Humanized or chimeric CD3 antibodies

Abstract

To provide humanized or chimeric antibodies binding CD3, useful in pharmaceutical compositions, treatment of diseases, diagnostic compositions and the like.SOLUTION: The present invention provides a humanized or chimeric antibody binding to human CD3, wherein the antibody comprises a binding region comprising heavy chain variable (VH) regions CDR1, CDR2, and CDR3 having specific sequences, respectively, and light chain variable (VL) regions CDR1, CDR2, and CDR3 having a specific sequence, the sequence GTN, and a specific sequence, respectively.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a humanized or chimeric antibody that binds to human CD3, and the humanized or chimeric antibody This relates to compositions containing such antibodies and the use of such humanized or chimeric antibodies in the treatment of diseases. [Background technology]

[0002] background Cluster of Differentiation 3 (CD3) has been known for a long time, and therefore This has become a subject of interest in many aspects. Specifically, antibodies produced against CD3 Alternatively, antibodies produced against T cell receptor complexes with CD3 as one component are known. (5) In vitro characterization of humanized OKT3 effector functional mutant antibodies is described. It has been done.[1]

[0003] Treatment with the anti-CD3 monoclonal antibody hOKT3 gamma 1 (Ala-Ala) is followed by continuous immunosuppressant therapy. Without administration of the drug, C-peptide response and clinical This results in improvements to floor parameters.[2]

[0004] A promising approach to improving targeted antibody therapy involves targeting cancer cells that express antigens, and then... This method involves specifically delivering toxic cells. It efficiently kills tumor cells. The concept of using T cells for this purpose has already been described [3]. However, in the early stages The clinical trials have been rather disappointing, mainly due to the low efficacy and seriousness of the bispecific antibodies. It had adverse effects (cytokine storm) and immunogenicity.[4] Bispecific antibodies Due to the progress of design and application, the first barrier of cytokine storm has been partially overcome, and the clinical efficacy has been improved with no dose-limiting toxicity [5]. For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been successful in treating cancer-related ascites after intraperitoneal application [6]. However, mouse / rat hybrids are immunogenic [7] and cannot be applied for long-term intravenous treatment in humans. Adverse events related to the high frequency of treatment caused by catumaxomab included cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) related to the effector function of the Fc region of catumaxomab [8]~[9]. Another antibody is ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in Phase II clinical development for metastatic breast cancer

[10] ~

[11] .

[0005] For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been successful in treating cancer-related ascites after intraperitoneal application [6]. However, mouse / rat hybrids are immunogenic [7] and cannot be applied for long-term intravenous treatment in humans. Adverse events related to the high frequency of treatment caused by catumaxomab included cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) related to the effector function of the Fc region of catumaxomab [8]~[9]. Another antibody is ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in Phase II clinical development for metastatic breast cancer

[10] ~

[11] . For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been successful in treating cancer-related ascites after intraperitoneal application [6]. However, mouse / rat hybrids are immunogenic [7] and cannot be applied for long-term intravenous treatment in humans. Adverse events related to the high frequency of treatment caused by catumaxomab included cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) related to the effector function of the Fc region of catumaxomab [8]~[9]. Another antibody is ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in Phase II clinical development for metastatic breast cancer

[10] ~

[11] . For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been successful in treating cancer-related ascites after intraperitoneal application [6]. However, mouse / rat hybrids are immunogenic [7] and cannot be applied for long-term intravenous treatment in humans. Adverse events related to the high frequency of treatment caused by catumaxomab included cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) related to the effector function of the Fc region of catumaxomab [8]~[9]. Another antibody is ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in Phase II clinical development for metastatic breast cancer

[10] ~

[11] . For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been successful in treating cancer-related ascites after intraperitoneal application [6]. However, mouse / rat hybrids are immunogenic [7] and cannot be applied for long-term intravenous treatment in humans. Adverse events related to the high frequency of treatment caused by catumaxomab included cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) related to the effector function of the Fc region of catumaxomab [8]~[9]. Another antibody is ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in Phase II clinical development for metastatic breast cancer

