen under typical physiological conditions for a significant half-life, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other functionally defined half-life (e.g., a time sufficient to elicit, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a time sufficient for the antibody to mobilize effector activity). The binding region that interacts with the antigen (sometimes referred to herein as a binding domain, both terms having the same meaning) comprises the variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant region of an antibody (Ab) may mediate the binding of the immunoglobulin to host tissues or host factors, such as various cells of the immune system (e.g., effector cells and T cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody, as used herein, includes fragments of antibodies that retain the ability to specifically interact with (e.g., bind to) an antigen, unless otherwise expressly stated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, V L , V H , C L and C H (ii) a monovalent fragment consisting of one domain, or a monovalent antibody as described in WO2007059782 (Genmab A / S); (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a V H Domain and C H(iv) an Fd fragment consisting essentially of one domain; and (iv) a V of a single arm of an antibody. L Domains and V H Furthermore, the two domains V of the Fv fragment are essentially the same. L and V H are encoded by separate genes, but using recombinant methods, they can be separated into V L Area and V H They may be joined by a synthetic linker that allows the domains to pair into a single protein chain forming a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv); see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by the context. While such fragments are generally included within the meaning of antibody, they, collectively and individually, are unique features of the present invention and exhibit distinct biological properties and utilities. These and other useful antibody fragments in the context of the present invention are discussed further herein. Unless otherwise specified, it should be understood that the term antibody also encompasses polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies, as well as antibody fragments (antigen-binding fragments) which retain the ability to specifically bind to antigens and which are obtained by any known method, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. The antibodies produced can be of any isotype.

[0026] As used herein, the terms "immunoglobulin heavy chain," "immunoglobulin heavy chain," or "heavy chain" refer to one of the immunoglobulin chains. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region may further include a hinge region. As used herein, the term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains: one pair of light (L) low-molecular-weight chains and one pair of heavy (H) chains, all four of which may be interconnected by disulfide bonds. The structure of immunoglobulins has been extensively characterized (see, e.g.,

[14] ). In the immunoglobulin structure, the two heavy chains are interconnected by disulfide bonds in the so-called "hinge region." Like heavy chains, each light chain typically consists of several regions: a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (i.e., regions where the sequence and / or the conformation of structurally defined loops may be hypervariable), also called complementarity-determining regions (CDRs), interspersed with highly conserved regions called framework regions (FRs). VH and VL each typically consist of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

[15] ). CDR sequences can be determined using methods provided by IMGT

[16] -

[17] .

[0027] As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, e.g., IgG1m(za) and IgG1m(f) [SEQ ID NO:15]), encoded by the heavy chain constant region genes. Thus, in one embodiment, the antibody comprises an immunoglobulin heavy chain of the IgG1 class or any allotype thereof. Furthermore, each heavy chain isotype can be combined with a kappa (κ) or lambda (λ) light chain.

[0028] As used herein, the term "chimeric antibody" refers to an antibody whose variable region originates from a non-human species (e.g., from a rodent) and whose constant region originates from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term broadly used to describe various types of antibody modification and is a process well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques, as described in

[18] . Chimeras can therefore be genetically or enzymatically engineered recombinant antibodies. Because the production of chimeric antibodies is known to those skilled in the art, chimeric antibodies of the present invention can also be produced by methods other than those described herein. Therapeutic chimeric monoclonal antibodies are developed to reduce antibody immunogenicity. They typically contain non-human (e.g., murine) variable regions specific for an antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" used in connection with chimeric antibodies refers to the region comprising the CDRs and framework regions of both the heavy and light chains of an immunoglobulin.

[0029] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, onto the homologous human acceptor framework regions (FRs) (see

[19] -

[20] ). To fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to substitute framework residues from the parent (i.e., non-human) into the human framework regions (backmutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily backmutations, may be applied to obtain a humanized antibody with desirable characteristics, such as affinity or biochemical properties.

[0030] A humanized or chimeric antibody according to any aspect or embodiment of the present invention may be referred to as a "humanized or chimeric CD3 antibody," "humanized or chimeric antibody of the invention," "CD3 antibody," or "CD3 antibody of the invention," all of which have the same meaning and purpose, except where the context indicates otherwise.

[0031] Because the amino acid sequences of antibodies of non-human origin differ from those of human origin, non-human antibodies are potentially immunogenic when administered to human patients. However, despite the antibody's non-human origin, its CDR segments are responsible for the antibody's ability to bind to its target antigen, and humanization aims to maintain the antibody's specificity and binding affinity. Thus, humanization of non-human therapeutic antibodies is performed to minimize its immunogenicity in humans while maintaining the specificity and binding affinity of the humanized antibody.

[0032] As used herein, the term "binding domain" refers to a region of an antibody that has the ability to bind to some molecule, such as a polypeptide present on a cell, bacterium, or virion.

[0033] As used herein, the term "binding" refers to the binding of an antibody to a predetermined antigen or target, where the binding to the antigen or target is typically about 10, as determined, for example, by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument, with the antigen as the ligand and the antibody as the analyte. -6 M or less, e.g. 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K below M D and is at least 10-fold, e.g., at least 100-fold, e.g., at least 1,000-fold, e.g., at least 10,000-fold, e.g., at least 100,000-fold, e.g., at least 100,000-fold lower than the affinity for binding to a non-specific antigen (e.g., BSA, casein) that is neither the predetermined antigen nor a closely related antigen. D The antibody binds to a predetermined antigen with an affinity equivalent to the K D Since it depends on the antibody's K D If K is very low (i.e., the antibody is highly specific), the affinity for the antigen can be 10,000 times lower or more than the affinity for a non-specific antigen. D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0034] As used herein, the term "human CD3" refers to the human cluster of differentiation 3 protein, which is part of the T cell coreceptor protein complex and is composed of four distinct chains. Because CD3 is also found in other species, the term "CD3" as used herein is not limited to human CD3, except where the context dictates otherwise. In mammals, this complex contains the CD3γ (gamma) chain (human CD3γ chain Swissprot P09693 or cynomolgus monkey CD3γ Swissprot Q95LI7), the CD3δ (delta) chain (human CD3δ Swissprot P04234 or cynomolgus monkey CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766, cynomolgus monkey CD3ε Swissprot Q95LI5, or rhesus monkey CD3ε Swissprot G7NCB9), and the CD3ζ (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0). These chains associate with a molecule known as the T cell receptor (TCR) to generate an activation signal in T lymphocytes. The TCR molecule and the CD3 molecule together make up the TCR complex.

[0035] Those skilled in the art know that amino acid sequences referred to as Swissprot numbers include signal peptides that are removed after translation of the protein. Thus, proteins such as CD3 present on the cell surface do not contain signal peptides. In particular, the amino acid sequences listed in Table 1 do not contain such signal peptides. Proteins such as those listed in Table 1 can be referred to as "mature proteins." Thus, SEQ ID NO:14 represents the amino acid sequence of mature human CD3δ (delta), SEQ ID NO:13 represents the amino acid sequence of mature human CD3ε (epsilon), SEQ ID NO:21 represents the amino acid sequence of mature cynomolgus monkey CD3ε, and SEQ ID NO:23 represents the amino acid sequence of mature rhesus monkey CD3ε. Thus, as used herein, the term "mature" refers to a protein that does not contain any signal or leader sequence.

[0036] It is well known that the homology, length, and cleavage site location of signal peptide sequences vary considerably among proteins. Signal peptides can be determined in different ways, for example, SEQ ID NO: 13 of the present invention was determined according to the SignalP application (available at http: / / www.cbs.dtu.dk / services / SignalP / ).

[0037] In certain specific embodiments, the humanized or chimeric antibodies of the invention bind to the epsilon chain of CD3, e.g., the epsilon chain of human CD3 (SEQ ID NO:13). In yet another specific embodiment, the humanized or chimeric antibodies bind to an epitope within amino acids 1-27 of the N-terminal portion of human CD3ε (epsilon) (SEQ ID NO:13). In certain such embodiments, the antibodies may further cross-react with other non-human primate species, such as cynomolgus monkeys (cynomolgus CD3 epsilon SEQ ID NO:21) and / or rhesus monkeys (rhesus CD3 epsilon SEQ ID NO:23).

[0038] As used herein, the term "cross-react" refers to the ability of an antibody, such as a humanized or chimeric antibody of the present invention, to bind to its target in a different species. Specifically, the humanized CD3 antibody exemplified in the Examples described herein has the ability to bind to human CD3 (Example 2), cynomolgus monkey CD3 (Example 2), and rhesus monkey CD3.

[0039] Antibodies of the present invention comprising CDR sequences as defined herein and further comprising framework regions may differ in sequence outside the CDR sequences, while still retaining full binding ability compared to the original antibody. Thus, the present invention also relates to antibodies comprising variable region amino acid sequences that have a certain sequence identity with any of the sequences described herein.

[0040] The term "sequence identity" as used in the context of the present invention refers to the percent identity between two sequences as a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap (i.e., % homology = 100 × number of identical positions / total number of positions). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller

[21] . In addition, the percent identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch

[22] . Multiple alignments are preferably performed using the Clustal W algorithm

[23] (e.g., as used in Vector NTI Advance® software version 11.5 (Invitrogen Inc.)).

[0041] Thus, in one embodiment, the VH region comprises: a) the VH sequence shown in SEQ ID NO:6; b) the VH sequence shown in SEQ ID NO:8; c) the VH sequence shown in SEQ ID NO:7; and d) VH sequence shown in SEQ ID NO:9 and (iii) a VH sequence selected from the group consisting of:

[0042] In one particular embodiment, the VH region comprises: a) the VH sequence shown in SEQ ID NO:6; b) the VH sequence shown in SEQ ID NO:8; c) the VH sequence shown in SEQ ID NO:7; and d) VH sequence shown in SEQ ID NO:9 and (iii) a VH sequence selected from the group consisting of:

[0043] In one embodiment, the VL region is a) the VL sequence shown in SEQ ID NO:10; b) the VL sequence shown in SEQ ID NO:11; and c) the VL sequence shown in SEQ ID NO: 12 and (iii) a VL sequence selected from the group consisting of:

[0044] In one particular embodiment, the VL region comprises: a) the VL sequence shown in SEQ ID NO:10; b) the VL sequence shown in SEQ ID NO:11; and c) the VL sequence shown in SEQ ID NO: 12 and (iii) a VL sequence selected from the group consisting of:

[0045] In one embodiment, the VH region comprises: a) the VH sequence shown in SEQ ID NO:6; b) the VH sequence shown in SEQ ID NO:8; c) the VH sequence shown in SEQ ID NO:7; and d) VH sequence shown in SEQ ID NO:9 is selected from the group consisting of:

[0046] In one embodiment, the VL region is a) the VL sequence shown in SEQ ID NO:10; b) the VL sequence shown in SEQ ID NO:11; and c) the VL sequence shown in SEQ ID NO: 12 is selected from the group consisting of:

[0047] In one embodiment, only one of the VH or VL sequences is 100% identical to one of the sequences disclosed herein, and the other has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to one of the sequences disclosed herein.

[0048] In one particular embodiment, the VH sequence is a) the VH sequence shown in SEQ ID NO:6; b) the VH sequence shown in SEQ ID NO:7; c) the VH sequence shown in SEQ ID NO:8; and d) VH sequence shown in SEQ ID NO:9 and the VL sequence has at least 97% amino acid sequence identity to at least one amino acid sequence set forth in the VH sequence selected from the group consisting of: i) the VL sequence shown in SEQ ID NO:10; ii) the VL sequence shown in SEQ ID NO:11; and iii) the VL sequence shown in SEQ ID NO: 12 and (iii) a VL sequence selected from the group consisting of:

[0049] In one embodiment, the VH and VL sequences are selected from the group consisting of the sequences shown below: a) VH and VL sequences having at least 90% identity to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; SEQ ID NOs: 7 and 10, respectively; SEQ ID NOs: 8 and 10, respectively; SEQ ID NOs: 9 and 10, respectively; SEQ ID NOs: 6 and 11, respectively; SEQ ID NOs: 7 and 11, respectively; SEQ ID NOs: 8 and 11, respectively; SEQ ID NOs: 9 and 11, respectively; SEQ ID NOs: 6 and 12, respectively; SEQ ID NOs: 7 and 12, respectively; SEQ ID NOs: 8 and 12, respectively; and VH and VL sequences having at least 90% identity to the sequences set forth in SEQ ID NOs: 9 and 12, respectively; b) VH and VL sequences having at least 95% identity to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; SEQ ID NOs: 7 and 10, respectively; SEQ ID NOs: 8 and 10, respectively; SEQ ID NOs: 9 and 10, respectively; SEQ ID NOs: 6 and 11, respectively; SEQ ID NOs: 7 and 11, respectively; SEQ ID NOs: 8 and 11, respectively; SEQ ID NOs: 9 and 11, respectively; SEQ ID NOs: 6 and 12, respectively; SEQ ID NOs: 7 and 12, respectively; SEQ ID NOs: 8 and 12, respectively; and VH and VL sequences having at least 95% identity to the sequences set forth in SEQ ID NOs: 9 and 12, respectively; c) SEQ ID NOs:6 and 10, respectively; SEQ ID NOs:7 and 10, respectively; SEQ ID NOs:8 and 10, respectively; SEQ ID NOs:9 and 10, respectively; SEQ ID NOs:6 and 11, respectively; SEQ ID NOs:7 and 11, respectively; SEQ ID NOs:8 and 11, respectively; SEQ ID NOs:9 and 11, respectively; SEQ ID NOs:6 and 12, respectively; SEQ ID NOs:7 and 12, respectively; SEQ ID NOs:8 and 12, respectively; and VH and VL sequences having at least 97% identity to the sequences set forth in SEQ ID NOs:9 and 12, respectively; d) SEQ ID NOs:6 and 10, respectively; SEQ ID NOs:7 and 10, respectively; SEQ ID NOs:8 and 10, respectively; SEQ ID NOs:9 and 10, respectively; SEQ ID NOs:6 and 11, respectively; SEQ ID NOs:7 and 11, respectively; SEQ ID NOs:8 and 11, respectively; SEQ ID NOs:9 and 11, respectively; SEQ ID NOs:6 and 12, respectively; SEQ ID NOs:7 and 12, respectively; SEQ ID NOs:8 and 12, respectively; and VH and VL sequences having at least 99% identity to the sequences set forth in SEQ ID NOs:9 and 12, respectively; e) VH and VL sequences having at least 100% identity to the sequences set forth in SEQ ID NOs: 6 and 10, respectively; SEQ ID NOs: 7 and 10, respectively; SEQ ID NOs: 8 and 10, respectively; SEQ ID NOs: 9 and 10, respectively; SEQ ID NOs: 6 and 11, respectively; SEQ ID NOs: 7 and 11, respectively; SEQ ID NOs: 8 and 11, respectively; SEQ ID NOs: 9 and 11, respectively; SEQ ID NOs: 6 and 12, respectively; SEQ ID NOs: 7 and 12, respectively; SEQ ID NOs: 8 and 12, respectively; and VH and VL sequences having at least 100% identity to the sequences set forth in SEQ ID NOs: 9 and 12, respectively; f) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; g) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; h) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; i) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; j) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; k) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 6 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; l) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; m) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 6 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; n) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 6 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; o) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; p) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 6 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; q) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; r) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; s) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 6 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; t) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; u) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; v) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; x) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; y) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 6 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; z) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO:6 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; aa) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ab) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:7 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; ac) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ad) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ae) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; af) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:7 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; ag) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ah) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:7 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; ai) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; aj) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO:7 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; a k) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; al) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; am) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; an) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ao) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; a) a VH sequence having at least 99% identity to the sequence shown in SEQ ID NO: 7 and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO: 10, 11, or 12; aq) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ar) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; as) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; at) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 7 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ba) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bb) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; bc) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; bd) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; be) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bf) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; bg) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; bh) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bi) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bj) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bk) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; bl) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bm) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bn) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; bo) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bp) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bq) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO:8 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; br) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bs) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; bt) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 8 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ca) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cb) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO:9 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; cc) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cd) a VH sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ce) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO: 9 and a VL sequence having at least 90% identity to the sequence shown in SEQ ID NO: 10, 11, or 12; cf) a VH sequence having at least 95% identity to the sequence shown in SEQ ID NO: 9 and a VL sequence having at least 97% identity to the sequence shown in SEQ ID NO: 10, 11, or 12; cg) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO:9 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12; ch) a VH sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ci) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cj) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; ck) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cl) a VH sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cm) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cn) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; co) a VH sequence having at least 99% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cp) a VH sequence having at least 99% identity to the sequence shown in SEQ ID NO: 9 and a VL sequence having at least 100% identity to the sequence shown in SEQ ID NO: 10, 11, or 12; cq) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cr) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 95% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; cs) a VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO: 9 and a VL sequence having at least 97% identity to the sequence set forth in SEQ ID NO: 10, 11, or 12; and ct) A VH sequence having at least 100% identity to the sequence set forth in SEQ ID NO:9 and a VL sequence having at least 99% identity to the sequence set forth in SEQ ID NO:10, 11, or 12.

[0050] In one embodiment, the binding region is a) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; b) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; c) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10; d) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:11; e) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12; f) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:10; g) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:11; h) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:12; i) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:11; j) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12; k) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:11; and l) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12 The VH and VL are selected from the group consisting of:

[0051] In certain embodiments, the binding region comprises: a) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; b) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; and c) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10 The VH and VL sequences are selected from the group consisting of:

[0052] The humanized antibodies of the present invention can be generated by comparing the heavy and light chain variable region amino acid sequences to a database of human germline variable region sequences to identify heavy and light chain human sequences with adequate homology for use as human variable framework regions. A series of humanized heavy and light chain variable regions can be designed, for example, by grafting mouse CDRs into the framework regions (identified as described above) and, if necessary, backmutating identified residues that may be critical for restoring antibody binding efficiency to their specific mouse sequences (mutating one or more of the human amino acid residues in the framework region back to the non-human amino acid at that specific position). Variant sequences with the lowest occurrence of potential T-cell epitopes, as determined by application of in silico techniques such as iTope™ and TCED™ (

[24] ,

[25] , and

[26] ), can then be selected.

[0053] Furthermore, the humanized antibodies of the present invention can also be "deimmunized." Deimmunization may be desirable because the presence of human T-cell epitopes within a protein sequence, such as the humanized antibodies of the present invention, may increase the immunogenicity risk profile due to their potential to activate helper T cells. Such activation of helper T cells can be avoided by deimmunization. Deimmunization can be performed by introducing mutations into the amino acid sequence of the humanized antibody to remove T-cell epitopes without significantly reducing the binding affinity of the antibody.