[10] ~

[11] . For example, certain bispecific antibodies that target antigens on tumor cells in one arm and, for example, CD3 on T cells in the other arm confer Fc receptor binding by the Fc region. Upon binding, a complex of T cells, tumor cells, and effector cells that bind to the antibody Fc region is formed, leading to the death of tumor cells [4]. Catumaxomab consists of a mouse IgG2a / rat IgG2b heterodimer and has been successful in treating cancer-related ascites after intraperitoneal application [6]. However, mouse / rat hybrids are immunogenic [7] and cannot be applied for long-term intravenous treatment in humans. Adverse events related to the high frequency of treatment caused by catumaxomab included cytokine release-related symptoms (i.e., fever, nausea, vomiting, chills, tachycardia, and hypotension) related to the effector function of the Fc region of catumaxomab [8]~[9]. Another antibody is ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines with low HER2 expression. Ertumaxomab is in Phase II clinical development for metastatic breast cancer

[10] ~

[11] . ​​​​​​​​​​​​​​​​​​​​​​​​​In this regard, the present invention relates to a humanized or chimeric antibody that binds to human CD3, wherein SEQ ID NO : Heavy chain variable (VH) regions CDR1, CDR2, and CDR, having sequences shown in 1, 2, and 3, respectively. 3, the sequence shown in SEQ ID NO:4, the sequence GTN, and the sequence shown in SEQ ID NO:5 or SEQ ID NO:60 The binding region includes light chain variable (VL) regions CDR1, CDR2, and CDR3, each having a specific sequence. Regarding antibodies.

[0008] In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, and SEQ The heavy chain variable (VH) regions CDR1, CDR2, and O have sequences shown in ID NO:1, 2, and 3, respectively. The sequence shown in CDR3, SEQ ID NO:4, sequence GTN, and SEQ ID NO:5 are used together. Regarding antibodies that contain binding regions including the light chain variable (VL) regions CDR1, CDR2, and CDR3, respectively. do.

[0009] In another aspect, the present invention relates to the first binding region of the antibody of the present invention and the first antigen-binding region. This relates to a bispecific antibody that includes a second binding region that binds to a different target.

[0010] In another aspect, the present invention relates to the nucleus encoding one or more amino acid sequences of the present invention. Regarding acid constructs.

[0011] In another aspect, the present invention relates to (i) the heavy chain sequence of the humanized or chimeric antibody of the present invention (ii) a nucleic acid sequence to encode the light chain sequence of the humanized or chimeric antibody of the present invention. This relates to a sequence, or an expression vector containing both (iii)(i) and (ii).

[0012] In another aspect, the present invention relates to a host cell containing the expression vector of the present invention.

[0013] In another aspect, the present invention relates to a composition comprising the antibody or bispecific antibody of the present invention. ru.

[0014] In another aspect, the present invention relates to the antibody or bispecific antibody of the present invention and pharmaceutically acceptable This relates to a pharmaceutical composition containing a carrier.

[0015] In another context, the present invention relates to the antibody or dual for use as a pharmaceutical. This relates to specific antibodies, compositions, or pharmaceutical compositions.

[0016] In another context, the present invention relates to the antibody or two of the present invention for use in treating diseases. This relates to a heavily specific antibody, composition, or pharmaceutical composition.

[0017] In another aspect, the present invention provides an antibody or two of the present invention to a subject requiring treatment for a disease. A method for treating a disease, comprising the step of administering a highly specific antibody, composition, or pharmaceutical composition. do.

[0018] In one aspect, the present invention diagnoses diseases characterized by the involvement or accumulation of CD3-expressing cells. Regarding the method, this method may involve the humanization of the present invention, which may be labeled with a detectable active substance. The process involves administering a chimeric antibody, the composition of the present invention, or the pharmaceutical composition of the present invention to a target. include.

[0019] In another aspect, the present invention relates to a method for producing the antibody or bispecific antibody of the present invention. Regarding the method, this method comprises a) a step of culturing the host cells of the present invention, and b) the step of using the culture medium. This includes a step for purifying antibodies.

[0020] In another aspect, the present invention relates to a diagnostic composition comprising the antibody or bispecific antibody of the present invention. Regarding.

[0021] In another aspect, the present invention relates to the presence of CD3 antigen in a sample or the presence of cells expressing CD3. Regarding a method for detecting the present invention, this method involves a) using the sample with the antibody or bispecificity of the present invention. The antibody is brought into contact with the aforementioned antibody or bispecific antibody and CD3 under conditions that allow for the formation of a complex between the antibody and CD3. The process includes a step of (b) analyzing whether a composite has been formed.

[0022] In another aspect, the present invention relates to i) the antibody or bispecific antibody of the present invention and ii) a kit This includes instructions for use and a method for detecting the presence of CD3 antigen or CD3-expressing cells in a sample. Regarding the kit for that purpose.

[0023] In another aspect, the present invention relates to an anti-idiotype antibody that binds to the antibody of the present invention. . [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1A shows the binding curves of the monospecific antibody mutant IgG1-huCD3 and Figure 1B shows the bispecific antibody mutant bsIgG1 huCD3×HER2 to the human T cell line Jurkat. The data shown are the average fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that yields half-maximal binding (EC50). [Figure 2] Figure 2A shows the binding curves of the monospecific antibody mutant IgG1-huCD3 and the bispecific antibody mutant bsIgG1 huCD3×HER2 to the cynomolgus monkey T cell line HSC-F, and Figure 2B shows the same curves. The data shown are the average fluorescence intensity (MFI) of a representative experiment, as described in Example 2. [Figure 3] T cell activation by IgG1-huCD3 antibody mutants. CD69 expression on human (Figure 3A) and cynomolgus monkey (Figure 3B) T cells 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 4] T cell proliferation induced by IgG1-huCD3 antibody mutants. Human (Figure 4A) or cynomolgus monkey (Figure 4B) PBMCs were incubated with IgG1-huCD3 antibody mutants for 3 days, and proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 5] The induction of human (Figure 5A) and cynomolgus monkey (Figure 5B) T cell-mediated cytotoxicity by huCD3 antibody mutants containing inactivating LFLEDA mutations was determined as described in Example 5. Representative results from two independent experiments conducted in pairs are shown. [Figure 6A] This table shows the binding curve of a deactivated monospecific antibody variant of IgG1-huCD3 to the human T cell line Jurkat. The data shown are the average fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that yields 50% maximum binding (EC50). [Figure 6B] This table shows the binding curve of the inactivated bispecific antibody mutant bsIgG1-huCD3×HER2 to the human T cell line Jurkat. The data shown are the average fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that yields 50% maximum binding (EC50). [Figure 7A] The binding curves of a deactivated monospecific antibody variant of IgG1-huCD3 to the cynomolgus monkey T cell line HSC-F are shown. The data shown are the average fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that yields 50% maximum binding (EC50). [Figure 7B]The binding curve of the inactivated bispecific antibody mutant bsIgG1-huCD3×HER2 to the cynomolgus monkey T cell line HSC-F is shown. The data shown are the average fluorescence intensity (MFI) of a representative experiment, as described in Example 2. The table shows the antibody concentration (μg / mL) that yields 50% maximum binding (EC50). [Figure 8A] T cell activation by inactivated 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 twice. Representative results from one experiment are shown. [Figure 8B] T cell activation by inactivated monospecific IgG1-huCD3. CD69 expression on cynomolgus monkey-derived T cells 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 by a non-activated bispecific bsIgG1-huCD3×HER2 antibody mutant. CD69 expression on human-derived T cells 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 8D] T cell activation by a non-activated bispecific bsIgG1-huCD3×HER2 antibody mutant. CD69 expression on cynomolgus monkey-derived T cells 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 9A] T cell proliferation induced by inactivated monospecific IgG1-huCD3. T cell proliferation was measured in human PBMCs. These PBMCs were incubated with various antibody variants for 3 days, and then 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 inactivated monospecific IgG1-huCD3. T cell proliferation was measured in cynomolgus monkey PBMCs. These PBMCs were incubated with various antibody variants for 3 days, and then 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 a non-activated, bispecific bsIgG1-huCD3×HER2 antibody mutant. T cell proliferation was measured in human PBMCs. These PBMCs were incubated with various antibody mutants for 3 days, and then proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 9D] T cell proliferation induced by a non-activated, bispecific bsIgG1-huCD3×HER2 antibody mutant. T cell proliferation was measured in cynomolgus monkey PBMCs. These PBMCs were incubated with various antibody mutants for 3 days, and then proliferation was measured by cell proliferation ELISA as described in Example 4. Representative results from two independent experiments are shown. [Figure 10] The induction of human (Figure 10A) and cynomolgus monkey (Figure 10B) T cell-mediated cytotoxicity by huCD3 antibody mutants containing inactivating LFLEDA mutations was determined as described in Example 5. Representative results from two independent experiments conducted in pairs are shown. [Figure 11] Activation of rhesus monkey T cells by IgG1-huCD3 antibody mutant. CD69 expression on rhesus monkey-derived T cells in PBMC culture was measured by FACS analysis, as described in