[0054] Thus, in one aspect of the invention, humanized antibodies can be produced by a method comprising the steps of: (i) comparing the entire non-human variable heavy and / or light chain sequence with a database of human germline sequences; (ii) obtaining a humanized sequence by selecting the human germline sequence with the highest homology to the non-human sequence; (iii) optimizing the humanized sequence by backmutation, if necessary; and (iv) expressing the sequence in a suitable expression system.

[0055] Thus, full-length antibodies of the present invention can be produced by a method comprising the steps of: (i) comparing the non-human variable heavy and light chain sequences with a database of human germline sequences; (ii) selecting the human germline sequence with the highest homology to the non-human sequence; (iii) obtaining humanized sequences by grafting non-human CDRs into the selected human germline sequences; (iv) optimizing the humanized sequences by backmutation, if necessary; (v) identifying constant heavy and light chain sequences; and (vi) expressing the complete heavy and light chain sequences in a suitable expression system. Thus, full-length antibodies of the present invention can be produced as described in Example 1. Producing full-length antibodies starting from either CDR sequences or entire variable region sequences is known to those skilled in the art. Thus, methods for making full-length antibodies of the present invention will be known to those skilled in the art.

[0056] As used herein, the term "complete heavy chain sequence" refers to a sequence consisting of a variable heavy chain sequence and a constant heavy chain sequence.

[0057] As used herein, the term "complete light chain sequence" refers to a sequence consisting of a variable light chain sequence and a constant light chain sequence.

[0058] Back mutations can be introduced by standard DNA mutagenesis methods. Standard techniques for such DNA mutagenesis are described in

[18] . Alternatively, the desired back mutations can be introduced using commercially available kits such as the Quickchange™ Site-Directed Mutagenesis Kit (Stratagene) or by de novo DNA synthesis.

[0059] Thus, in one embodiment the antibody is a humanized antibody.

[0060] Chimeric antibodies can be generated by replacing all of the constant region sequences of a non-human (e.g., murine) antibody with constant region sequences of human origin. Thus, the chimeric antibody retains its entire non-human variable region sequence. Thus, the chimeric antibodies of the present invention can be produced by a method comprising expressing a non-human variable heavy chain (SEQ ID NO:27), a non-human variable light chain sequence (SEQ ID NO:28), a human constant heavy chain sequence, and a human constant light chain sequence in a suitable expression system, thereby producing a full-length chimeric antibody. Alternative methods can also be used. Such methods for producing chimeric antibodies are known to those skilled in the art, and therefore, methods for producing the chimeric antibodies of the present invention will be apparent to those skilled in the art.

[0061] Thus, in one embodiment the antibody is a chimeric antibody.

[0062] In one embodiment, the antibody is a full-length antibody. As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent antibody or a variant antibody) that contains all of the heavy and light chain constant and variable domains that correspond to those normally found in a wild-type antibody of that isotype.

[0063] In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

[0064] As used herein, the term "Fc region" refers to a region comprising, from the N-terminus to the C-terminus, at least a hinge region, a CH2 region, and a CH3 region. The Fc region may further comprise a CH1 region N-terminal to the hinge region.

[0065] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to the Eu numbering system as set forth in Kabat.

[0066] Unless otherwise stated or contradicted by context, the amino acids of constant region sequences are numbered according to the Eu-index of numbering (described in

[27] ), and are referred to herein as "Eu numbering as described in Kabat," "Eu numbering of Kabat," or "in the Eu numbering system."

[0067] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 in the EU numbering system. However, the CH1 region may be of any of the other subtypes described herein.

[0068] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 in the EU numbering system. However, the CH2 region may be of any of the other subtypes described herein.

[0069] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 in the EU numbering system. However, the CH3 region may be of any of the other subtypes described herein.

[0070] In one embodiment, the immunoglobulin heavy chain isotype is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain can be of any allotype within each immunoglobulin class, such as IgG1m(f) (SEQ ID NO:15). Thus, in one particular embodiment, the immunoglobulin heavy chain isotype is IgG1 or any allotype thereof, for example, IgG1m(f) (SEQ ID NO:15).

[0071] When targeting the antigen CD3, which is part of the T cell receptor (TCR), a T cell-specific cell killing mechanism is desirable. Other effector functions, such as complement activation, may not be necessary, so reducing effector functions is desirable. C1q binding is the first step in the complement cascade, so it serves as an indicator of the antibody's complement-dependent cytotoxicity (CDC) ability. Avoiding C1q binding to antibodies also avoids activation of the complement cascade.

[0072] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% compared to a wild-type antibody, wherein C1q binding is determined by ELISA.

[0073] As used herein, the term "modified" refers to an Fc region whose amino acid sequence is not identical to that of a wild-type Fc region. That is, amino acid residues at specific positions in the wild-type Fc region have been substituted, deleted, or inserted to alter, for example, the C1q binding site, the binding site for other effector molecules, or binding to an Fc receptor (FcR). Such modifications of the amino acid sequence can be prepared by substituting one or more amino acids with conservative amino acids, or by substituting one or more amino acids with alternative amino acids that are physically and / or functionally similar to those present in the wild-type. Substitutions can also be prepared by substituting non-conservative amino acids.

[0074] In the context of the present invention, amino acids can be described as conservative or non-conservative amino acids, and can therefore be classified accordingly. Amino acid residues can also be classified into classes defined by alternative physical and functional properties. Thus, amino acid classes can be reflected in one or both of the following tables:

[0075] Conserved classes of amino acid residues TIFF0007757247000001.tif41128Alternative physical and functional classifications of amino acid residues TIFF0007757247000002.tif74144

[0076] In the context of the present invention, substitutions in antibodies, such as humanized or chimeric antibodies, include: Original amino acid - position - replacement amino acid It is shown in the form:

[0077] With reference to well-known amino acid nomenclature, three-letter or one-letter symbols are used to designate any amino acid residue, including the symbols Xaa and X. Thus, the designation "L234F" or "Leu234Phe" means that the antibody contains a substitution of leucine with phenylalanine at amino acid position 234.

[0078] Substitution of an amino acid at a given position with any other amino acid is Original amino acid position, i.e., for example, "L234" It is mentioned that:

[0079] For modifications in which the original and / or replacement amino acids may include two or more amino acids, but not all amino acids, those two or more amino acids can be separated by "," or " / ." For example, substitution of leucine with phenylalanine, arginine, lysine, or tryptophan at position 234 is "Leu234Phe,Arg,Lys,Trp" or "Leu234Phe / Arg / Lys / Trp" or "L234F,R,K,W" or "L234F / R / K / W" or "L234→F,R,K or W."

[0080] For the purposes of this invention, such designations may be used interchangeably and have the same meaning and purpose.

[0081] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids, or with other amino acids, e.g., unnatural amino acids. For example, substitution of amino acid L at position 234 includes each of the following substitutions: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. Note that this is equivalent to designating 234X, where X designates any amino acid other than the original amino acid. These substitutions can also be designated as L234A, L234C, etc., or L234A,C, etc., or L234A / C / , etc. The same applies equally to any and all positions referred to herein, and any one of such substitutions is specifically included herein.

[0082] Antibodies of the invention may also include deletions of amino acid residues. Such deletions are designated by "del" and include, for example, designations such as L234del. Thus, in such embodiments, the leucine at position 234 has been deleted from the amino acid sequence.

[0083] The terms "amino acid" and "amino acid residue" may be used interchangeably herein.

[0084] As used herein, the term "C1q binding" refers to the binding of C1q to an antibody when the antibody is bound to its antigen. As used herein, the term "binding to its antigen" refers to the binding of an antibody to its antigen, whether in vivo or in vitro.

[0085] As used herein, the term "reduce" when referring to C1q binding refers to the ability of the antibodies of the invention to reduce, minimize, or completely inhibit C1q binding to the antibody when compared to C1q binding to a wild-type antibody.

[0086] As used herein, the term "wild-type antibody" with respect to use of the antibodies of the invention in comparative assays refers to an antibody that is identical to a test antibody except that it is not inactive. In this context, the term "inactive" refers to a modified Fc region that has reduced or absent C1q binding (i.e., when C1q binding is determined by ELISA) as determined in Example 10; a modified Fc region that has reduced or absent Fc-mediated T cell proliferation (i.e., when T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay) as determined in Example 4; and / or a modified Fc region that has reduced or absent Fc-mediated CD69 expression (i.e., when Fc-mediated CD69 expression is determined in a PBMC-based functional assay) as determined in Example 3. A wild-type antibody thus contains naturally occurring amino acids in its immunoglobulin heavy chain, i.e., an antibody that does not contain any amino acid modifications that might alter or reduce the antibody's ability to interact with, for example, C1q, an Fc receptor, etc. Such a wild-type antibody will therefore remain an activated antibody capable of binding, for example, C1q. Wild-type antibodies and antibodies of the invention may contain other amino acid modifications that do not affect the ability of the antibody to elicit effector function, such as to make the antibody a bispecific antibody.

[0087] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay (ELISA), a test that uses antibodies and a color change to identify a substance. e nzyme- l inked i mmuno s Orbent aELISA refers to a method for assaying antibodies of the present invention. A first specific antibody is attached to the surface of a plate. Proteins from a sample are then added to test for binding to the first specific antibody. A second antibody that binds to the antibody from the sample is added. The second antibody is linked to an enzyme, and in the final step, a substance containing a substrate for the enzyme is added. The subsequent reaction produces a detectable signal (most commonly a color change in the substrate). The concept of ELISA is well known in the art, and various methods for performing ELISA are considered to be part of the method for evaluating the antibodies of the present invention. Therefore, this description should not be construed as limiting, as various forms of ELISA can be performed, as described in Example 4.

[0088] Specifically, the ability of the antibodies of the present invention to bind to C1q can be determined by the following steps: (i) coating the antibodies onto a 96-well plate; (ii) adding 3% serum; (iii) adding an anti-human C1q antibody; (iv) developing the plate; and (v) measuring OD 405nm Thus, in one embodiment, the antibody comprises an Fc region modified such that C1q binding to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type antibody, wherein C1q binding is determined by (i) coating the antibody to a 96-well plate, (ii) adding 3% serum, (iii) adding an anti-human C1q antibody, (iv) developing the plate, and (v) measuring OD 405nm Thus, in certain embodiments, C1q binding is assessed as described in Example 10.

[0089] As used herein, the term "Fc receptor" or "FcR" refers to a protein found on the surface of certain cells. FcRs bind to the Fc region of an antibody. There are several different types of FcRs, classified based on the type of antibody they recognize. For example, Fcγ (gamma) receptors bind to antibodies of the IgG class.

[0090] As used herein, the terms "Fcγ receptor," "Fc gamma receptor," or "FcγR" refer to a group of Fc receptors that belong to the immunoglobulin superfamily and are the Fc receptors most important for inducing phagocytosis of opsonized (coated) microorganisms. This family includes several members, FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), which have different molecular structures and therefore different antibody affinities.

[0091] Fc-mediated effector functions form part of the biological activity of human immunoglobulin G (IgG) molecules. Examples of such effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are triggered by the binding of various effector molecules to the Fc region. In the context of the present invention, "Fc binding," "Fc receptor binding," "FcR binding," and "binding of an antibody Fc region to an FcR" refer to the binding of the Fc region to an Fc receptor (FcR) or an effector molecule. The terms "FcγR binding" and "FcγRI binding" refer to the binding of the Fc region to an Fc gamma receptor and an Fc gamma receptor I, respectively. When a CD3 antibody binds to a T cell, the wild-type Fc region of the CD3 antibody binds to FcRs present on other cells, such as monocytes, leading to nonspecific Fc-mediated activation of the T cell. Such nonspecific Fc-mediated activation of T cells would be undesirable. T cells can also be activated by targeted (or target-specific) T cell activation. Such targeted T cell activation would be highly desirable for the treatment of a range of indications, including cancer. As used herein, the term "targeted T cell activation" refers to directing T cells to specific cells, such as tumor cells, by using a bispecific antibody comprising a first binding region that binds to a specific target, such as a tumor target on tumor cells, and a second binding region that binds to a T cell-specific target, such as CD3. Thus, targeting of T cells to specific cells, such as tumor cells, can be facilitated by using a bispecific antibody in which one binding region binds to CD3 present on T cells and the other binding region binds to a target-specific antigen, for example, on tumor cells. Because nonspecific Fc-mediated T cell activation is still possible, such undesired nonspecific Fc-mediated T cell activation through Fc-mediated crosslinking should be avoided and can be neutralized by rendering the Fc region inactive with respect to such activity. This prevents interaction between the inactive Fc region and any Fc receptors present. The humanized antibodies of the present invention were tested in several different assays and found to be inactive. See Examples 3-5.Another test CD3 antibody, huCLB-T3 / 4, with amino acid modifications in the Fc region was also found to be inactive when tested in a different assay (see Examples 7-10). Humanized CD3 antibodies of the present invention, including the amino acid substitutions L234F, L235E, and D265A as described in the Examples, exhibited low levels of CD69 expression on T cells (Example 3), blockage of Fc-mediated T cell proliferation (Example 4), and no nonspecific target killing in the form of a bispecific antibody (Example 5). Thus, the humanized antibodies of the present invention demonstrate superior results in several assays compared to wild-type antibodies.

[0092] The antibodies of the present invention may contain modifications in the Fc region. When an antibody contains such modifications, it may be an inactive or non-activated antibody. As used herein, the terms "inactive," "inactive," or "non-activating" refer to an Fc region that is at least incapable of binding to any Fcγ receptor, incapable of inducing Fc-mediated cross-linking of FcRs, incapable of inducing FcR-mediated cross-linking of target antigens by the two Fc regions of individual antibodies, or incapable of binding to C1q. The inactivity of the Fc region of a humanized or chimeric CD3 antibody is conveniently tested using an antibody in a monospecific format, and the inactive Fc region thus identified can be used in bispecific or other humanized or chimeric multispecific CD3 antibodies.

[0093] For the purpose of therapeutic antibody development, several mutants can be constructed to render the Fc region of the antibody inactive for interaction with Fc gamma receptors and C1q, examples of such mutants are described herein.

[0094] Thus, in one embodiment, the antibody comprises an Fc region that has been modified such that the antibody mediates Fc-mediated T cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% compared to a wild-type antibody, wherein the T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0095] As used herein, the term "reduce" refers to a reduction in activity or expression compared to a control protein, such as an antibody. Particularly in reference to T cell proliferation, the term "reduce" refers to the ability of an antibody of the invention to reduce, minimize, or completely inhibit T cell proliferation compared to the proliferation of T cells bound by a wild-type antibody. The ability of an antibody to reduce T cell proliferation can be assessed by a PBMC-based functional assay, as described in Examples 4 and 8. In one embodiment, the assay is performed on human PBMCs. In another embodiment, the assay is performed on cynomolgus monkey PBMCs. In yet another embodiment, the assay is performed on rhesus monkey PBMCs. Because the antibodies of the invention are cross-reactive, the PBMC-based assays described herein can be performed using PBMCs from any species, e.g., human, cynomolgus monkey, or rhesus monkey, to demonstrate a reduction in T cell proliferation, as long as the PBMCs of the species used are within the cross-reactivity spectrum of the antibody.

[0096] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to assess the functional characteristics of an antibody of the invention, such as the ability of the antibody to affect T-cell proliferation or CD69 expression, in which the only cells present are peripheral blood mononuclear cells. Thus, in one embodiment, T cell proliferation is measured by a method comprising incubating PBMCs with an antibody in the range of 1-1000 ng / mL for 3 days at 37°C in a 5% (vol / vol) CO2 humidified incubator, adding a chemical compound such as BrdU that is incorporated into the DNA of proliferating cells, incubating for 5 hours, pelleting the cells, drying the cells, optionally storing the cells at 4°C, coating the cells to an ELISA plate, incubating with anti-BrdU-peroxidase for 90 minutes at room temperature, developing with 1 mg / mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) for approximately 30 minutes, adding 100 μL of 2% oxalic acid to stop the reaction, and measuring absorbance at 405 nm in a suitable microplate reader.

[0097] As used herein, the term "proliferation" refers to cell growth through cell division.

[0098] As used herein, the term "BrdU" refers to 5-bromo-2'-deoxyuridine, a thymidine homolog. When BrdU is added to cell culture for a limited period (e.g., 4 hours), it is incorporated into the DNA of proliferating cells. After fixing the cells, the detection of incorporated BrdU can be performed in an ELISA using anti-BrdU-peroxidase. Therefore, BrdU incorporation is a measure of proliferation.

[0099] In one embodiment, the antibody comprises an Fc region that has been modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% when compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is measured in a PBMC-based functional assay.

[0100] As used herein, the term "reduce" refers to a reduction in activity or expression compared to a control protein, such as an antibody. Particularly when referring to the expression level of the T cell activation marker CD69, the term "reduce" refers to a reduction in the expression level of CD69 compared to the expression level of CD69 when a wild-type antibody binds to a T cell, assuming that both binding regions of the antibody bind to CD3. The ability of an antibody to reduce CD69 expression can be assessed by a PBMC-based functional assay, as described in Examples 3 and 7. Thus, in one embodiment, CD69 expression is measured by a method comprising incubating PBMCs with an antibody in the range of 1 to 1000 ng / mL for 16 to 24 hours at 37°C in a 5% (vol / vol) CO2 humidified incubator, washing the cells, staining the cells with mouse anti-human CD28-PE antibody and mouse anti-human CD69-APC antibody at 4°C, and determining CD69 expression on CD28-positive cells by flow cytometry.

[0101] As used herein, the term "CD69" refers to Cluster of Differentiation 69, a human transmembrane C-type lectin protein encoded by the CD69 gene. Activation of T lymphocytes and natural killer (NK) cells induces CD69 expression both in vivo and in vitro. CD69 functions as a signaling receptor involved in cell activation events such as proliferation in lymphocytes, e.g., natural killer cells, and platelets, and in the induction of specific genes.