Example 6. [Figure 12] T cell activation by inactivating mutants of the huCLB-T3 / 4 antibody. IgG1-huCLB-T3 / 4 mutants were titrated onto PBMCs. CD69 expression on T cells in PBMC cultures was measured by FACS analysis, as described in Example 7. Representative examples from three experiments are shown. [Figure 13]T cell proliferation by inactivating mutants of the huCLB-T3 / 4 antibody. PBMCs were incubated with the antibody for 3 days, and 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 deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 14B] In vitro T cell-mediated cytotoxicity induced by deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 14C] In vitro T cell-mediated cytotoxicity induced by deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 14D] In vitro T cell-mediated cytotoxicity induced by deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 14E] In vitro T cell-mediated cytotoxicity induced by deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 14F] In vitro T cell-mediated cytotoxicity induced by deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 14G] In vitro T cell-mediated cytotoxicity induced by deactivating antibody variants of the CD3 antibody. The induction of T cell-mediated cytotoxicity by antibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [Figure 14A-G]) was determined as described in Example 9. The average from two experiments conducted in pairs is shown. [Figure 15] In vitro T cell-mediated cytotoxicity induced by inactivated huCLB-T3 / 4 mutants. The induction of T cell-mediated cytotoxicity by the antibody mutant (LFLEDA LAL [Figure 15A~C]) was determined as described in Example 9. The average values ​​from a single experiment conducted in pairs are shown. [Figure 16A] Evaluation of C1q binding to inactivated huCLB-T3 / 4 antibody variants. The binding of C1q to single-specificity IgG1 huCLB-T3 / 4 (Figure 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (Figure B-D), as well as their inactivated antibody variants, was evaluated by ELISA as described in Example 10. The results in the graph are representative examples from experiments with n=2. [Figure 16B] Evaluation of C1q binding to inactivated huCLB-T3 / 4 antibody variants. The binding of C1q to single-specificity IgG1 huCLB-T3 / 4 (Figure 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (Figure B-D), as well as their inactivated antibody variants, was evaluated by ELISA as described in Example 10. The results in the graph are representative examples from experiments with n=2. [Figure 16C]Evaluation of C1q binding to inactivated huCLB-T3 / 4 antibody variants. The binding of C1q to single-specificity IgG1 huCLB-T3 / 4 (Figure 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (Figure B-D), as well as their inactivated antibody variants, was evaluated by ELISA as described in Example 10. The results in the graph are representative examples from experiments with n=2. [Figure 16D] Evaluation of C1q binding to inactivated huCLB-T3 / 4 antibody variants. The binding of C1q to single-specificity IgG1 huCLB-T3 / 4 (Figure 16A-C) and bsIgG1-huCLB-T3 / 4×HER2 (Figure B-D), as well as their inactivated antibody variants, was evaluated by ELISA as described in Example 10. The results in the graph are representative examples from experiments with n=2. [Figure 17] The pharmacokinetic (PK) analysis of the inactivated 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 (Figure 17A). The plasma clearance rate, calculated as described in Figure 11, is shown (Figure 17B). The horizontal dotted line represents the mean clearance rate of human IgG1 antibody in SCID mice (10 mL / day / kg). [Figure 18] Frequency of positive T cell response in HLA-typed healthy donors. A positive response was defined as an SI index of ≥1.9 in both proliferation assays and IL-2 secretion assays. Humanized A33 was used as a clinical benchmark control antibody, exhibiting high levels of immunogenicity clinically and consistently inducing a 20–30% T cell response in EpiScreen assays. KLH response was included to check the quality (after thawing) of PBMCs. [Figure 19] Sequence alignment of the heavy chain (VH) and light chain (VL) variable regions of the humanized CD3 antibody of the present invention. [Figure 20] Expression levels of the IgG1-huCD3-H1L1 mutant in the Expi293F supernatant, measured by Octet RED using an anti-human IgG immobilized sensor. [Figure 21]Binding of IgG1-huCD3-H1L1-LFLEDA mutant to Jurkat cells. [Figure 22] Binding of CD3 bispecific antibody variants to human T cells. Circles indicate binding of the huCD3-H1L1-LFLEDA variant, triangles indicate binding of the huCD3-H1L1-LFLEDA-LK55N variant, and squares indicate binding of the CD3-hmAb286-LFLEDA variant. [Figure 23] Cytotoxicity of A431 cells (low-HER2 expression cells) (Figure 23A) and AU565 cells (high-HER2 expression cells) (Figure 23B) treated with CD3 and HER2 affinity mutants by huCD3×HER2 antibody affinity mutants. [Figure 24] Tumor size reduction in NOD-SCID mice induced by huCD3-H1L1×HER2-LFLEDA 7, 14, 21, and 28 days (Figure 24A) and 29 days (Figure 24B) after tumor inoculation. [Figure 25] Tumor size reduction in NOD-SCID mice induced by huCD3-H1L1×CD20-LFLEDA 7, 14, and 21 days (Figure 25A) and 21 day (Figure 25B) after tumor inoculation. [Modes for carrying out the invention]

[0025] Detailed explanation In one aspect, the present invention relates to a humanized or chimeric antibody that binds to human CD3, and SEQ The heavy chain variable (VH) regions CDR1, CDR2, and O have sequences shown in ID NO:1, 2, and 3, respectively. and CDR3, the sequence shown in SEQ ID NO:4, sequence GTN, and SEQ ID NO:5 or SEQ ID NO: An antibody comprising light chain variable (VL) regions CDR1, CDR2, and CDR3, each having the sequence shown in 60. Regarding.

[0026] In one embodiment, the present invention is a humanized or chimeric antibody that binds to human CD3, and SEQ The heavy chain variable (VH) regions CDR1, CDR2, and O have sequences shown in ID NO:1, 2, and 3, respectively. The sequence shown in CDR3, SEQ ID NO:4, sequence GTN, and SEQ ID NO:5 are used together. Regarding antibodies that contain binding regions including the light chain variable (VL) regions CDR1, CDR2, and CDR3, respectively. do.

[0027] As used in this specification, the term "antibody" refers to an immunoglobulin molecule. A fragment of, or a derivative thereof, which, under typical physiological conditions, is considerably The half-life over the period is, for example, at least about 30 minutes, at least about 45 minutes, at least about 1 hour. At least approximately 2 hours, at least approximately 4 hours, at least approximately 8 hours, at least approximately 12 hours, approximately 2 Half-lives of 4 hours or more, approximately 48 hours or more, approximately 3, 4, 5, 6, or 7 days or more, or other times A functionally defined period of intent (for example, a period that induces a physiological response related to antibody binding to an antigen) , sufficient time to promote, enhance, and / or adjust, and / or the antibody has an effect It has a half-life (sufficient time to mobilize antigenic activity) and the ability to specifically bind to the antigen. This refers to a binding region that interacts with an antigen (referred to as a binding domain in this specification). (Sometimes the term is used, and both terms have the same meaning) refers to immunoglobulin molecules. It includes variable regions of both the heavy and light chains. The constant region of the antibody (Ab) is host tissue or host factors. Children, for example, various cells of the immune system (e.g., effector cells and T cells) and complements To the components of the system, such as C1q, the first component in the classical pathway of complement activation. It can mediate the binding of immunoglobulins. As shown above, the anti used herein The term "body" includes fragments of an antibody that retain the ability to specifically interact (e.g., bind) with an antigen, unless otherwise stated or clearly inconsistent in context. The antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of binding fragments included within the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of the V , V L , V H , C L and C H 1 domains, or monovalent antibodies described in WO20070 59782 (Genmab A / S); (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region; (iii ) Fd fragments consisting essentially of the V domain and the C H 1 domain; and (iv) Fv fragments consisting essentially of the V H domain of a single arm of an antibody and the V domain and the V L domain. H Furthermore, the two domains V and V L of the Fv fragment, although encoded by separate genes, can be joined by a synthetic linker that enables them to pair and form a single protein chain that forms a monovalent H unit (known as a single-chain antibody or single-chain Fv (scFv); e.g., Bir d et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-588 L V regions and V H regions to form a single protein chain that forms a monovalent [[ID=第46]]unit (known as a single-chain antibody or single-chain Fv (scFv); e.g., Bir d et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-588 d et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-588 See 3 (1988). Such single-chain antibodies, unless otherwise noted, or in context Unless explicitly stated above, such fragments are included in the term antibody. Generally, the term "antibody" is included in the meaning of the present invention, but they are used both collectively and individually. This is a unique characteristic, exhibiting different biological properties and usefulness. In relation to the present invention... These antibody fragments and other useful antibody fragments are described herein. Further discussion will follow. Unless otherwise specified, the term antibody refers to polyclonal Antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, as well as enzyme cleavage, Antigens obtained by any known method, such as peptide synthesis and recombinant techniques. This also includes antibody fragments (antigen-binding fragments) that retain the ability to bind specifically. It should be understood that the antibodies produced can be of any isotype. .