[0102] As used herein, the term "peripheral blood mononuclear cell (PBMC)-based functional assay" refers to an assay used to evaluate the functional characteristics of an antibody of the invention, such as its ability to affect T cell proliferation or CD69 expression, in which the only cells present are peripheral blood mononuclear cells. The PBMC-based functional assay described in Examples 3, 4, 5, and 7, when assessing CD69 expression, includes (i) incubating PBMCs with antibody for approximately 16-24 hours at 37°C in a 5% (vol / vol) CO2 humidified incubator, (ii) washing the cells, (iii) staining the cells with mouse anti-human CD28-PE antibody and mouse anti-human CD69-APC antibody at 4°C, and (iv) determining CD69 expression on CD28-positive cells by flow cytometry. Thus, in one embodiment, CD69 expression can be determined as described in Examples 3, 4, 5, or 7.

[0103] Thus, amino acids in the Fc region that play a major role in the interaction with C1q and Fc gamma receptors can be modified. Examples of amino acid positions that can be modified include positions L234, L235, and P331. Combinations thereof, such as L234F / L235E / P331S, can cause a significant decrease in binding to human CD64, CD32A, CD16, and C1q.

[0104] Thus, in one embodiment, the amino acids at at least one position corresponding to L234, L235, and P331 can be A, A, and S, respectively ([1],

[28] ). Furthermore, the L234F and L235E amino acid substitutions can result in an Fc region in which interaction with Fc gamma receptors and C1q is blocked (

[29] -

[30] ). Thus, in one embodiment, the amino acids at positions corresponding to L234 and L235 can be F and E, respectively. The D265A amino acid substitution can reduce binding to all Fc gamma receptors and prevent ADCC (

[31] ). Thus, in one embodiment, the amino acid at position corresponding to D265 can be A. Binding to C1q can be blocked by mutating positions D270, K322, P329, and P331. Mutation of these positions to either D270A, K322A, P329A, or P331A can render the antibody CDC-deficient (

[32] ). Thus, in one embodiment, the amino acids at at least one position corresponding to D270, K322, P329, and P331 can be A, A, A, and A, respectively.

[0105] An alternative approach to minimize the interaction of the Fc region with Fc gamma receptor and C1q is to remove the glycosylation site of the antibody. By mutating position N297 to, for example, Q, A, and E, the glycosylation site that is crucial for IgG-Fc gamma receptor interaction is removed. Thus, in one embodiment, the amino acid at the position corresponding to N297 can be G, Q, A, or E (

[33] ). Another alternative approach to minimize the interaction of the Fc region with Fc gamma receptor can be obtained by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (

[31] ).

[0106] Alternatively, human IgG2 and IgG4 subclasses are thought to be inherently impaired in their interactions with C1q and Fc gamma receptors, although interactions with Fc gamma receptors (Fc gamma receptors) have also been reported (

[34] -

[35] ). In both isotypes, mutations can be engineered to disrupt these residual interactions and reduce unwanted side effects associated with FcR binding. For IgG2, these include L234A and G237A, and for IgG4, they include L235E. Thus, in one embodiment, the amino acids at positions corresponding to L234 and G237 in a human IgG2 heavy chain can be A and A, respectively. In one embodiment, the amino acid at position corresponding to L235 in a human IgG4 heavy chain can be E.

[0107] Other approaches to further minimize interactions with Fc gamma receptors and C1q in IgG2 antibodies include those described in

[36] and

[37] .

[0108] The hinge region of an antibody may also be important for interactions with Fc gamma receptors and complement (

[38] -

[39] ). Therefore, mutations in or deletion of the hinge region can affect the effector functions of antibodies.

[0109] As used herein, the term "cross-linking" refers to the indirect cross-linking of antibody Fab arms (monovalent or bivalent) that are bound to a target antigen by an FcR-bearing cell via binding to the antibody Fc region. Thus, an antibody that binds to that target antigen on a target antigen-bearing cell can cross-link another cell that expresses an FcR.

[0110] As used herein, the term "non-specific killing" refers to the killing of cells by the cytotoxic function of T cells or other effector cells, which is activated independent of the tumor target antigen. Thus, non-specific killing means that tumor target-bearing cells can be killed, for example, by cytotoxic T cells, rather than by antibodies that bind to the tumor target, such as by inducing CDC.

[0111] The present inventors have shown that a non-activated Fc region can be obtained by modifying one or more of at least five specific amino acid positions in the Fc region (Examples 3-5, 7-10).

[0112] Thus, in one embodiment, the antibody comprises a first and a second immunoglobulin heavy chain, wherein in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0113] In one embodiment, in both the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0114] In another embodiment, in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively, and the amino acids at positions corresponding to N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0115] As used herein, the term "amino acid corresponding to position" refers to the amino acid position number in the human IgG1 heavy chain. Unless otherwise stated or contradicted by context, amino acids in constant region sequences are numbered herein according to the Eu numbering index (described in

[27] ). Thus, an amino acid or segment in one sequence "corresponding to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain, e.g., when using standard sequence alignment programs such as ALIGN, ClustalW, or others, typically with default settings. Methods for aligning sequences or segments in sequences and thereby determining positions in sequences that correspond to the amino acid positions of the present invention are believed to be well known in the art.

[0116] In the present invention, amino acids can be defined as above.

[0117] The term "amino acid is not" or similar expressions, with respect to an amino acid in a heavy chain, should be understood to mean that the amino acid is any other amino acid except the particular amino acid mentioned. For example, the amino acid at the position corresponding to L234 in the human IgG1 heavy chain is not L means that the amino acid can be either another natural amino acid other than L or an unnatural amino acid.

[0118] In one embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0119] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0120] In one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is a hydrophobic amino acid or a polar amino acid.

[0121] The term "hydrophobic" as used herein in reference to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0122] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0123] In another embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is an aliphatic uncharged amino acid, an aromatic amino acid or an acidic amino acid.

[0124] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L, and V.

[0125] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T, and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of F, T, and W.

[0126] The term "acidic", as used herein with respect to an amino acid residue, refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at a position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of D and E.

[0127] In certain embodiments, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0128] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0129] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0130] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is a hydrophobic amino acid or a polar amino acid.

[0131] The term "hydrophobic" as used herein in reference to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y.

[0132] The term "polar" as used herein in reference to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T. In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0133] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0134] In another embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is an aliphatic uncharged amino acid, an aromatic amino acid or an acidic amino acid.

[0135] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L, and V. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L, and V.

[0136] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T, and W. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of F, T, and W.

[0137] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of D and E.

[0138] In a particular embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0139] In a further embodiment, in at least one of said first and second heavy chains the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0140] In one aspect, in at least one of the first and second heavy chains, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0141] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N.

[0142] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain is not N and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain is P.

[0143] In a further embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0144] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0145] In one embodiment, in at least one of said first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.

[0146] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0147] The term "hydrophobic" as used herein in reference to amino acid residues refers to amino acid residues selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0148] The term "polar" as used herein in reference to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively.

[0149] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively.

[0150] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0151] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0152] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0153] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0154] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively.

[0155] In certain embodiments, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively.

[0156] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are aliphatic uncharged amino acids, aromatic amino acids or acidic amino acids.

[0157] The term "aliphatic uncharged" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, G, I, and V, respectively.

[0158] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T, and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of F, T, and W, respectively.

[0159] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of D and E, respectively.

[0160] In certain embodiments, the amino acids at positions corresponding to L234 and L235 in at least one of the first and second heavy chains are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0161] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively.

[0162] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0163] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively.

[0164] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0165] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0166] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0167] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0168] In one embodiment, in both said first and second heavy chains, at least the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively.

[0169] In one embodiment in at least one of said first and second heavy chains the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively.

[0170] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0171] In one embodiment, in at least one of said first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acid corresponding to position D265 is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

[0172] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0173] The term "hydrophobic" as used herein in reference to an amino acid residue refers to an amino acid residue selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0174] The term "polar" as used herein in reference to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively, and the amino acid at position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0175] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively, and the amino acid at position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0176] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to L234, L235 and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

[0177] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W and Y, respectively.

[0178] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively, and the amino acid at position corresponding to position D265 in a human heavy chain is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0179] In certain embodiments, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively, and the amino acid at position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0180] In another embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged amino acids, aromatic amino acids or acidic amino acids.

[0181] The term "aliphatic uncharged" as used herein in reference to an amino acid residue refers to any amino acid residue selected from the group consisting of A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L, and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I, and V.

[0182] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T, and W. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are selected from the group consisting of F, T, and W, respectively.

[0183] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E. Thus, in one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are selected from the group consisting of D and E, respectively.

[0184] In a particular embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0185] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are not L, L and D, respectively.

[0186] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0187] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are aliphatic uncharged amino acids, aromatic amino acids, or acidic amino acids.

[0188] In one embodiment, in both said first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, G, I, L, and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of A, G, I, and V.

[0189] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are selected from the group consisting of D and E, respectively.

[0190] In a particular embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0191] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively.

[0192] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0193] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, or A, A and A, respectively.

[0194] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain are N and P, respectively.

[0195] In a particular embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0196] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are F, E and A, respectively.

[0197] In one embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0198] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are A, A and A, respectively.

[0199] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively.

[0200] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain are F, E, A, Q and S, respectively.

[0201] In certain embodiments, an antibody of the invention comprises a VH sequence as shown in SEQ ID NO:8, a VL sequence as shown in SEQ ID NO:10, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0202] In another embodiment, an antibody of the invention comprises the VH sequence shown in SEQ ID NO:8, the VL sequence shown in SEQ ID NO:12, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0203] In another embodiment, an antibody of the invention comprises the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0204] In another embodiment, an antibody of the invention comprises the VH sequence shown in SEQ ID NO:6, the VL sequence shown in SEQ ID NO:12, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0205] In another embodiment, an antibody of the invention comprises the VH sequence shown in SEQ ID NO:9, the VL sequence shown in SEQ ID NO:10, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0206] In another embodiment, an antibody of the invention comprises the VH sequence shown in SEQ ID NO:9, the VL sequence shown in SEQ ID NO:12, and in at least one of the heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0207] In one aspect, the present invention relates to a multispecific antibody comprising at least a first binding region of an antibody according to any aspect or embodiment described herein and one or more binding regions that bind to one or more targets different from the first binding region. Such a multispecific antibody can be a bispecific antibody.

[0208] Thus, in one aspect, the invention relates to a bispecific antibody comprising a first binding region of an antibody according to any aspect or embodiment described herein and a second binding region that binds to a different target than the first binding region.

[0209] The term "multispecific antibody" refers to an antibody having specificity for at least two different, e.g., at least three, typically non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. If the epitopes are on different targets, such targets may be on the same cell or cell type or on different cells or cell types.

[0210] The term "bispecific antibody" refers to an antibody having specificity for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. If the epitopes are on different targets, such targets may be on the same cell or cell type or on different cells or cell types.

[0211] In one embodiment, the bispecific antibody comprises a first heavy chain and a second heavy chain.

[0212] Embodiments relating to modifications of the Fc region and to specific amino acid substitutions are considered to be part of any bispecific antibody of the invention. Thus, in one embodiment, at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any embodiment described herein, such as those described with respect to providing an inactive Fc region. In one embodiment, both the first and second heavy chains comprise one or more amino acids modified as defined in any embodiment described herein, such as those described with respect to providing an inactive Fc region. Thus, a bispecific antibody comprises an Fc region modified according to any aspect or embodiment described herein, or at least one of the first and second heavy chains comprises one or more amino acids modified as defined in any aspect or embodiment described herein.

[0213] Thus, in one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, wherein the Fc region has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody as determined by ELISA.

[0214] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:12, wherein the Fc region has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody as determined by ELISA.

[0215] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, wherein the Fc region has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody as determined by ELISA.

[0216] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:6 and the VL sequence set forth in SEQ ID NO:12, wherein the Fc region has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody as determined by ELISA.

[0217] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:10, wherein the Fc region has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody as determined by ELISA.

[0218] In one embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:12, wherein the Fc region has been modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type antibody as determined by ELISA.

[0219] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:10, and wherein the Fc region is modified such that the antibody mediates Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0220] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:12, and wherein the Fc region is modified such that the antibody mediates Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0221] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:6 and the VL sequence set forth in SEQ ID NO:10, and wherein the Fc region is modified such that the antibody mediates Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0222] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:6 and the VL sequence set forth in SEQ ID NO:12, and wherein the Fc region is modified such that the antibody mediates Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0223] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:10, and wherein the Fc region is modified such that the antibody mediates Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0224] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:12, and wherein the Fc region is modified such that the antibody mediates Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0225] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:10, and wherein the Fc region is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0226] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:12, and wherein the Fc region is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0227] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:6 and the VL sequence set forth in SEQ ID NO:10, and wherein the Fc region is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0228] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:6 and the VL sequence set forth in SEQ ID NO:12, and wherein the Fc region is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0229] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:10, and wherein the Fc region is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0230] In one embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence set forth in SEQ ID NO:9 and the VL sequence set forth in SEQ ID NO:12, and wherein the Fc region is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody, wherein the Fc-mediated CD69 expression is determined in a PBMC-based functional assay.

[0231] In certain embodiments, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0232] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12, and in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0233] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, and in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0234] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12, and in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0235] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10, and in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0236] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12, and in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0237] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody with two arms containing different antigen-binding regions; (ii) a single-chain antibody with specificity for two different epitopes, for example, due to two scFvs linked in tandem by an additional peptide linker; (iii) a dual-variable domain antibody (DVD-Ig™) (

[40] ), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond; (iv) chemically linked bispecific (Fab')2 fragments; (v) a TandAb®, which is a fusion of two single-chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of an scFv and a diabody resulting in a multivalent molecule; and (vii) a so-called "dock and lock" antibody based on the "dimerization and docking domain" in protein kinase A. These include "Dock-and-Lock®" molecules, which when applied to Fab can give trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments, (viii) so-called Scorpion molecules, which contain, for example, two scFvs fused to either end of a human Fab arm, and (ix) diabodies.

[0238] In one embodiment, the bispecific antibody of the invention is a diabody, a crossbody, or a bispecific antibody obtained by controlled Fab arm exchange, such as a DuoBody® as described in the present invention (e.g., as described in

[41] ).

[0239] (ii) recombinant IgG-like dual targeting molecules, in which each side of the molecule contains an Fab fragment or portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or stabilized diabody is fused to a heavy-chain constant domain, Fc region, or portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together or to a heavy-chain constant domain, Fc region, or portion thereof; and (vi) ScFv and diabody-based antibodies and heavy-chain antibodies (e.g., domain antibodies, Nanobodies®), in which different single-chain Fv molecules, or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or to another protein or carrier molecule fused to a heavy-chain constant domain, Fc region, or portion thereof.

[0240] Examples of IgG-like molecules with complementary CH3 domain molecules include Triomab® (Trion Pharma / Fresenius Biotech,

[42] ), Knobs-into-Holes (Genentech,

[43] ), CrossMAb (Roche,

[44] ) and electrostatically matched (Amgen,

[45] -

[46] ; Chugai,

[47] ; Oncomed,

[48] ), LUZ-Y (Genentech), DIG-body and PIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono,

[49] ), Biclonic (Merus), FcΔAdp (Regeneron,

[50] ), bispecific IgG1 and IgG2 (Pfizer / Rinat,

[51] ), Azymetric scaffolds (Zymeworks / Merck,

[52] ), mAb-Fv (Xencor,

[53] ), bivalent bispecific antibodies (Roche) and DuoBody® molecules (Genmab A / S,

[41] ).

[0241] Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one antibody (Genentech), Cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

[54] ), Zybodies™ (Zyngenia), common light chain approach (Crucell / Merus,

[55] ), κλBody (NovImmune), and CovX-body® (CovX / Pfizer).

[0242] Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD)-Ig™ (Abbott,

[56] ), Dual domain double head antibodies (Unilever; Sanofi Aventis,

[57] ), IgG-like bispecifics (ImClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec,

[58] ), scFv fusions (Novartis), scFv fusions (Changzhou Adam Biotech Inc.,

[59] ) and TvAb (Roche,

[59] ,

[60] ).

[0243] Examples of Fc-fusion molecules include, but are not limited to, ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics,

[62] ,

[63] ), and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine-China).

[0244] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0245] Examples of ScFv-based antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Bispecific T cell Engager (BiTE®) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), Single-chain Diabody (Academic), TCR-like antibody (AIT, ReceptorLogics), human serum albumin ScFv fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies® (Ablynx), dual targeting heavy chain only domain antibody.

[0246] Furthermore, it is believed that any monospecific antibody that satisfies the assay conditions described herein can form the basis for a bispecific antibody. That is, a bispecific antibody in which one of the binding regions binds to CD3 can be derived from any monospecific CD3 antibody that is tested in a functional assay and meets the requirements set forth herein. Such bispecific antibodies can be obtained by the methods described in

[41] , which is incorporated herein by reference.

[0247] Thus, in a particular embodiment, the first and second heavy chains each comprise at least a hinge region, a CH2 region, and a CH3 region, wherein the first heavy chain comprises a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and the second heavy chain comprises a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and wherein the substitutions are not at the same positions in the first and second heavy chains. In this context, the term "substituted" refers to the substitution of the amino acid at a particular amino acid position with another natural or unnatural amino acid. Thus, a "substituted" amino acid at a position corresponding to a position in a human IgG1 heavy chain means that the amino acid at that particular position is different from the naturally occurring amino acid in the IgG1 heavy chain.

[0248] In one embodiment, in said first heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is not K, L or M, and optionally the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is F; and in said second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405 and Y407 in the human IgG1 heavy chain has been substituted.

[0249] In one aspect, in said first heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and in said second heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is not F, and optionally the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is K.

[0250] In one embodiment, in said first heavy chain, the amino acid at a position corresponding to F405 in a human IgG1 heavy chain is not F, R, or G, and in said second heavy chain, an amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, Y407, and K409 in a human IgG1 heavy chain has been substituted.

[0251] In one embodiment, in said first heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is not K, L or M, and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is not F.

[0252] In yet another embodiment, in said first heavy chain the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L and in said second heavy chain the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R, or vice versa.

[0253] Thus, in one embodiment, in the first heavy chain the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R and in the second heavy chain the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L.