[0028] The terms "immunoglobulin heavy chain" and "heavy chain of immunoglobulin" used in this specification are... Or, "heavy chain" refers to one of the chains of immunoglobulin. A heavy chain is typically a heavy chain. The variable region of the chain (abbreviated as VH in this specification) and the immunoglobulin isotype are defined. It consists of a heavy chain steady region (abbreviated as CH in this specification) and a typical heavy chain steady region. In essence, it consists of three domains CH1, CH2, and CH3. The heavy chain constant region is further... It may include the region. The term "immunoglobulin" as used herein refers to two pairs of polypeptides. This refers to a class of structurally related glycoproteins consisting of butyl chains, where one pair is lighter. (L) Low molecular weight chain, one pair is a heavy (H) chain, all four are interconnected by disulfide bonds. They can be linked to

[14] . The structure of immunoglobulins has been characterized in detail (see, for example,

[14] ). ). In the structure of immunoglobulins, disulfide bonds in the so-called "hinge region" The two heavy chains are interconnected. Like the heavy chains, each light chain is typically composed of several regions. That is, the light chain variable region (abbreviated as VL in this specification) and the light chain steady region (CL in this specification). It consists of (abbreviated as ). The light chain constant region is typically composed of one domain CL. This is achieved. Furthermore, the VH and VL regions are also known as the Complementarity Determination Region (CDR), a highly variable region. (That is, the form of the array and / or structurally defined loops can be hypervariable) The variable region can be further subdivided, and between them are framework regions (FRs). There are scattered areas with high levels of preservation, known as VH and VL. Typically, three CDRs are... It is composed of four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus. The sequences are arranged as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see

[15] ). The CDR sequence is I The determination can be made using the methods provided by the MGT.

[16]

[17]

[0029] As used herein, the term "isotype" refers to a gene that is coded by a heavy chain constant region. The immunoglobulin classes being tested (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, and also (IgM) or any allotype thereof, e.g., IgG1m(za) and IgG1m(f) [SEQ ID NO :15]) refers to. Therefore, in one aspect, the antibody is of the IgG1 class or any of its alloclasses. It contains immunoglobulin heavy chains of various types. Furthermore, each heavy chain isotype is further classified into kappa (κ) light chains. Alternatively, it can be combined with a lambda (λ) light chain.

[0030] As used herein, the term "chimeric antibody" refers to an antibody whose variable region originates from a non-human species (for example) Chimeric antibodies refer to antibodies whose constant region originates from a different species, such as humans, but whose normal region originates from a rodent. It can be produced by antibody engineering. "Antibody engineering" refers to the process of producing various types of antibodies. This is a widely used term and a process well known to those skilled in the art. In particular, chimeric antibodies are...

[18] It can be created using the standard DNA techniques described. Therefore, the chimera is inherited. These may be recombinant antibodies that have been genetically or enzymatically modified. The preparation of chimeric antibodies is known to those skilled in the art. Therefore, the production of the chimeric antibody of the present invention is carried out by a method other than the method described herein. It is also possible. Therapeutic chimeric monoclonal antibodies are developed to reduce antibody immunogenicity. These are typically non-human (e.g., mouse) variable antigens specific to the antigen of interest. It may contain a region and human constant antibody heavy chain and light chain domains. Used in relation to chimeric antibodies. The term "variable region" or "variable domain" used here refers to the heavy and light chains of immunoglobulins. This refers to the area encompassing both CDR and framework domains.