[0254] In yet another embodiment, a humanized or chimeric CD3 antibody of the invention comprises in at least one of the first and second heavy chains an inactivating substitution as disclosed in any one of the above embodiments, e.g., one or more of L234F, L235E, and D265A, and the amino acid at the position corresponding to F405 is not F. In one embodiment, a humanized or chimeric CD3 antibody of the invention comprises in at least one of the first and second heavy chains an inactivating substitution as disclosed in any one of the above embodiments, e.g., one or more of L234F, L235E, and D265A, and an additional substitution at position K409, e.g., K409R. In particular, in one embodiment, a humanized or chimeric CD3 antibody of the invention comprises, in both the first and second heavy chains, an inactivating substitution as disclosed in any one of the above embodiments, e.g., one or more of L234F, L235E, and D265A, and a substitution at position F405, e.g., F405L. In one embodiment, a humanized or chimeric CD3 antibody of the invention comprises, in both the first and second heavy chains, an inactivating substitution as disclosed in any one of the above embodiments, e.g., one or more of L234F, L235E, and D265A, and an additional substitution at position K409, e.g., K409R. Such antibodies are useful for generating bispecific antibodies.

[0255] Thus, in yet another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to F405 in the human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at position corresponding to K409 in the human IgG1 heavy chain is R.

[0256] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P, respectively; in the first heavy chain, the amino acid at position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at position corresponding to K409 in a human IgG1 heavy chain is R.

[0257] In an alternative embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to K409 in the human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at position corresponding to F405 in the human IgG1 heavy chain is L.

[0258] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P, respectively; in the first heavy chain, the amino acid at position corresponding to K409 in a human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at position corresponding to F405 in a human IgG1 heavy chain is L.

[0259] In another embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at position corresponding to K409 in a human IgG1 heavy chain is R.

[0260] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P, respectively; in the first heavy chain, the amino acid at the position corresponding to F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at the position corresponding to K409 in a human IgG1 heavy chain is R.

[0261] In an alternative embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to K409 in the human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at position corresponding to F405 in the human IgG1 heavy chain is L.

[0262] In one embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P, respectively; in the first heavy chain, the amino acid at position corresponding to K409 in a human IgG1 heavy chain is R; and in the second heavy chain, the amino acid at position corresponding to F405 in a human IgG1 heavy chain is L.

[0263] As described herein, recruitment of T cells to specific target cells, such as cancer or tumor cells, provides a method for killing the target cells. The inventors have shown that a bispecific CD3xHER2 antibody containing the specific amino acid substitutions L234F, L235E, and D265A in both heavy chains can induce killing of AU565 cells, as described in Example 5. T cell-mediated killing can be achieved by a bispecific antibody that targets CD3 with a first binding region and another target with a second binding region. Thus, in one embodiment, the first binding region is according to any of the embodiments described herein for a humanized or chimeric CD3 antibody, and the second binding region binds to a different target than the first binding region. It should be understood that when an antibody is a bispecific antibody, at least one half of the antibody, i.e., one of the heavy and light chain pairs of the antibody, is a humanized or chimeric antibody as described herein. Thus, one half of the bispecific antibody can be a humanized or chimeric antibody of the invention that binds to CD3, and the other half can be humanized, chimeric, fully non-human, or fully human that binds to a second target. Thus, in one embodiment, the antibody comprises first and second heavy chains and first and second light chains, wherein the first heavy chain and the first light chain are humanized or chimeric and connected by a disulfide bridge to form a first binding region, and the second heavy chain and the second light chain are fully human and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and wherein the second binding region binds to a different target. In one embodiment, the antibody comprises first and second heavy chains, first and second light chains, wherein the first heavy chain and the first light chain are humanized or chimeric and connected by a disulfide bridge to form a first binding region, and the second heavy chain and the second light chain are humanized or chimeric and connected by a disulfide bridge to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and wherein the second binding region binds to a different CD3 epitope than the first binding region.

[0264] Thus, in one embodiment a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0265] In one embodiment a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0266] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0267] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0268] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0269] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0270] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0271] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0272] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0273] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0274] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0275] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0276] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0277] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0278] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0279] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0280] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0281] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0282] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0283] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0284] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in at least one of the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0285] In another embodiment, a bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain, the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain, the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0286] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0287] In another embodiment, the bispecific antibody comprises a first binding region comprising the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12, and a second binding region comprising the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30, wherein in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; in the first heavy chain the amino acid at position corresponding to position F405 in a human IgG1 heavy chain is L; and in the second heavy chain the amino acid at position corresponding to position K409 in a human IgG1 heavy chain is R.

[0288] As used herein, the term "disulfide bridge" refers to a covalent bond between two cysteine ​​residues, i.e., this interaction can also be referred to as a Cys-Cys interaction.

[0289] As used herein, the term "target" refers to a molecule to which the binding region of an antibody of the invention binds. When used in reference to antibody binding, the term encompasses any antigen against which the antibody is raised.

[0290] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric and are connected by disulfide bridges to form a first binding region, and the second heavy chain and the second light chain are fully human and are connected by disulfide bridges to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein and the second binding region binds to a different target, and in at least one of the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0291] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric and are connected by disulfide bridges to form a first binding region, and the second heavy chain and the second light chain are fully human and are connected by disulfide bridges to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, the second binding region binds to a different CD3 epitope than the first binding region, and in at least one of the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0292] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric and are connected by disulfide bridges to form a first binding region, and the second heavy chain and the second light chain are fully human and are connected by disulfide bridges to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein and the second binding region binds to a different target, and in both the first and second heavy chains the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0293] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric and are connected by disulfide bridges to form a first binding region, and the second heavy chain and the second light chain are fully human and are connected by disulfide bridges to form a second binding region, wherein the first binding region is according to any aspect or embodiment described herein, and the second binding region binds to a different CD3 epitope than the first binding region, and in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0294] Nucleic acid constructs, expression vectors, and host cells In one aspect, the present invention relates to nucleic acid constructs encoding one or more of the sequences set forth in Table 1. Accordingly, the present invention relates to nucleic acid constructs encoding any one of the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26.

[0295] In yet another aspect, the present invention relates to a nucleic acid construct encoding the sequence of a humanized or chimeric CD3 antibody of the invention, an expression vector comprising the nucleic acid construct of the invention, a host cell comprising said expression vector, and a method for producing the antibody by culturing said host cell under appropriate conditions, whereby the antibody is produced and, optionally, recovered. The humanized CD3 antibody may also be referred to as "huCD3."

[0296] In one aspect, the invention provides an expression vector comprising (i) a nucleic acid sequence encoding the heavy chain sequence of a humanized or chimeric antibody of the invention, (ii) a nucleic acid sequence encoding the light chain sequence of a humanized or chimeric antibody of the invention, or (iii) both (i) and (ii).Accordingly, the expression vector comprises one or more nucleic acid constructs or nucleic acid sequences according to any aspect or embodiment described herein.

[0297] In one embodiment, an expression vector of the invention comprises a nucleic acid sequence encoding one or more of the heavy and light chain CDR sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, and 5, and the sequence GTN.

[0298] In one embodiment, the invention provides an expression vector comprising a nucleic acid sequence encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs:6, 7, 8, 9, 10, 11, 12, 19, 20, 27, 28, 29, and 30, or any combination thereof. In another embodiment, the expression vector comprises a nucleic acid sequence encoding the VH CDR3 amino acid sequence set forth in SEQ ID NO:3. In another embodiment, the expression vector comprises a nucleic acid sequence encoding a VH amino acid sequence selected from SEQ ID NOs:6, 7, 8, 9, 19, 27, and 29. In another embodiment, the expression vector comprises a nucleic acid sequence encoding a VL amino acid sequence selected from SEQ ID NOs:10, 11, 12, 20, 28, and 30. In another embodiment, the expression vector comprises a nucleic acid sequence encoding the constant region of a human antibody light chain, a human antibody heavy chain, or both. In another embodiment, the invention provides an expression vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NOs:15, 16, 23, 24, 25, and 26.

[0299] In certain embodiments, the expression vector comprises a nucleic acid sequence encoding one or more variants of the above amino acid sequences, the variants having up to 25 amino acid modifications, for example, up to 20, for example, up to 15, 14, 13, 12, or 11 amino acid modifications, for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, for example, conservative or non-conservative substitutions, or at least 80% identity, for example, at least 85% identity, 90% identity, or 95% identity, for example, 96% identity, 97% identity, 98% identity, or 99% identity, to any of the above amino acid sequences. The present invention also relates to nucleic acid sequences that differ from the above-mentioned nucleic acid sequences but encode the same amino acid sequence as the antibody of the present invention due to the variability of the genetic code. For example, variations in the nucleic acid sequence can result in an amino acid sequence identical to any of the amino acid sequences described herein. Methods for identifying such additional nucleic acid sequences based on the genetic code are well known to those skilled in the art.

[0300] In yet another embodiment, the expression vector further comprises a nucleic acid sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chains of an antibody, eg, a human antibody.

[0301] Expression vectors such as those described above can be used for the recombinant production of antibodies of the invention.

[0302] In the context of the present invention, an expression vector can be any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the humanized or chimeric CD3 antibody is expressed in a naked DNA or RNA vector, such as a linear expression element (e.g., as described in

[64] ), a compacted nucleic acid vector (e.g., as described in

[65] and / or

[66] ), a plasmid vector such as pBR322, pUC19 / 18, or pUC118 / 119, a "midge" minimal size nucleic acid vector (e.g., as described in

[67] ), or CaP04 - It is included in a precipitating nucleic acid vector construct, such as a precipitating construct (e.g., those described in

[68] ,

[69] ,

[70] , and

[71] ). Such nucleic acid vectors and methods for their use are well known in the art (see, e.g.,

[72] and

[73] ).

[0303] In one embodiment, a vector is suitable for expression of a humanized or chimeric CD3 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (

[74] ), and pET vectors (Novagen, Madison, Wisconsin).

[0304] Additionally or alternatively, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in

[75] and

[76] ).

[0305] The nucleic acid construct and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence capable of targeting a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell culture medium. Such sequences are known in the art and include secretion leader or signal peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transport sequences, chloroplast transport sequences), membrane localization / anchor sequences (e.g., membrane permeabilization sequences, GPI anchor sequences), and the like, all of which are well known in the art.

[0306] In the expression vectors of the present invention, the sequence encoding the humanized or chimeric CD3 antibody can comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer, and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an efficient poly(A) termination sequence, an origin of replication for plasmid production in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid construct and / or vector can also comprise an inducible promoter, such as CMV IE, rather than a constitutive promoter (those skilled in the art will recognize that these terms are descriptive of the degree of gene expression under certain conditions).

[0307] In one embodiment, an expression vector encoding a humanized or chimeric CD3 antibody can be placed into and / or delivered to a host cell or animal by a viral vector.

[0308] Such expression vectors can be used for the recombinant production of humanized or chimeric CD3 antibodies.

[0309] In one aspect, the invention provides a host cell comprising an expression vector of the invention.

[0310] In one aspect, a humanized or chimeric CD3 antibody of any aspect or embodiment described herein is provided using a recombinant eukaryotic, prokaryotic, or microbial host cell that produces the antibody. Accordingly, the present invention provides a recombinant eukaryotic, prokaryotic, or microbial host cell that produces a humanized or chimeric CD3 antibody or immunoglobulin as defined herein. Exemplary host cells include yeast, bacterial, and mammalian cells, such as CHO or HEK-293 cells. For example, in one embodiment, the host cell contains a nucleic acid sequence stably integrated into the cellular genome that contains a sequence encoding expression of a humanized or chimeric CD3 antibody described herein. In another embodiment, the host cell contains a non-integrated nucleic acid sequence, such as a plasmid, cosmid, phagemid, or linear expression element, that contains a sequence encoding expression of a humanized or chimeric CD3 antibody described herein.

[0311] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which an expression vector or nucleic acid construct or nucleic acid sequence has been introduced. It should be understood that such terms refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur over successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still encompassed within the term "host cell" as used herein. Recombinant host cells include, for example, eukaryotic host cells such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells, prokaryotic cells such as E. coli, and other eukaryotic hosts such as plant cells and fungi.

[0312] In yet another aspect, the present invention provides a method for producing a humanized or chimeric CD3 antibody of the invention, comprising the steps of: a) culturing the host cell of the present invention as described above, and b) recovering and / or purifying the antibody of the invention from the culture medium The present invention relates to a method comprising:

[0313] In yet another aspect, the nucleotide sequence encoding the humanized or chimeric CD3 antibody sequence encodes a second moiety, such as a therapeutic polypeptide. Exemplary therapeutic polypeptides are described elsewhere herein. In one embodiment, the invention provides a method for producing a humanized or chimeric CD3 antibody fusion protein, comprising: a) culturing a host cell containing an expression vector comprising such a nucleotide sequence; and b) recovering and / or purifying the humanized or chimeric CD3 antibody fusion protein from the culture medium The present invention relates to a method comprising:

[0314] composition In one aspect, the invention provides a composition comprising an antibody or bispecific antibody according to any aspect and embodiment described herein.

[0315] In one aspect, the invention provides a pharmaceutical composition comprising an antibody or bispecific antibody as defined in any one of the aspects and embodiments described herein and a pharmaceutically acceptable carrier.

[0316] Pharmaceutical compositions can be formulated using pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients according to conventional techniques, such as those disclosed in

[77] .

[0317] Pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, should be suitable for the humanized or chimeric antibody of the present invention and the selected mode of administration. The suitability of carriers and other components of pharmaceutical compositions is determined based on the lack of a significant negative effect (e.g., no significant effect (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on the desired biological properties of the selected compound of the present invention or pharmaceutical composition with respect to antigen binding.

[0318] Pharmaceutical compositions of the present invention may also include diluents, bulking agents, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0319] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention or its amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and other factors well known in the medical arts.

[0320] The pharmaceutical composition can be administered by any suitable route and manner. Suitable routes for administering the humanized or chimeric antibodies of the invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0321] In one embodiment, the pharmaceutical compositions of the present invention are administered parenterally.

[0322] As used herein, the expressions "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.

[0323] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0324] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with the humanized or chimeric antibodies of the present invention.

[0325] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0326] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for extemporaneously preparing sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Unless conventional media or agents are incompatible with active compounds, they can be used in the pharmaceutical compositions of the present invention.When referring to "active compounds", it is also intended to refer to the humanized or chimeric antibodies of the present invention.

[0327] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0328] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0329] The pharmaceutical compositions of the present invention may also contain isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0330] Pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives, or buffers that may enhance the shelf life or effectiveness of the pharmaceutical composition. Humanized or chimeric antibodies of the present invention can be prepared with carriers that will protect the compound against rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with waxes, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art (see, e.g.,

[78] ).

[0331] In one embodiment, the humanized or chimeric antibody of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Conventional media or agents are contemplated for use in the pharmaceutical compositions of the present invention, unless they are incompatible with the active compounds. Other active or therapeutic compounds can also be incorporated into the compositions.

[0332] Pharmaceutical compositions for injections typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or nonaqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as monostearate salts and gelatin. Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or a combination of the ingredients listed above as needed, and then sterilizing by microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients, such as those listed above.For the preparation of sterile powder for preparing sterile injectable solution, examples of preparation methods are vacuum drying and freeze-drying (lyophilization), which produces a powder of active ingredient and any additional ingredients desired from the solution that has been previously sterilized and filtered.

[0333] Sterile injectable solution can be prepared by incorporating the required amount of active ingredient into suitable solvent with one or combination of the above-listed ingredients as needed, and then sterilizing by microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile medium that contains a basic dispersion medium and other necessary ingredients, such as those listed above.For the preparation of sterile powder for preparing sterile injectable solution, examples of preparation methods are vacuum drying and freeze-drying (lyophilization), which produces a powder of active ingredient and any additional ingredients desired from the solution that has been previously sterilized and filtered.

[0334] Therapeutic applications In another aspect, the present invention relates to a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any aspect or embodiment described herein, for use as a medicament.

[0335] In another aspect, the invention relates to a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any aspect or embodiment described herein for use in the treatment of a disease.

[0336] The humanized or chimeric antibody or pharmaceutical composition of the present invention can be used to treat any cancer in which the effector mechanism of cytotoxic T cells is desired.For example, the humanized or chimeric antibody can be administered to cultured cells in vitro or ex vivo, or administered to a human subject, for example, in vivo, to treat or prevent disorders such as cancer, inflammatory disorders, or autoimmune disorders.As used herein, the term "subject" typically refers to a human who responds to the humanized or chimeric antibody or pharmaceutical composition.The subject can include, for example, a human patient with a disorder that can be corrected or improved by adjusting target function or directly or indirectly leading to cell killing.

[0337] In another aspect, the present invention provides a method for treating or preventing a disorder, such as cancer, in which T cell recruitment contributes to the treatment or prevention, comprising administering to a subject in need thereof a therapeutically effective amount of a humanized or chimeric antibody or pharmaceutical composition of the present invention. In this method, the humanized or chimeric antibody is typically administered to the subject in an amount effective to treat or prevent the disorder.

[0338] In one particular aspect, the present invention relates to a method for treating cancer, comprising administering to a subject in need thereof a humanized or chimeric antibody or a pharmaceutical composition of the invention as defined in any of the aspects and embodiments described herein.

[0339] In another aspect, the present invention relates to a use or a method as defined in any aspect or embodiment described herein, wherein the humanized or chimeric antibody is a bispecific antibody that specifically binds to both CD3 and a cancer-specific target, or a target overexpressed in or associated with cancer, such as HER2, CD19, EpCAM, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-MAA), and the disease is cancer, such as breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer, and squamous cell carcinoma of the head and neck, cervical cancer, pancreatic cancer, testicular cancer, malignant melanoma, soft tissue cancer (e.g. synovial sarcoma), low-grade or high-grade B-cell lymphoma, chronic lymphocytic leukemia or acute lymphocytic leukemia.

[0340] The effective dosage and dosage regimen of the humanized or chimeric antibody depends on the disease or condition being treated and can be determined by one skilled in the art.

[0341] A physician skilled in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician can start the dosage of the humanized or chimeric antibody used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Generally, an appropriate dosage of the composition of the present invention is the amount of humanized or chimeric antibody that is the minimum dose effective to produce a therapeutic effect in a particular dosage regimen. Such an effective dosage will generally depend on the factors described above.

[0342] For example, an "effective amount" for therapeutic use can be measured by its ability to stabilize disease progression. The ability of a compound to inhibit cancer can be evaluated, for example, in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of a humanized or chimeric antibody to inhibit cell growth or induce cytotoxicity in in vitro assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound, i.e., a therapeutic humanized or chimeric antibody or pharmaceutical composition of the present invention, can reduce tumor size or otherwise ameliorate symptoms in a subject. One skilled in the art would be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0343] An exemplary non-limiting range for a therapeutically effective amount of a humanized or chimeric antibody of the present invention is about 0.001 to 30 mg / kg, such as about 0.001 to 20 mg / kg, for example, about 0.001 to 10 mg / kg, for example, about 0.001 to 5 mg / kg, for example, about 0.001 to 2 mg / kg, for example, about 0.001 to 1 mg / kg, for example, about 0.001, about 0.01, about 0.1, about 1, about 5, about 8, about 10, about 12, about 15, or about 18 mg / kg.

[0344] Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, eg, administration near the target site.

[0345] Dosage regimens in the above-described methods and uses of treatment are adjusted to provide the optimum desired response (e.g., a therapeutic response), for example, by administering a single bolus or several divided doses over time, or the dose can be proportionally reduced or increased as required by the therapeutic situation.

[0346] In one embodiment, the efficacy of treatment is monitored, eg, at predetermined time points during therapy.

[0347] If desired, the effective daily dose of the pharmaceutical composition may be divided into two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In another embodiment, to minimize any undesirable side effects, the humanized or chimeric antibody or pharmaceutical composition is administered by slow continuous infusion over an extended period of time, e.g., 24 hours or more.

[0348] While it is possible for the humanized or chimeric antibodies of the present invention to be administered alone, it is preferable to administer the humanized or chimeric antibodies as a pharmaceutical composition as described above.

[0349] An effective dose of a humanized or chimeric antibody of the invention can also be administered using a dosing period of once per week, once per two weeks, or once per three weeks. The dosing period can be limited to, for example, 8 weeks, 12 weeks, or until clinical progression is established. Alternatively, an effective dose of a humanized or chimeric antibody of the invention can be administered every two weeks, every three weeks, or every four weeks.

[0350] In one embodiment, the humanized or chimeric antibody is administered in mg / m 2The humanized or chimeric antibody can be administered by infusion in a weekly dose calculated as follows: Dose (mg / kg) x 70:1.8. Such dosage can be based on the mg / kg dosage above, for example, according to the following formula: Dose (mg / kg) x 70:1.8. Such administration can be repeated, for example, 1 to 8 times, for example, 3 to 5 times. Administration can be by continuous infusion over a period of 2 to 24 hours, for example, 2 to 12 hours. In one embodiment, to reduce toxic side effects, the humanized or chimeric antibody can be administered by slow continuous infusion over an extended period of time, for example, 24 hours or more.

[0351] In one embodiment, the humanized or chimeric antibody can be administered in a weekly dose calculated as a fixed dose, up to eight times, e.g., four to six times, when administered once a week. Such a regimen can be repeated one or more times as needed, e.g., after six or twelve months. Such fixed doses can be based on the mg / kg dosages described above, assuming, for example, a body weight of 70 kg. Dosage can be determined or adjusted by measuring the amount of the humanized or chimeric antibody of the invention in the blood after administration, e.g., by collecting a biological sample and using an anti-idiotypic antibody targeting the binding region of the humanized or chimeric antibody of the invention.

[0352] In one embodiment, the humanized or chimeric antibody can be administered in a maintenance therapy, for example, once a week for a period of six months or more.

[0353] Humanized or chimeric antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of certain events in the progression of cancer, and / or reduce the risk of recurrence if the cancer is in remission.

[0354] Parenteral compositions can be formulated into dosage unit form to facilitate administration and ensure uniform dosage.Dosage unit form as used herein refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect together with necessary pharmaceutical carrier.The specifications of the dosage unit form of the present invention are determined by or directly depend on (a) the specific characteristics of active compound and the specific therapeutic effect to be achieved, and (b) the constraints inherent in the art when formulating such active compound for the treatment of susceptibility in individuals.

[0355] Humanized or chimeric antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of certain events in cancer progression, and / or reduce the risk of recurrence if the cancer is in remission. This can be particularly useful in patients where other biological factors make it difficult to locate a tumor known to be present.

[0356] Diagnostic Applications The humanized or chimeric antibodies of the present invention can also be used for diagnostic purposes, using compositions comprising the humanized or chimeric antibodies described herein. Accordingly, the present invention provides diagnostic methods and compositions that use the humanized or chimeric antibodies described herein. Such methods and compositions can be used for purely diagnostic purposes, such as detecting or identifying disease, or for purposes such as monitoring the progress of therapeutic treatment, monitoring disease progression, assessing post-treatment status, monitoring disease recurrence, and assessing the risk of developing disease.

[0357] In one aspect, the present invention relates to a method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, the method comprising administering to a subject a humanized or chimeric antibody of the invention, a composition of the invention, or a pharmaceutical composition of the invention, wherein optionally the humanized or chimeric antibody is labeled with a detectable agent.

[0358] In one aspect, the humanized or chimeric antibodies of the present invention are used ex vivo in the diagnosis of diseases, for example, where cells expressing the specific target of interest to which the humanized or chimeric antibody binds are indicative of the disease or involved in a pathogenesis process, by detecting the level of the target in a sample taken from a patient, or the level of cells expressing the target of interest on their cell surface. This can be achieved, for example, by contacting the test sample, optionally together with a control sample, with a humanized or chimeric antibody of the present invention under conditions that allow binding of the antibody to the target. Complex formation can then be detected (e.g., using ELISA). When a control sample is used along with the test sample, the levels of the humanized or chimeric antibody or antibody-target complex are analyzed in both samples, and a statistically significantly higher level of the humanized or chimeric antibody or antibody-target complex in the test sample indicates that the target in the test sample is present at a higher level compared to the control sample.

[0359] Examples of conventional immunoassays in which the humanized or chimeric antibodies of the present invention can be used include, but are not limited to, ELISA, RIA, FACS assay, plasmon resonance assay, chromatographic assay, tissue immunohistochemistry, Western blot, and / or immunoprecipitation.

[0360] Accordingly, in one embodiment, the invention relates to a method of diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, the method comprising administering to a subject an antibody, bispecific antibody, composition or pharmaceutical composition according to any aspect or embodiment described herein, wherein optionally the antibody is labeled with a detectable label.

[0361] In one aspect, the present invention provides a method for detecting the presence of a target or a cell expressing a target in a sample, comprising: contacting the sample with a humanized or chimeric antibody of the invention under conditions that allow binding of the humanized or chimeric antibody to a target in the sample; and a step of analyzing whether a complex has been formed The sample is typically a biological sample.

[0362] In one embodiment, the sample is a tissue sample known or suspected to contain a specific target and / or cells expressing said target. For example, in situ detection of target expression can be achieved by removing a histological specimen from a patient and administering a humanized or chimeric antibody of the present invention to such a specimen. The humanized or chimeric antibody can be administered by applying or overlaying the humanized or chimeric antibody to the specimen, which is then detected using appropriate means. Not only the presence of the target or target-expressing cells, but also the distribution of the target or target-expressing cells in the tissue under test can then be determined (e.g., in connection with assessing the spread of cancer cells). Those skilled in the art will readily appreciate that, using the present invention, any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0363] In the above assays, the humanized or chimeric antibody can be labeled with a detectable substance so that the bound antibody can be detected. Alternatively, the bound (primary) specific humanized or chimeric antibody can be detected with an antibody labeled with a detectable substance that binds to the primary specific humanized or chimeric antibody. Furthermore, the above assays can use diagnostic compositions comprising an antibody or bispecific antibody according to any aspect or embodiment described herein. Thus, in one aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to any aspect or embodiment described herein.

[0364] The level of a target in a sample can also be estimated by a competitive immunoassay that utilizes a target standard labeled with a detectable substance and an unlabeled target-specific humanized or chimeric antibody. In this type of assay, the biological sample, the labeled target standard, and the target-specific humanized or chimeric antibody are combined, and the amount of labeled target standard bound to the unlabeled target-specific humanized or chimeric antibody is determined. The amount of target in the biological sample is inversely proportional to the amount of labeled target standard bound to the target-specific humanized or chimeric antibody.

[0365] Suitable labels for target-specific humanized or chimeric antibodies, secondary antibodies, and / or target standards used in in vitro diagnostic techniques include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. An example of a luminescent material is luminol. Examples of suitable radioactive materials include fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. 125 I, 131 I, 35 S, and 3 H is one example.

[0366] In one aspect, the target-specific humanized or chimeric antibodies of the invention are used for in vivo imaging of target-expressing tissues, such as tumors. For in vivo methods, antibody fragments, such as (Fab')2, Fab, and Fab' fragments, are particularly advantageous because they have rapid distribution kinetics.

[0367] In vivo imaging can be performed by any suitable technique, for example, 99 Tc, 131 I, 111 Target-specific humanized or chimeric antibodies (e.g., antibodies or fragments) labeled with In or other gamma-emitting isotopes can be used to image the accumulation or distribution of the target-specific antibody in target-expressing tissues, such as tumors, with a gamma scintillation camera (e.g., Elscint Apex 409ECT device), typically using a low-energy high-resolution collimator or a low-energy universal collimator. 89 Zr, 76 Br, 18Labeling with F or other positron-emitting radionuclides can also be used to image the distribution of target-specific humanized or chimeric antibodies or antibody fragments in tumors using positron emission tomography (PET). Images obtained using such techniques can be used to assess the biodistribution of targets in patients, mammals, or tissues, for example, when using the target as a biomarker for the presence of cancer / tumor cells. Variations on this technique include the use of magnetic resonance imaging (MRI) to improve imaging over gamma camera techniques. Traditional immunoscintigraphy methods and principles are described, for example, in

[79] ,

[80] , and

[81] . Additionally or alternatively, such images can serve as the basis for surgical techniques to remove tumors. Furthermore, such in vivo imaging techniques allow for the identification and localization of tumors in situations where a patient is confirmed to have a tumor (e.g., by the presence of other biomarkers, metastases, etc.), but the tumor cannot be identified by traditional analytical techniques. All of these methods are features of the present invention.

[0368] The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful for detecting micrometastases in human patients (e.g., patients who have not previously been diagnosed with cancer or who are in recovery / remission from cancer).

[0369] In one aspect, the invention provides an in vivo imaging method in which a target-specific humanized or chimeric antibody of the invention is conjugated to a radiopaque substance to facilitate detection, the conjugated humanized or chimeric antibody is administered to a host, such as by injection into the bloodstream, and the presence and location of the labeled humanized or chimeric antibody in the host is assayed. By this technique and the other diagnostic methods described herein, the invention provides methods for screening for the presence of disease-associated cells in human patients or biological samples taken from human patients and / or for assessing the distribution of target-specific humanized or chimeric antibodies prior to target-specific ADC therapy.

[0370] For diagnostic imaging, radioisotopes can be attached to target-specific humanized or chimeric antibodies either directly or indirectly using intermediate functional groups. Useful intermediate functional groups include ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, e.g.,

[82] ).

[0371] In addition to radioisotopes and radiopaque substances, diagnostic methods can also be performed using target-specific antibodies conjugated to dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancing agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions) (see, e.g.,

[83] , which describes MRI techniques and the preparation of antibodies conjugated to MRI-enhancing agents). Such diagnostic / detection agents can be selected from agents and fluorescent compounds used in MRI. Loading a target-specific humanized or chimeric antibody with a radiometal or paramagnetic ion may require reacting it with a reagent bearing a long tail to which multiple chelating groups for binding the ions can be attached. Such tails can be polymers such as polylysine, polysaccharides, or other derivatized or derivatizable chains bearing pendant groups to which chelating groups can be attached, such as porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and other compounds known to be useful for this purpose. The chelate can be coupled to the target-specific humanized or chimeric antibody using standard chemistry.

[0372] Thus, the present invention provides diagnostic target-specific humanized or chimeric antibodies, wherein the target-specific humanized or chimeric antibodies are conjugated to an imaging agent (e.g., a magnetic resonance imaging, computed tomography, or ultrasound contrast enhancement agent) or a radionuclide, which may be, for example, a gamma-, beta-, alpha-, Auger-, or positron-emitting isotope.

[0373] In one aspect, the invention relates to a diagnostic composition comprising an antibody or bispecific antibody of the invention.

[0374] In yet another aspect, the present invention provides a kit for detecting the presence of a target antigen or a cell expressing a target antigen in a sample, comprising: a target-specific humanized or chimeric antibody of the present invention, and Instructions for use of the kit The present invention relates to a kit comprising:

[0375] Thus, in one aspect, the present invention comprises: a) contacting a sample with an antibody or bispecific antibody of the invention under conditions that allow for the formation of a complex between the antibody or bispecific antibody and CD3; and b) analyzing whether a complex is formed The present invention provides a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample, comprising:

[0376] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a target-specific humanized or chimeric antibody and one or more reagents for detecting the binding of the target-specific humanized or chimeric antibody to a target. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, where the enzymatic reaction produces a visualized product. In one embodiment, the present invention provides a diagnostic kit comprising one or more target-specific humanized or chimeric antibodies of the present invention, labeled or unlabeled, in a suitable container, incubation reagents for an indirect assay, and, depending on the nature of the label, a substrate or derivatizing agent for detection in such an assay. Control reagents and instructions for use may also be included.

[0377] Diagnostic kits for use with target-specific humanized or chimeric antibodies, such as labeled target-specific antibodies, to detect the presence of a target in a tissue sample or host can also be provided. In such diagnostic kits, as well as kits for therapeutic use described elsewhere herein, the target-specific humanized or chimeric antibody is typically provided in a container in lyophilized form, either alone or together with additional antibodies specific for the target cell or target peptide. A pharmaceutically acceptable carrier (e.g., an inert diluent) and / or its components, such as Tris, phosphate, or carbonate buffer, stabilizers, preservatives, biocides, inert proteins such as serum albumin, etc. (typically in a separate container for mixing), and additional reagents (also typically in separate containers) are typically also included. Certain kits also include a second antibody capable of binding to the target-specific humanized or chimeric antibody, typically in a separate container. The second antibody is typically conjugated to a label and formulated in a manner similar to the target-specific humanized or chimeric antibody of the present invention. Using the methods described above and elsewhere herein, target-specific humanized or chimeric antibodies can be used to define subsets of cancer / tumor cells and characterize such cells and associated tumor tissue.

[0378] Anti-idiotype antibodies In yet another aspect, the present invention relates to anti-idiotypic antibodies that bind to the humanized or chimeric antibodies of the invention described herein.

[0379] Anti-idiotypic (Id) antibodies are antibodies that generally recognize unique determinants associated with the antigen-binding site of an antibody. Anti-Id antibodies can be prepared by immunizing an animal of the same species and genotype as the source of the CD3 monoclonal antibody with the monoclonal antibody from which the anti-Id is to be prepared. The immunized animal is typically able to recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibodies that respond to these idiotypic determinants (anti-Id antibodies). Such antibodies are described, for example, in U.S. Pat. No. 4,699,880. Such antibodies are a further feature of the present invention.

[0380] Anti-Id antibodies can also be used as "immunogens" to elicit immune responses in yet another animal to produce so-called anti-anti-Id antibodies. Anti-anti-Id antibodies can be epitopically identical to the original monoclonal antibody that induced the anti-Id. Thus, by using antibodies directed against the idiotypic determinants of a monoclonal antibody, it is possible to identify other clones expressing antibodies with the same specificity. Anti-Id antibodies can be altered (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein with respect to the CD3-specific antibodies of the present invention. For example, a monoclonal anti-Id antibody can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Serum from these mice will typically contain anti-anti-Id antibodies with binding characteristics similar, if not identical, to those of the original / parent CD3 antibody.

[0381] array [Table 1] TIFF0007757247000004.tif226170TIFF0007757247000005.tif240170TIFF0007757247000006.tif46170 [Example]

[0382] Example 1: Generation of humanized CD3 antibodies and non-activating antibody variants Humanization of CD3 antibody Humanization of the murine CD3 antibody (US Pat. No. 8,236,308, herein referred to as IgG1-CD3) was performed by Antitope (Cambridge, UK) using their modified germline humanization (CDR grafting) technology (EP 0629240). Using this technology, four different VH chains (SEQ ID NOs: 6, 7, 8, and 9) and three different VL chains (SEQ ID NOs: 10, 11, and 12) were designed. By combining these four VH with three VL chains, 12 different antibodies were generated. These humanized variants are referred to herein as huCD3. Thus, a humanized variant comprising a VH and a VL of the present invention would be designated, for example, as IgG1-huCD3-H1L1, meaning that the particular variant is of the IgG1 isotype, is a humanized CD3, and comprises a VH amino acid sequence designated "H1" and defined in SEQ ID NO:6, and a VL amino acid sequence designated "L1" and defined in SEQ ID NO:10. Thus, for example, H1 would refer to the variable heavy chain region, VH1, and L1 would refer to the variable light chain region, VL1.

[0383] Specifically, in the Examples described herein, variants IgG1-huCD3-H1L1 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:6 and the VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H1L2 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:6 and the VL2 sequence shown in SEQ ID NO:11), IgG1-huCD3-H1L3 (humanized CD3 comprising the VH1 sequence shown in SEQ ID NO:6 and the VL3 sequence shown in SEQ ID NO:12), IgG1-huCD3-H3L3 (humanized CD3 comprising the VH3 sequence shown in SEQ ID NO:8 and the VL3 sequence shown in SEQ ID NO:12), IgG1-huCD3-H4L1 (humanized CD3 comprising the VH4 sequence shown in SEQ ID NO:9 and the VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H3L1 (humanized CD3 comprising the VH4 sequence shown in SEQ ID NO:11 and the VL2 sequence shown in SEQ ID NO:11), and IgG1-huCD3-H3L1 (humanized CD3 comprising the VH4 sequence shown in SEQ ID NO:11 and the VL2 sequence shown in SEQ ID NO:11). The following humanized CD3s were generated and tested: IgG1-huCD3-H3L3 (a humanized CD3 comprising the VH3 sequence shown in SEQ ID NO:8 and the VL1 sequence shown in SEQ ID NO:10), IgG1-huCD3-H3L3 (a humanized CD3 comprising the VH3 sequence shown in SEQ ID NO:8 and the VL3 sequence shown in SEQ ID NO:12), and IgG1-huCD3-H4L3 (a humanized CD3 comprising the VH4 sequence shown in SEQ ID NO:9 and the VL3 sequence shown in SEQ ID NO:12).

[0384] In some examples, an antibody containing the heavy and light chain variable region sequences of huCLB-T3 / 4 (SEQ ID NOs: 17 and 18, respectively) was used as a control antibody (Labrijn et al., PNAS 2013, 110:5145-50) and to test different combinations of inactivating mutations in the Fc region (see Examples 8-10). huCBL-T3 / 4 is a humanized version of the murine CD3 antibody CLB-T3 / 4 (Parren et al., Res Immunol. 1991, 142(9)749-63). Both sequences (SEQ ID NOs: 17 and 18) were cloned into the appropriate pcDNA3.3 (Invitrogen) expression vector and expressed by co-transfection into HEK293F cells. The resulting control antibody is designated IgG1-huCLB-T3 / 4.

[0385] In some examples, an antibody containing the heavy and light chain variable region sequences of the CD20 antibody 7D8 (SEQ ID NO:29 corresponding to the VH sequence and SEQ ID NO:30 corresponding to the VL sequence) was used as a positive control, which is designated "IgG1-CD20" when used in connection with the positive control.

[0386] These IgG1-CD3 (i.e., chimeric parent CD3 antibody), IgG1-huCD3 and IgG1-huCLB-T3 / 4 antibodies were used in monospecific and bispecific formats, and bispecific antibodies were generated as described below.

[0387] HER2 antibody In some examples, an antibody against HER2 was used. The VH and VL sequences (SEQ ID NOs: 19 and 20, respectively) of this HER2-specific antibody (antibody 169) have been previously described (WO2012 / 143524 [Genmab]; Labrijn et al., PNAS 2013, 110:5145-50). This antibody was used in both monospecific and bispecific formats and is designated "IgG1-HER2."

[0388] b12 antibody In some examples, antibody b12, a gp120-specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3)812-23), was used as a negative control and is designated "IgG1-b12."

[0389] Expression Antibodies were expressed as IgG1,κ or IgG1,λ with or without inactivating mutations as described below and mutations in the CH3 domain that allow for the generation of bispecific antibodies by the method described below: IgG1-HER2-K409R, IgG1-b12-K409R, IgG1-CD3-F405L. Plasmid DNA mixtures encoding both the heavy and light chains of the antibodies were transiently transfected into Freestyle HEK293F cells (Invitrogen, USA) using 293fectin (Invitrogen, USA) essentially as described by the manufacturer.

[0390] Antibody purification The culture supernatant was filtered through a 0.2 μm dead-end filter, loaded onto a 5 mL MabSelect SuRe column (GE Health Care), and eluted with 0.1 M sodium citrate-NaOH (pH 3). The eluate was immediately neutralized with 2 M Tris-HCl (pH 9) and dialyzed overnight against 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). Alternatively, following purification, the eluate was loaded onto a Hi Prep desalting column, and the antibody was exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun) buffer. After dialysis or buffer exchange, the sample was sterile filtered through a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE, and concentration was measured by absorbance at 280 nm. The purified antibody was stored at 2–8°C.

[0391] Generation of bispecific antibodies Bispecific antibodies were generated in vitro using DuoBody® platform technology, i.e., 2-MEA-induced Fab arm exchange as described in WO2011 / 147986 and Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2013, 5:962-973). To enable the production of bispecific antibodies by this method, IgG1 molecules were engineered to carry a single mutation in the CH3 domain: an F405L mutation in one parental IgG1 antibody (i.e., IgG1-CD3 antibody) and a K409R mutation in the other parental IgG1 antibody (i.e., HER2 or b12 antibody). To generate bispecific antibodies, these two parent antibodies were incubated with 25 mM or 75 mM 2-mercaptoethylamine-HCl (2-MEA) at a final concentration of 0.5 mg / mL for 5 hours at 31°C in a total volume of 500 μL of TE. The reduction reaction was stopped when the reducing agent 2-MEA was removed using a PD-10 column (GE Healthcare, Product No. 17-0851-01) equilibrated with 25 mL of PBS. Prior to desalting, 2 mL of PBS (B. Braun, Product No. 3623140) was added to the sample to adjust the volume to 2.5 mL. Elution was performed with 3.5 mL of PBS. The sample was collected in an Amicon Ultra centrifuge unit (30 kD MWCO, Millipore, Product No. UFC803096) and concentrated by centrifugation at 3000 × g for 8 minutes. The volume was adjusted to 500 μL with PBS (if necessary) and the sample was sterile filtered through a 0.2 μm filter (Millex-GV, product number SLGV004SL). The bispecific product was stored at 2-8°C.

[0392] In an alternative method yielding the same bispecific antibody, 100 μg of the two parent antibodies were mixed and incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 400 μL of PBS (B. Braun, product number 3623140) for 5 hours at 31°C to generate the bispecific antibody. The reduction reaction was stopped when the reducing agent 2-MEA was removed by washing four times with 400 μL of PBS by centrifugation at 13,000 × g for 10 minutes in an Amicon Ultra 0.5 ml centrifuge unit (10 kD MWCO, Millipore, product number UFC501096). The sample was collected in a new tube by inverting the filter and centrifuging at 1,000 g for 2 minutes. The volume was adjusted to 200 μL with PBS (if necessary). The absorbance at 280 nm (A280) of the bispecific product was measured to determine the final concentration. HPLC cation exchange chromatography (HPLC-CEX) (described in WO2013 / 060867) was performed to determine the amount of bispecific product. Samples were stored at 2-8°C.

[0393] The bispecific antibodies produced are referred to below as "K409R IgG1 backbone" and "F405L IgG1 backbone".

[0394] Deactivating mutations We generated several antibody variants with one or more amino acid substitutions in the Fc region. A non-activating Fc region prevents the antibody from interacting with Fc receptors present on blood cells such as monocytes or from interacting with C1q to activate the classical complement pathway. We tested antibody variants containing different combinations of amino acid substitutions in the Fc region to determine whether they reduced Fc activity. Up to five amino acid substitutions were introduced, including the mutations N297Q, L234A, L235A, L234F, L235E, D265A, and P331S. Substitutions at one or more of these five amino acid positions were introduced into the K409R and / or F405L IgG1 backbone. The following Fc region mutants of the huCLB-T3 / 4 antibody were generated: N297Q (referring to the N297Q substitution; designated IgG1-huCLB-T3 / 4-N297Q), LFLE (referring to the L234F / L235E substitution; designated IgG1-huCLB-T3 / 4-LFLE), LALA (referring to the L234A / L235A substitution; designated IgG1-huCLB-T3 / 4-LALA), LFLENQ (referring to the L234F / L235E / N297Q substitution; designated IgG1-huCLB-T3 / 4-LFLENQ), and LFLEDA (referring to the L234F / L235E / D265A substitution; designated IgG1-huCLB-T3 / 4-LFLEDA). ), DA (referring to a D265A substitution; termed IgG1-huCLB-T3 / 4-DA), DAPS (referring to a D265A / P331S substitution; termed IgG1-huCLB-T3 / 4-DAPS), DANQ (referring to a D265A / N297Q substitution; termed IgG1-huCLB-T3 / 4-DANQ), LFLEPS (referring to a L234F / L235E / P331S substitution; termed IgG1-huCLB-T3 / 4-LFLEPS), and LFLEDANQPS (referring to a L234F / L235E / D265A / N297Q / P331S substitution; termed IgG1-huCLB-T3 / 4-LFLEDANQPS).

[0395] Specifically, we engineered an IgG1-huCD3 antibody mutant with a non-activating Fc region by introducing a combination of three amino acid substitutions, designated LFLEDA, containing the mutations L234F, L235E, and D265A, into the K409R and F405L IgG1 backbone. The resulting non-activating antibody mutant is designated with the suffix "-LFLEDA."

[0396] Example 2: Binding of humanized CD3 antibodies and their non-activating mutants to CD3-expressing human and cynomolgus monkey T cell lines Purified variants of the humanized CD3 (huCD3) antibody and bispecific (bs) IgG1-huCD3xHER2 molecule with or without the LFLEDA mutation in the Fc region (see Example 1) were analyzed for binding to the human T cell line Jurkat (clone E6-1, ATCC® TIB-152™, LGC Standards GmbH, Wesel, Germany) or the cynomolgus monkey T cell line HSC-F (catalog number JCRB1164; Human Science Research Resources Bank, Osaka, Japan) by FACS analysis. In addition to the inactivating mutation LFLEDA, the antibody variants contained the F405L or K409R mutations as described in Example 1.

[0397] cells (1×10 5 cells / well) were incubated in polystyrene 96-well round-bottom plates (Greiner bio-one 650101) with serial dilutions of antibody preparations (ranging from 5 to 10,000 ng / mL in 3-fold dilutions) in 100 μL of PBS / 0.1% BSA / 0.02% azide for 30 min at 4°C.

[0398] After washing twice in PBS / 0.1% BSA / 0.02% azide, cells were incubated with 100 μL of secondary antibody at 4°C for 30 minutes. The secondary antibody used in all experiments was R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:100 in PBS / 0.1% BSA / 0.02% azide. Cells were then washed twice with PBS / 0.1% BSA / 0.02% azide, resuspended in 150 μL of PBS / 0.1% BSA / 0.02% azide, and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed by nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V5.04 software (GraphPad Software, San Diego, CA, USA).

[0399] FIG. 1A shows that binding of the IgG1λ-huCD3 mutants IgG1-huCD3-H1L1 (SEQ ID NOs: 6 and 10, respectively), IgG1-huCD3-H1L2 (SEQ ID NOs: 6 and 11, respectively), IgG1-huCD3-H1L3 (SEQ ID NOs: 6 and 12, respectively), IgG1-huCD3-H3L3 (SEQ ID NOs: 8 and 12, respectively), and IgG1-huCD3-H4L1 (SEQ ID NOs: 9 and 10, respectively) with wild-type Fc regions to Jurkat cells was observed for all mutants, and that the binding ability of IgG1-CD3-LFLEDA (the parent CD3 antibody described in Example 1 with a non-activating LFLEDA mutation) and IgG1-huCD3-H3L1-LFLEDA with a non-activating LFLEDA mutation was similar to that of the huCD3 mutants with wild-type Fc regions. Binding of IgG1-huCLB-T3 / 4, included as a positive control, was stronger compared to the IgG1-huCD3 variant to Jurkat cells. No binding was observed with the negative control antibody IgG1-b12.

[0400] Figure 6A shows that the binding of IgG1-CD3-LFLEDA (the parent CD3 antibody described in Example 1 with a non-activating LFLEDA mutation), IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA to Jurkat cells was similar to that of huCD3 mutants with wild-type Fc regions. Binding of IgG1-huCLB-T3 / 4, included as a positive control, to Jurkat cells was stronger than that of the IgG1-huCD3 mutants at low antibody concentrations but similar at high antibody concentrations. Overall, the humanized CD3 mutants maintained the CD3 binding ability of the IgG1-CD3 antibody. No binding was observed with the negative control antibody IgG1-b12.

[0401] Figure 1B shows that the bispecific antibody variants bsIgG1 CD3xHER2, bsIgG1 CD3xb12-LFLEDA, and bsIgG1 huCD3-H3L1xHER2-LFLEDA also bind to Jurkat cells. The maximum binding values ​​of these bispecific antibodies are higher than the maximum binding values ​​of the monospecific antibodies. The EC50 concentrations of the bispecific antibodies are 6- to 10-fold higher. Again, no binding was observed with the negative control antibody IgG1-b12.

[0402] Figure 6B shows that the bispecific non-activating Fc antibody variants bsIgG1 huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA also bind to Jurkat cells. The maximum binding values ​​of these bispecific antibodies are higher than those of the monospecific antibodies. The EC50 concentrations of the bispecific antibodies are 4- to 10-fold higher. The binding values ​​are higher for the bispecific antibodies because monovalent binding allows more antibody to accumulate on the cell surface. Again, no binding was observed for the negative control antibody IgG1-b12.

[0403] Figure 2A shows that the IgG1-huCD3 mutants with wild-type Fc regions, IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1, as well as IgG1-CD3-LFLEDA and IgG1-huCD3-H3L1-LFLEDA, showed similar binding to the cynomolgus T cell line HSC-F. No binding was observed for the control antibody huCLB-T3 / 4, which does not cross-react with cynomolgus CD3, or the negative control antibody IgG1-b12.

[0404] Figure 7A shows that the IgG1-huCD3 mutants IgG1-CD3-LFLEDA, IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA had similar binding to the cynomolgus monkey T cell line HSC-F. No binding was observed with the negative control antibody IgG1-b12.

[0405] Figure 2B shows that the bispecific antibody variants bsIgG1 CD3xHER2 and bsIgG1 huCD3-H3L1-LFLEDA also bind to HSC-F cells. The maximum binding values ​​of these bispecific antibodies are higher than those of the monospecific anti-CD3 variants. The EC50 concentrations of the bispecific antibodies are 10- to 12-fold higher than those of the monospecific anti-CD3 antibodies. Again, no binding was observed with the negative control antibody IgG1-b12.

[0406] Figure 7B shows that the bispecific non-activating Fc antibody variants bsIgG1 huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA also bind to HSC-F cells. The maximum binding values ​​of these bispecific antibodies are higher than those of the monospecific anti-CD3 variants. The EC50 concentrations of the bispecific antibodies are 3- to 6-fold higher than those of the monospecific anti-huCD3 antibodies. Again, no binding was observed with the negative control antibody IgG1-b12.

[0407] Example 3: T cell activation by humanized CD3 antibody variants CD69 expression is an early marker of T cell activation. CD3 antibodies can mediate cross-linking between T cells and immune cells by binding CD3 expressed by T cells and Fc receptors expressed by immune cells via the antibody's Fc region (e.g., IgG1 Fc region). This can lead to T cell activation and induction of CD69. Antibody variants containing a non-activating Fc region (LFLEDA mutation) do not bind to Fc receptors. Therefore, it was predicted that non-activating CD3 antibodies would not induce T cell activation and CD69 expression because the non-activating Fc region does not bind to immune cells expressing Fc receptors and therefore cannot cross-link T cells and immune cells.

[0408] To determine the initial activation of T cells after incubation with humanized CD3 (huCD3) variants with and without LFLEDA mutations in the Fc region, CD69 expression on T cells was assessed by FACS analysis. The LFLEDA variants contain the F405L or K409R mutations described in Example 1 in addition to the non-activating mutations.

[0409] PBMCs were isolated from whole blood or buffy coat by density gradient separation using Leucosep tubes (no. 227290; Greiner Bio-one, Alphen aan den Rijn, The Netherlands), washed with PBS, and resuspended in culture medium.

[0410] Dose-response series of huCD3 antibody variants, negative control (IgG1-b12), and positive controls (IgE-huCD3 and parental IgG1-CD3) were prepared in culture medium (10-fold dilutions ranging from 0.1 to 1,000 ng / mL) and added to wells of 96-well round-bottom plates containing human or cynomolgus monkey PBMCs. After 16–24 h of incubation, cells were pelleted by centrifugation, and the supernatants (containing cytokines) were collected and stored at −20°C. Cells were then washed with PBS / 0.1% BSA / 0.02% azide and stained for 30 min at 4°C with mouse anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, The Netherlands; a T cell marker) and mouse anti-human CD69-APC (340560; BD Biosciences, Franklin Lakes, NJ), which cross-react with cynomolgus monkey CD28 and CD69. Unbound antibody was removed by washing twice with PBS / 0.1% BSA / 0.02% azide. Cells were resuspended in 150 μL / well, and CD69 expression on CD28-positive cells was measured using a FACS Canto II (BD Biosciences).

[0411] Figure 3 shows that IgG1-CD3 with a wild-type IgG1 Fc region (as described in Example 1) and the humanized IgG1-huCD3 variant induced similar levels of CD69 expression in human-derived (Figure 3A) and cynomolgus monkey-derived (Figure 3B) T cells. The non-activating (LFLEDA) IgG1-CD3-LFLEDA and IgG1-huCD3-H3L1 variants induced low levels of CD69 expression in human T cells. No CD69 expression was induced by the non-activating IgG1-huCD3 variant in cynomolgus monkey T cells. The control antibody IgG1-b12 also did not induce CD69 expression in human or cynomolgus monkey T cells.

[0412] Figure 8 shows that the non-activating (LFLEDA) IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA mutants induced low levels of CD69 expression in human T cells. Figures 8A and 8B show the induction of CD69 expression in T cells derived from cynomolgus monkeys. The observed slight activation by the non-activating mutants may be due to cross-linking of CD3 molecules due to bivalent binding of the CD3 antibody. This explanation is supported by the observation that activation was reduced at the highest concentration where antibody binding was monovalent. The control antibody IgG1-b12 also did not induce CD69 expression in human or cynomolgus monkey T cells.

[0413] Figures 8C and 8D show that the nonactivating bispecific antibody variants bsIgG1-huCD3-H3L1xHER2-LFLEDA, bsIgG1-huCD3-H3L3xHER2-LFLEDA, bsIgG1-huCD3-H1L1xHER2-LFLEDA, bsIgG1-huCD3-H1L3xHER2-LFLEDA, bsIgG1-huCD3-H4L1xHER2-LFLEDA, and bsIgG1-huCD3-H4L3xHER2-LFLEDA do not induce CD69 expression in T cells from humans (Figure 8C) or cynomolgus monkeys (Figure 8D), although some induction of CD69 expression was observed at higher antibody concentrations.

[0414] Example 4: T cell proliferation induced by humanized CD3 antibody variants The effect of humanized CD3 (huCD3) antibody variants (described in Example 1) on the proliferation of human and cynomolgus T cells was assessed using the Roche Applied Science Cell Proliferation ELISA Kit (BrdU Kit, no. 11647229001; Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.

[0415] Human or cynomolgus monkey PBMCs isolated from whole blood or buffy coats were incubated in 96-well culture plates with serial dilutions of IgG1 huCD3 antibody variants (ranging from 0.1 to 1,000 ng / mL in 10-fold dilutions). IgE-CD3 and IgG1-huCLB-T3 / 4 were included as positive controls, and IgG1-b12 was included as a negative control. After 3 days of incubation with the antibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added to the medium and the plates were incubated for 5 hours. Cells were then pelleted by centrifugation, and the supernatants were collected and stored at -20°C. The plates were then dried and stored at 4°C until ELISA was performed.

[0416] BrdU incorporation into DNA was determined by ELISA according to the manufacturer's instructions (Roche Applied Science). After fixing the cells to the plate, the plate was incubated with a peroxidase-conjugated anti-BrdU antibody for 90 minutes at room temperature. The plate was washed with PBST, and binding was detected using ABTS buffer (rather than the TMB solution provided with the kit). Color development was stopped after 30 minutes by adding 2% oxalic acid to the wells. OD405nm was then measured using an EL808 ELISA reader.

[0417] Figure 4 shows that incubation of PBMCs with parental IgG1-CD3 and humanized IgG1-huCD3 variants with wild-type IgG1 Fc regions induced robust proliferation of human T cells (Figure 4A) and cynomolgus T cells (Figure 4B) even at extremely low antibody concentrations. Incubation with the non-activating LFLEDA variants of the IgG1-huCD3 antibody did not induce proliferation of human T cells (Figures 4A and 9A) or cynomolgus T cells (Figures 4B and 9B). Thus, although the non-activating variants of the IgG1-huCD3 antibody induced low levels of CD69 expression in human T cells (as shown in Example 3), these non-activating IgG1-huCD3 variants did not induce human T cell proliferation.

[0418] Figures 9C and 9D show that the non-activating bispecific antibody variants bsIgG1-huCD3-H3L1xHER2-LFLEDA, bsIgG1-huCD3-H3L3xHER2-LFLEDA, bsIgG1-huCD3-H1L1xHER2-LFLEDA, bsIgG1-huCD3-H1L3xHER2-LFLEDA, bsIgG1-huCD3-H4L1xHER2-LFLEDA, and bsIgG1-huCD3-H4L3xHER2-LFLEDA do not induce proliferation of T cells isolated from humans (Figure 9C) and cynomolgus monkeys (Figure 9D).

[0419] Example 5: In vitro T cell-mediated cytotoxicity induced by humanized CD3 antibody variants Tumor-specific T cell-mediated cytotoxicity can be mediated by bispecific antibodies that bind to CD3 with one arm and to a tumor-specific target such as HER2 with the other. Simultaneous binding of a bispecific antibody to both T cells and tumor cells will lead to T cell activation and tumor cell-specific cytotoxicity. In this example, T cell-mediated cytotoxicity against HER2-positive tumor cells was evaluated using a bispecific antibody against CD3 (humanized variant) and HER2.

[0420] Therefore, AU565 (human breast cancer) cells were cultured in RPMI 1640 supplemented with 10% (vol / vol) heat-inactivated CCS, 1.5 g / L sodium bicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g / L glucose (Sigma), 50 IU / mL penicillin, and 50 μg / mL streptomycin. The cell line was maintained at 37°C in a 5% (vol / vol) CO2 humidified incubator. After culturing AU565 cells until near confluence, the cells were trypsinized, resuspended in culture medium, and passed through a cell strainer to obtain a single-cell suspension. 5 × 10 cells were placed in each well of a 96-well culture plate. 4 Cells were seeded and allowed to adhere to the plates by incubating at least 3 hours at 37°C and 5% CO2.

[0421] Human or cynomolgus monkey PBMCs were isolated from whole blood or buffy coats. Isolated PBMCs were washed with PBS, resuspended in culture medium, and added to AU565 tumor cells at a 1:1 ratio in a 96-well plate. The percentage of T cells present in PBMCs was measured by FACS analysis using a mouse anti-human CD3-PerCP (BD, no. 345766) antibody (for T cell staining), which cross-reacts with cynomolgus monkey CD3. The T cell content in the PBMC populations used was typically 50-60%.

[0422] Dilution series of the bispecific antibody variants bsIgG1 CD3xHER2-LFLEDA, bsIgG1 CD3xb12-LFLEDA, bsIgG1 huCD3-H3L1xHER2-LFLEDA, bsIgG1-huCD3-H3L3xHER2-LFLEDA, bsIgG1-huCD3-H1L1xHER2-LFLEDA, bsIgG1-huCD3-H1L3xHER2-LFLEDA, bsIgG1-huCD3-H4L1xHER2-LFLEDA, and bsIgG1-huCD3-H4L3xHER2-LFLEDA (final concentrations ranging from 0.001 ng / mL to 10,000 ng / mL) were prepared in culture medium and added to the plates. IgG1-HER2-LFLEDA and IgG1-b12 were included as controls. In addition to the inactivating mutation, the LFLEDA antibody mutants contain the F405L or K409R mutation for preparation in a bispecific format (see Example 1). Plates were incubated at 37°C and 5% CO2 for 3 days. As a benchmark for 100% tumor cell killing, cells were incubated with 1 μM staurosporine (product number S6942-200, Sigma). Plates were washed twice with PBS, and 150 μL of culture medium containing 10% Alamar Blue was added to each well. Plates were incubated at 37°C and 5% CO2 for 4 hours. Absorbance at 590 nm was measured (Envision, Perkin Elmer, Waltham, MA).

[0423] The bispecific CD3×HER2-LFLEDA antibody variants (bsIgG1-huCLB-T3 / 4×HER2-LFLEDA and bsIgG1-CD3×HER2-LFLEDA) induced killing of AU565 cells at low concentrations using either human effector cells (Figure 5A) or cynomolgus effector cells (Figure 5B). The CD3 bispecific control antibody huCLB-T3 / 4×HER2-LFLEDA, which does not cross-react with cynomolgus CD3, induced killing of AU565 cells only when human PBMCs were used (Figure 5A). Accordingly, target cell killing was not observed when cynomolgus effector cells were used in the assay (Figure 5B). Incubation with the monospecific IGG1-b12 or IgG1-HER2-LFLEDA or bsIgG1-CD3×b12-LFLEDA antibodies did not induce nonspecific killing of target cells.

[0424] The bispecific antibody variants bsIgG1 huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA induced killing of AU565 cells at low concentrations using human effector cells (Figure 10A) or cynomolgus monkey effector cells (Figure 10B). Incubation with monospecific IgG1-b12 or IgG1-HER2-LFLEDA antibodies did not induce nonspecific target cell killing (Figures 10A and B). Thus, humanized CD3 variants containing a nonactivating Fc region do not induce nonspecific target cell killing. This demonstrates that variants containing a non-activating Fc region can be used to ensure targeted T cell activation and thus avoid non-targeted T cell activation.

[0425] Example 6: Activation of Rhesus T Cells by Humanized CD3 Antibody Variants To determine the initial activation of T cells after incubation with humanized CD3 (huCD3) antibody variants with wild-type IgG1 Fc regions, CD69 expression on rhesus T cells was assessed. Isolation of rhesus PBMCs and assessment of CD69 expression by flow cytometry were performed as described in Example 3.

[0426] Figure 11 shows that the humanized CD3 antibody variants IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, IgG1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1 induced CD69 expression in T cells from rhesus monkeys to the same level as IgG1-CD3 (described in Example 1). The negative control antibody IgG1-b12 did not induce CD69 expression in rhesus monkey T cells. Therefore, the huCD3 variants of the present invention can be used in experiments using rhesus monkey CD3. The huCD3 variants cross-react with rhesus monkey CD3.

[0427] Example 7: T cell activation by non-activating mutants of huCLB-T3 / 4 To determine the initial activation of T cells after incubation with IgG1-huCLB-T3 / 4 mutants with mutations in the Fc region (see Example 1), CD69 expression on T cells was assessed by FACS analysis.

[0428] PBMCs were isolated from whole blood or buffy coat by density gradient separation using Leucosep tubes (no. 227290; Greiner Bio-one, Alphen aan den Rijn, The Netherlands), washed with PBS, and resuspended in culture medium.

[0429] Dose-response series of IgG1-huCLB-T3 / 4 mutants, a negative control (IgG1-huCLB-T3 / 4-Fab), and a positive control (IgE-huCLB-T3 / 4) were prepared in culture medium (3-fold dilutions ranging from 1 to 1,000 ng / mL) and added to wells of a 96-well round-bottom plate containing PBMCs. After 16–24 h of incubation, cells were pelleted by centrifugation, and the supernatant (containing cytokines) was collected and stored at −20°C. Cells were then washed with PBS / 0.1% BSA / 0.02% azide and stained with mouse anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, The Netherlands; a T cell marker) and mouse anti-human CD69-APC (340560; BD Biosciences, Franklin Lakes, NJ) for 30 min at 4°C. Unbound antibody was removed by washing twice with PBS / 0.1% BSA / 0.02% azide. Cells were resuspended in 150 μL / well, and CD69 expression on CD28-positive cells was measured using a FACS Canto II (BD Biosciences).

[0430] Figure 12A shows that cells incubated with IgE-huCLB-T3 / 4, IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA, and IgG1-huCLB-T3 / 4-DAPS expressed high levels of CD69. Incubation with IgG1-huCLB-T3 / 4-N297Q induced somewhat lower levels of CD69 compared with wild-type IgG1-huCLB-T3 / 4, and incubation with IgG1-huCLB-T3 / 4-LFLE and IgG1-huCLB-T3 / 4-LFLEPS induced even lower levels of CD69. Incubation of PBMCs with IgG1-CD3 Fab, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1-huCLB-T3 / 4-DANQ, and IgG1-huCLB-T3 / 4-LFLEDANQPS antibodies did not induce any CD69 expression on T cells.

[0431] Figure 12B shows that cells incubated with IgE-huCLB-T3 / 4 and IgG1-huCLB-T3 / 4 showed high CD69 expression. Incubation with IgG1-huCLB-T3 / 4-LALA induced somewhat lower levels of CD69 compared with wild-type IgG1-huCLB-T3 / 4, and incubation with IgG1-huCLB-T3 / 4-LFLEDA and IgG1-b12 (negative control) did not induce any CD69 expression on T cells.

[0432] Example 8: T cell proliferation by inactivating mutants of huCLB-T3 / 4 The effect of huCLB-T3 / 4 mutants (described in Example 1) on T cell proliferation was assessed using a Roche Applied Science Cell Proliferation ELISA Kit (BrdU Kit, no. 11647229001; Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions.

[0433] PBMCs isolated from whole blood or buffy coats were incubated with serial dilutions of IgG1-CD3 mutants (ranging from 0.1 to 1,000 ng / mL) in 96-well culture plates. IgE-CD3 and IgG1-CD3 were included as positive controls, and IgG1-b12 (with the K409R mutation to generate a bispecific antibody) was included as a negative control. After 3 days of incubation with the antibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added to the medium and the plates were incubated for 5 hours. Cells were then pelleted by centrifugation, and the supernatants were collected and stored at -20°C. The plates were then dried and stored at 4°C until ELISA was performed.

[0434] BrdU incorporation into DNA was determined by ELISA according to the manufacturer's instructions (Cell Proliferation ELISA, BrdU Kit, No. 11647229001; Roche Applied Science). After fixing the cells to the plate, the plate was incubated with a peroxidase-conjugated anti-BrdU antibody for 90 minutes at room temperature (RT). The plate was washed with PBST, and binding was detected using ABTS buffer (rather than the TMB solution provided with the kit). Color development was stopped after 30 minutes by adding 2% oxalic acid to the wells. OD405nm was then measured using an EL808 ELISA reader.

[0435] Figure 13A shows that incubation of PBMCs with IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA, and IgG1-huCLB-T3 / 4-DAPS induced robust T cell proliferation, even at very low antibody concentrations. Incubation with IgG1-huCLB-T3 / 4-N297Q induced dose-dependent proliferation comparable to the IgE-huCLB-T3 / 4 positive control. Incubation of PBMCs with IgG1-huCLB-T3 / 4-Fab, IgG1-b12-N297Q, IgG1-huCLB-T3 / 4-LFLE, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1-huCLB-T3 / 4-LFLEPS, IgG1-huCLB-T3 / 4-DANQ, and IgG1-huCLB-T3 / 4-LFLEDANQPS antibodies did not induce T cell proliferation.

[0436] Figure 13B shows that incubation of PBMCs with IgG1-huCLB-T3 / 4 induced robust T cell proliferation, even at very low antibody concentrations. Incubation with IgE-huCLB-T3 / 4 (positive control) and IgG1-huCLB-T3 / 4-LALA induced dose-dependent proliferation. Incubation of PBMCs with IgG1-huCLB-T3 / 4-LFLEDA did not induce T cell proliferation.

[0437] Based on the results of Examples 7 and 8, the subset of mutants that appeared to be least activating were subjected to further analysis.

[0438] Example 9: In vitro T cell-mediated cytotoxicity induced by the non-activating antibody variant huCLB-T3 / 4 AU565 (human breast cancer) cells were cultured in RPMI 1640 supplemented with 10% (vol / vol) heat-inactivated CCS, 1.5 g / L sodium bicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g / L glucose (Sigma), 50 IU / mL penicillin, and 50 μg / mL streptomycin. The cell line was maintained at 37°C in a 5% (vol / vol) CO2 humidified incubator. AU565 cells were cultured until near confluence. Cells were trypsinized, resuspended in culture medium, and passed through a cell strainer to obtain a single-cell suspension. 5 × 10 cells were added to each well of a 96-well culture plate. 4 Cells were seeded and allowed to adhere to the plates by incubating at least 3 hours at 37°C and 5% CO2.

[0439] Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteers using Leucosep 30 mL tubes according to the manufacturer's protocol (Greiner Bio-one). Isolated PBMCs were washed with PBS, resuspended in culture medium, and added to AU565 tumor cells at a 1:1 ratio in a 96-well plate. The percentage of T cells present in PBMCs was measured by FACS analysis using mouse anti-human CD3-PerCP (BD, no. 345766) antibody (for T cell staining). The T cell content in the PBMC population used was typically 50-60%.

[0440] Dilution series (final concentrations ranging from 0.004 ng / mL to 1000 ng / mL) of IgG1-b12, IgG1-huCLB-T3 / 4, IgG1-HER2, and bispecific huCLB-T3 / 4×b12 and huCLB-T3 / 4×HER2 antibodies expressed as different Fc variants, wild-type, N297Q, LFLE, LALA, LFLENQ, LFLEDA, DANQ, and LFLEDENQPS, were prepared in culture medium and added to the plates. Plates were incubated at 37°C and 5% CO for 3 days. As a standard for 100% tumor cell killing, cells were incubated with 1 μM staurosporine (Sigma-Aldrich S6942-200).

[0441] After incubation, the supernatant was removed and stored at -20°C. The plates were washed twice with PBS, and 150 μL of culture medium containing 10% Alamar Blue was added to each well. The plates were incubated at 37°C and 5% CO2 for 4 hours. The absorbance at 590 nm was measured (Envision, Perkin Elmer, Waltham, MA).

[0442] Two experiments were performed using PBMCs from different donors. In the first experiment, the Fc variants N297Q, LFLE, LFLENQ, LFLEDA, DANQ, and LFLEDANQPS were tested (Figures 14A-G). In the second experiment, the Fc variants LFLEDA and LALA were tested (Figures 15A-C). Both experiments included an antibody with a wild-type Fc domain as a reference. Incubation with wild-type monospecific IgG1-huCLB-T3 / 4 or bispecific huCLB-T3 / 4xb12 antibodies induced nonspecific killing of target cells (Figures 14A-G and 15A-C). Monospecific IgG1-huCLB-T3 / 4 and the bsIgG1-huCLB-T3 / 4xb12 mutants N297Q (Figure 14A-G) and LALA (Figure 15A-C) still induced some nonspecific target cell killing, but to a lesser extent than the wild-type antibodies tested in the same experiments. None of the other IgG1-huCLB-T3 / 4 or bsIgG1-huCLB-T3 / 4xb12 antibodies tested with deactivating mutations induced nonspecific target cell killing (Figures 14A-G and 15A-C).

[0443] All bispecific huCLB-T3 / 4×HER2 antibodies induced dose-dependent killing of AU565 cells with at least comparable potency compared to wild-type bispecific huCLB-T3 / 4×HER2 antibodies lacking deactivating mutations (Figures 14A-G and 15A-C). Maximal killing occurred at very low concentrations.

[0444] Wild-type or inactivating mutants of the monospecific b12 or HER2 antibodies did not induce cytotoxicity (FIGS. 14A-G and 15A-C), as expected.

[0445] Example 10: Evaluation of C1q binding to non-activating antibody mutants of huCLB-T3 / 4 The interaction of C1q with antibody bound to target cells is the first step in the classical pathway of complement activation. Because wild-type IgG1 harbors an interaction site for C1q, we assessed the interaction of C1q with these non-activating IgG1 mutants by ELISA.

[0446] Dilution series (ranging from 7 to 30,000 ng / mL at 4-fold dilutions) of IgG1-huCLB-T3 / 4, bsIgG1-huCLB-T3 / 4×HER2, and IgG1-CD20 (positive control) and their non-activating antibody variants described in Example 1 above were coated onto 96-well Microlon ELISA plates (Greiner, Germany) overnight at 4° C. Plates were washed and blocked with PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. Plates were sequentially incubated with 3% pooled human serum (Sanquin, Product No. M0008) at 37°C for 1 hour, 100 μL / well of rabbit anti-human C1q (DAKO, Product No. A0136, 1:4,000) at room temperature for 1 hour, and 100 μL / well of swine anti-rabbit IgG-HRP (DAKO, Product No. P0399, 1:10,000) as the detection antibody for 1 hour at room temperature, with washing between incubations. Detection was performed by adding 1 mg / mL of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) for approximately 30 minutes. The reaction was stopped by adding 100 μL of 2% oxalic acid. Absorbance at 405 nm was measured using a microplate reader (Biotek, Winooski, VT). Log-transformed data were analyzed by fitting to a variable slope sigmoidal dose-response curve using GraphPad Prism software.

[0447] Figure 16A shows that antibodies IgG1-CD20 and IgG1-huCLB-T3 / 4, which contain wild-type IgG1 Fc regions, exhibited C1q binding. None of the antibody variants containing inactivating mutations evaluated (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS, LFLEDANQPS, LALA) exhibited detectable C1q binding.

[0448] Figure 16B shows that the antibody bsIgG1-huCLB-T3 / 4xHER2, which contains a wild-type IgG1 Fc region, exhibited C1q binding. None of the antibody variants with inactivating mutations evaluated (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS, LFLEDANQPS, LALA) exhibited detectable C1q binding.

[0449] Figures 16C and 16D show that antibodies IgG1-CD20, IgG1-huCLB-T3 / 4, and bsIgG1-huCLB-T3 / 4×HER2, which contain wild-type IgG1 Fc regions, exhibited C1q binding. No C1q binding was detected for antibody mutants with deactivating mutations (LFLEDA and LALA).

[0450] Example 11: Pharmacokinetic (PK) analysis of non-activating antibody mutants Mice in this study were housed in a barrier unit at the Central Laboratory Animal Facility (Utrecht, The Netherlands) in filter-top cages with free access to water and food. All experiments were approved by the Utrecht University Animal Ethics Committee. Three mice per group were housed in 7- to 10-week-old CB-17 SCID mice (CB-17 / Icr-Prkdc <scid>Mice (IcrIcoCrl, Charles River) were intravenously injected with 100 μg of wild-type antibody (IgG1-huCLB-T3 / 4, IgG1-HER2, or bsIgG-huCLB-T3 / 4×HER2) or its inactivating mutant (LALA, LFLEDA, LFLENQ, DANQ, or LFLEDANQPS). 50 μL blood samples were collected from the saphenous vein 10 min, 4 h, 1 day, 2 days, 7 days, 14 days, and 21 days after antibody administration. Blood was collected into heparin-containing vials and centrifuged at 10,000 × g for 5 min. Plasma was stored at -20°C until antibody concentration was determined.

[0451] Human IgG concentrations were determined using a total hIgG sandwich ELISA. For this assay, mouse mAb anti-human IgG-kappa clone MH16 (no. M1268, CLB Sanquin, The Netherlands) was used as the capture antibody, coated at 2 μg / mL onto 96-well Microlon ELISA plates (Greiner, Germany). After blocking the plates with PBS supplemented with 0.2% bovine serum albumin, serially diluted samples were added in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin) and incubated for 1 h at room temperature (RT) on a plate shaker. The plates were then incubated with goat anti-human IgG immunoglobulin (no. 109-035-098, Jackson, West Grace, PA) and developed with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was stopped after 30 min by adding 2% oxalic acid to the wells. The absorbance at 405 nm was measured in a microplate reader (Biotek, Winooski, VT).

[0452] Plasma clearance rate (mL / day / kg) was calculated based on the area under the curve (AUC) according to the following equation: TIFF0007757247000007.tif10128

[0453] Data analysis was performed using Graphpad prism software.

[0454] Figure 17A shows that antibody variants N297Q, DANQ, LFLENQ, and LFLEDANQPS produced lower plasma human IgG concentrations compared to the wild-type antibody, suggesting more rapid clearance. Antibody variants LFLEDA and LALA produced plasma human IgG concentrations similar to those of the wild-type antibody.

[0455] Figure 17B shows that the plasma clearance rates of antibody variants N297Q, DANQ, and LFLENQ were 2-3 fold higher than that of the wild-type antibody. The clearance rate of antibody variant LFLEDANQPS was 3-5 fold higher than that of the wild-type antibody. The plasma clearance rates of antibody variants LFLEDA and LALA were similar to that of the wild-type antibody.

[0456] Example 12: In vitro immunogenicity evaluation of the IgG1-LFLEDA backbone To determine the clinical immunogenic potential of the IgG1-LFLEDA-K409R backbone, Antitope's EpiScreen™ platform was applied to IgG1-HER2-LFLEDA. Briefly, PBMCs were isolated from a cohort of 50 HLA-typed healthy donors representative of European and North American populations. Following CD8+ T cell depletion, PBMC preparations were individually frozen and stored. Thawed PBMCs were then cultured and co-cultured with IgG1-HER2-LFLEDA-K409R or one of the control samples (IgG1-HER2 or IgG1-HER2-LFLEDA-K409R) for 5–8 days. The ability of the samples to elicit CD4+ T cell responses was assessed by measuring cell proliferation ([3 H ]-thymidine incorporation) and IL-2 production (ELISpot assay). Donors were considered positive if they showed a response with a stimulation index (SI; signal / baseline signal) of ≥ 1.9 in both assays.

[0457] EpiScreen™ analysis revealed that IgG1-HER2-LFLEDA produced positive CD4+ T cell responses in four donors (8%), comparable to the four (8%) and three (6%) positive responses for IgG1-HER2 and IgG1-HER2-LFLE, respectively (Figure 18). Thus, IgG1-HER2-LFLEDA-K409R (as well as IgG1-HER2 and IgG1-HER2-LFLE-K409R) demonstrated low immunogenic potential, with response frequencies below 10%. The positive control, humanized A33 (e.g.,

[84] ), was used as a clinical benchmark control antibody. This humanized A33 has demonstrated high levels of immunogenicity in the clinic, typically eliciting 20–30% T cell responses in the EpiScreen assay. In one aspect, the present invention provides: [Item 1] A humanized or chimeric antibody that binds to human CD3, comprising a binding region comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NO:4, sequence GTN, and sequence shown in SEQ ID NO:5, respectively. [Item 2] The VH region is (a) the VH sequence shown in SEQ ID NO:6; (b) the VH sequence shown in SEQ ID NO:8; (c) the VH sequence shown in SEQ ID NO:7, and (d) VH sequence shown in SEQ ID NO:9 2. The antibody of item 1, having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in a VH sequence selected from the group consisting of: [Item 3] The VL region is (a) the VL sequence shown in SEQ ID NO: 10; (b) the VL sequence shown in SEQ ID NO:11, and (c) VL sequence shown in SEQ ID NO: 12 The antibody of any one of the preceding claims, having at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in a VL sequence selected from the group consisting of: [Item 4] The VH region is (a) the VH sequence shown in SEQ ID NO:6; (b) the VH sequence shown in SEQ ID NO:8; (c) the VH sequence shown in SEQ ID NO:7, and (d) VH sequence shown in SEQ ID NO:9 The antibody of any one of the preceding claims, selected from the group consisting of: [Item 5] The VL region is (a) the VL sequence shown in SEQ ID NO: 10; (b) the VL sequence shown in SEQ ID NO:11, and (c) VL sequence shown in SEQ ID NO: 12 The antibody of any one of the preceding claims, selected from the group consisting of: [Item 6] The VH region and the VL region are (a) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; (b) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10; (c) the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10; (d) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:11; (e) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:12; (f) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:10; (g) the VH sequence shown in SEQ ID NO: 7 and the VL sequence shown in SEQ ID NO: 11; (h) the VH sequence shown in SEQ ID NO:7 and the VL sequence shown in SEQ ID NO:12; (i) a VH sequence shown in SEQ ID NO:8 and a VL sequence shown in SEQ ID NO:11; (j) a VH sequence shown in SEQ ID NO: 8 and a VL sequence shown in SEQ ID NO: 12; (k) the VH sequence shown in SEQ ID NO: 9 and the VL sequence shown in SEQ ID NO: 11; and (l) The antibody of any one of the preceding claims, selected from the group consisting of the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:12. [Item 7] The binding region is (a) the VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; (b) the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:10, and (c) the antibody of any one of the preceding claims, comprising a VH sequence and a VL sequence selected from the group consisting of the VH sequence shown in SEQ ID NO:9 and the VL sequence shown in SEQ ID NO:10. [Item 8] The antibody of any one of the preceding claims, which is a humanized antibody. [Item 9] 2. The antibody of item 1, which is a chimeric antibody. [Item 10] The antibody of any one of the preceding claims, which is a full-length antibody. [Item 11] The antibody of any one of the preceding claims, comprising an Fc region comprising a first and a second immunoglobulin heavy chain. [Item 12] 10. The antibody of claim 1, wherein the first and second heavy chains are of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [Item 13] comprising an Fc region modified such that binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type antibody; C1q binding determined by ELISA, An antibody according to any one of the preceding claims. [Item 14] the antibody comprises an Fc region modified such that the antibody mediates Fc-mediated T cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to a wild-type antibody; The antibody of any one of the preceding claims, wherein the T cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay. [Item 15] the antibody comprises an Fc region that has been modified to reduce Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% when compared to a wild-type antibody; The antibody of any one of the preceding claims, wherein Fc-mediated CD69 expression is determined in a PBMC-based functional assay. [Item 16] comprising a first and a second immunoglobulin heavy chain; in at least one of said first and said second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively; An antibody according to any one of the preceding claims. [Item 17] 17. The antibody of item 16, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D. [Item 18] 17. The antibody of item 16, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain is not N. [Item 19] 17. The antibody of item 16, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively. [Item 20] 20. The antibody of any one of items 16 and 19, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, or A and A, respectively. [Item 21] 21. The antibody of item 20, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively. [Item 22] 21. The antibody of item 20, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively. [Item 23] 17. The antibody of any one of items 1 to 16, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively. [Item 24] 24. The antibody of item 23, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively. [Item 25] 25. The antibody of item 24, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively. [Item 26] 25. The antibody of item 24, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are A, A, and A, respectively. [Item 27] 17. The antibody of item 16, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively. [Item 28] A bispecific antibody comprising a first binding region of the antibody according to any one of Items 1 to 12 and a second binding region that binds to a target different from the first antigen-binding region. [Item 29] 29. The bispecific antibody of item 28, comprising a first and a second heavy chain. [Item 30] (a) the bispecific antibody comprises an Fc region modified according to any one of items 13 to 15; or (b) at least one of the first and second heavy chains comprises one or more amino acids modified as described in any one of items 16 to 27; 29. The bispecific antibody of item 29. [Item 31] 31. The bispecific antibody according to any one of Aspects 28 to 30, wherein each of the first and second heavy chains comprises at least a hinge region, a CH2 region, and a CH3 region; wherein the first heavy chain comprises at least one amino acid substitution at a position corresponding to a position in a human IgG1 heavy chain selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409; and the second heavy chain comprises at least one amino acid substitution at a position corresponding to a position in a human IgG1 heavy chain selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409; and wherein the first and second heavy chains do not comprise substitutions at the same positions. [Item 32] 32. The bispecific antibody of item 31, wherein the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L in the first heavy chain and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R in the second heavy chain, or vice versa. [Item 33] 33. The bispecific antibody according to any one of items 28 to 32, wherein the first binding region is according to any one of items 1 to 7, and the second binding region binds to a target different from the first binding region. [Item 34] A nucleic acid construct encoding one or more of the amino acid sequences set forth in Table 1. [Item 35] (i) a nucleic acid sequence encoding the heavy chain sequence of the humanized antibody or chimeric antibody according to any one of items 1 to 33; (ii) a nucleic acid sequence encoding the light chain sequence of the humanized or chimeric antibody according to any one of items 1 to 33; or (iii) Both (i) and (ii) An expression vector comprising: [Item 36] A host cell comprising the expression vector of item 35. [Item 37] 37. The host cell of item 36, which is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell. [Item 38] A composition comprising the antibody of any one of items 1 to 27 or the bispecific antibody of any one of items 28 to 33. [Item 39] A pharmaceutical composition comprising the antibody of any one of items 1 to 27 or the bispecific antibody of any one of items 28 to 33, and a pharmaceutically acceptable carrier. [Item 40] The antibody of any one of items 1 to 27, the bispecific antibody of any one of items 28 to 33, the composition of item 38, or the pharmaceutical composition of item 39 for use as a medicament. [Item 41] The antibody of any one of items 1 to 27, the bispecific antibody of any one of items 28 to 33, the composition of item 38, or the pharmaceutical composition of item 39 for use in treating a disease. [Item 42] A method for treating a disease, comprising a step of administering to a subject in need thereof the antibody according to any one of items 1 to 27, the bispecific antibody according to any one of items 28 to 33, the composition according to item 38, or the pharmaceutical composition according to item 39. [Item 43] 43. The use or method according to any one of items 40 to 42, wherein the disease is cancer, an infectious disease, or an autoimmune disease. [Item 44] A method for diagnosing a disease characterized by the involvement or accumulation of CD3-expressing cells, comprising administering to a subject the antibody of any one of items 1 to 27, the bispecific antibody of any one of items 28 to 33, the composition of item 38, or the pharmaceutical composition of item 39, wherein optionally the antibody or the bispecific antibody is labeled with a detectable agent. [Item 45] (a) culturing the host cell according to any one of items 36 to 37; and (b) Purifying the antibody from the culture medium 34. A method for producing the antibody according to any one of items 1 to 27 or the bispecific antibody according to any one of items 28 to 33, comprising: [Item 46] A diagnostic composition comprising the antibody according to any one of items 1 to 27 or the bispecific antibody according to any one of items 28 to 33. [Item 47] (a) contacting a sample with the antibody according to any one of items 1 to 27 or the bispecific antibody according to any one of items 28 to 33 under conditions allowing the formation of a complex between the antibody or the bispecific antibody and CD3; and (b) analyzing whether a complex is formed; 1. A method for detecting the presence of CD3 antigen or the presence of cells expressing CD3 in a sample, comprising: [Item 48] (i) the antibody according to any one of items 1 to 27 or the bispecific antibody according to any one of items 28 to 33, and (ii) Instructions for use of the kit 1. A kit for detecting the presence of CD3 antigen or the presence of cells expressing CD3 in a sample, comprising: [Item 49] An anti-idiotype antibody that binds to the antibody according to any one of items 1 to 27.

[0458] List of References TIFF0007757247000008.tif172163TIFF0007757247000009.tif236163TIFF0007757247000010.tif204163

[0459] Sequence information SEQUENCE LISTING <110> Genmab A / S <120> Humanized or chimeric CD3 antibodies <150> PCT / EP2013 / 064330 <151> 2013-07-05 <150> PCT / EP2014 / 050340 <151> 2014-01-09 <150> DK PA 2014 00009 <151> 2014-01-09 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> homo sapiens <400> 1 Gly Phe Thr Phe Asn Thr Tyr Ala 1 5 <210> 2 <211> 10 <212> PRT <213> homo sapiens <400> 2 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr 1 5 10 <210> 3 <211> 16 <212> PRT <213> homo sapiens <400> 3 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 15 <210> 4 <211> 9 <212> PRT <213> homo sapiens <400> 4 Thr Gly Ala Val Thr Thr Ser Asn Tyr 1 5 <210> 5 <211> 9 <212> PRT <213> homo sapiens <400> 5 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 6 <211> 125 <212> PRT <213> homo sapiens <400> 6 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 125 <212> PRT <213> homo sapiens <400> 7 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 8 <211> 125 <212> PRT <213> homo sapiens <400> 8 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 9 <211> 125 <212> PRT <213> Homo sapiens <400> 9 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 109 <212> PRT <213> homo sapiens <400> 10 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 11 <211> 109 <212> PRT <213> homo sapiens <400> 11 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 12 <211> 109 <212> PRT <213> homo sapiens <400> 12 Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 13 <211> 186 <212> PRT <213> homo sapiens <400> 13 Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30 Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45 Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60 Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg 65 70 75 80 Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg 85 90 95 Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile 100 105 110 Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr 115 120 125 Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly 130 135 140 Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro 145 150 155 160 Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp 165 170 175 Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 180 185 <210> 14 <211> 150 <212> PRT <213> homo sapiens <400> 14 Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys 1 5 10 15 Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser 20 25 30 ...

Claims

1. A humanized antibody that binds to human CD3 and is an IgG1 isotype antibody, comprising a binding region including the heavy chain variable (VH) sequence shown in SEQ ID NO: 6 and the light chain variable (VL) sequence shown in SEQ ID NO:

10.

2. 2. The antibody of claim 1, comprising a first and a second heavy chain, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively, and the amino acid numbers are numbered according to the Eu numbering index.

3. 3. The antibody of claim 1, comprising a first and a second heavy chain, wherein in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively, and the amino acid numbers are numbered according to the Eu numbering index.

4. 4. The antibody of claim 2 or 3, wherein the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively, and the amino acid numbers are numbered according to the Eu numbering index.

5. The antibody of any one of claims 1 to 4, which is an antibody of the IgG1m(f) allotype and comprises a heavy chain constant region having the sequence shown in SEQ ID NO: 15, 25, or 26.

6. The antibody of any one of claims 2 to 5, wherein each heavy chain is combined with a kappa (κ) light chain.

7. The antibody of any one of claims 2 to 5, wherein each heavy chain is combined with a lambda (λ) light chain.

8. The antibody according to any one of claims 1 to 7, which is a full-length antibody.

9. A bispecific antibody comprising a first binding region and a second binding region, wherein the first binding region is a binding region of the antibody of any one of claims 1 to 8, and the second binding region binds to a target different from that of the first antigen-binding region.

10. 10. The bispecific antibody of claim 9, wherein the bispecific antibody comprises first and second heavy chains and first and second light chains, wherein the first heavy chain and the first light chain are humanized and linked by a disulfide bond to form a first binding region, and the second heavy chain and the second light chain are fully human and linked by a disulfide bond to form a second binding region.

11. 11. The antibody of claim 9 or 10, comprising first and second heavy chains, wherein in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; the amino acid at position K409 in the human IgG1 heavy chain in the first heavy chain is R; and the amino acid at position F405 in the human IgG1 heavy chain in the second heavy chain is L, wherein the amino acid numbers are numbered according to the Eu numbering index.

12. 11. The antibody of claim 9 or 10, comprising first and second heavy chains, wherein in at least one of the first and second heavy chains, the amino acids at positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; the amino acid at position F405 in the human IgG1 heavy chain in the first heavy chain is L; and the amino acid at position K409 in the human IgG1 heavy chain in the second heavy chain is R, wherein the amino acid numbers are numbered according to the Eu numbering index.

13. 13. The bispecific antibody of claim 10, wherein in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively, and the amino acid numbers are numbered according to the Eu numbering index.

14. 14. The bispecific antibody of claim 11, wherein the amino acids at positions corresponding to positions N297 and P331 in the human IgG1 heavy chain are N and P, respectively, and the amino acid numbers are numbered according to the Eu numbering index.

15. (i) a nucleic acid sequence encoding the heavy chain sequence of the humanized or chimeric antibody of any one of claims 1 to 8, and (ii) a nucleic acid sequence encoding the light chain sequence of the humanized or chimeric antibody according to any one of claims 1 to 8 or a set of an expression vector containing the nucleic acid sequence of (i) and an expression vector containing the nucleic acid sequence of (ii).

16. A host cell comprising the expression vector of claim 14.

17. 16. The host cell of claim 15, which is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell.

18. A composition comprising an antibody according to any one of claims 1 to 8 or a bispecific antibody according to any one of claims 9 to 14.

19. A pharmaceutical composition comprising the antibody of any one of claims 1 to 8 or the bispecific antibody of any one of claims 9 to 14, and a pharmaceutically acceptable carrier.

20. 20. The antibody of any one of claims 1 to 8, the bispecific antibody of any one of claims 9 to 14, the composition of claim 18, or the pharmaceutical composition of claim 19, for use as a medicament.

21. 20. The antibody of any one of claims 1 to 8, the bispecific antibody of any one of claims 9 to 14, the composition of claim 18, or the pharmaceutical composition of claim 19 for use in the treatment of a disease.

22. 22. The antibody, bispecific antibody, composition, or pharmaceutical composition of claim 21, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

Images