[0031] As used herein, the term "humanized antibody" refers to a human antibody with a constant domain and a human variable domain. A non-human variable domain modified to have a high level of sequence homology to the domain This refers to the genetically modified non-human antibodies it contains. These, as a whole, form antigen-binding sites. Six non-human antibody complementarity-determining regions (CDRs) are used in homologous human acceptor framework regions. This can be achieved by transplanting onto (FR) (see

[19] ~

[20] ). Parental opposition To completely reconstitute the binding affinity and specificity of the body, parental antibodies (i.e., non-human) are used. Replacing framework residues from antibodies into the human framework region (reverse mutation) ) may be necessary. Structural homology modeling is an important framework for antibody binding properties. It can be useful in identifying amino acid residues in the muwerk region. Therefore, humanized antibodies are non Human CDR sequences, optionally containing one or more amino acid return mutations to non-human amino acid sequences. This may include the human framework domain and the fully human steady-state domain. Optionally, To obtain humanized antibodies with desirable characteristics such as affinity and biochemical properties, it is essential to Alternatively, additional amino acid modifications that are not revertant mutations may be applied.

[0032] Humanized or chimeric antibodies according to any aspect or embodiment of the present invention are "humanized or "Chimeric CD3 antibody," "Humanized or chimeric antibody of the present invention," "CD3 antibody," or "The present invention" These can all be called "CD3 antibodies," and unless there is a contextual inconsistency, they all mean the same thing. It has taste and purpose.

[0033] The amino acid sequences of non-human antibodies differ from those of human antibodies, therefore, when administered to human patients... In that case, non-human antibodies are potentially immunogenic. However, even if the antibody is of non-human origin... Nevertheless, the CDR segment is responsible for the antibody's ability to bind to its target antigen. Humanization aims to maintain the specificity and binding affinity of the antibody. Thus, the humanization of non-human therapeutic antibodies minimizes their immunogenicity in humans. At the same time, the humanized antibody maintains the specificity and binding affinity of the non-human antibody. It is carried out in such a way.

[0034] As used herein, the term "binding region" refers to a region of an antibody, for example, a cell. The ability to bind to some molecule, such as polypeptides present on bacteria or virions. This refers to a region that possesses [a certain characteristic].

[0035] As used herein, the term "binding" refers to a predetermined antigen or target. This refers to the binding of an antibody to an antigen or target, such as surface plasmons. Using SPR technology, the BIAcore 3000 instrument uses an antigen as a ligand and separates the antibody. When determined as a precipitate, it is typically about 10 -6 M or less, for example, 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 Affini equivalent to A nonspecific antigen that occurs in the tee and is neither a pre-determined antigen nor a closely related antigen (for example) Its affinity for binding to BSA (casein) is less than one-tenth, for example, 100 minutes. K is less than or equal to 1, for example, less than 1 / 1000, for example, less than 1 / 10,000, for example, less than 1 / 100,000 D It binds to an antigen determined in advance with an affinity corresponding to [the specified affinity]. The degree of lowness depends on the K of that antibody. D Because it depends on the antibody K D If the antibody level is very low (i.e., the antibody level is (In the case of highly specific antigens), low affinity for the antigen is indicative of low affinity for nonspecific antigens. Compared to the affinity, it can be less than 1 / 10,000. The term “K D(M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.

[0036] As used herein, the term "human CD3" refers to one of the T cell co-receptor protein complexes. CD3 refers to the human differentiation antigen group 3 protein, which is composed of four different chains. Since it is also found in other species, the term "CD3" used herein may not be contextually consistent. Unless otherwise specified, this is not limited to human CD3. In mammals, this complex is CD3γ (cancer (human CD3γ chain Swissprot P09693 or cynomolgus monkey CD3γ Swissprot Q95LI7), C D3δ (delta) chain (human CD3δ Swissprot P04234 or cynomolgus monkey CD3δ Swissprot Q95) LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766, or cynomolgus) The CD3ε Swissprot Q95LI5, or the rhesus (CD3ε Swissprot G7NCB9) and CD3ζ( Contains zeta chains (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TK0) These chains associate with molecules known as T cell receptors (TCRs), and It generates an activation signal in lymphocytes. Both TCR and CD3 molecules constitute the TCR complex. ru.

[0037] The amino acid sequence referred to as the Swissprot number is removed after protein translation. It is known to those skilled in the art that it contains nal peptides. Therefore, it is present on the cell surface. Proteins such as CD3 do not contain signal peptides. In particular, the amino acid combinations listed in Table 1 The column does not contain such signal peptides. Proteins such as those listed in Table 1. This can be called a "mature protein." Therefore, SEQ ID NO:14 is mature human CD3δ. This represents the amino acid sequence of (delta), and SEQ ID NO:13 is the amino acid sequence of mature human CD3ε (epsilon). The SEQ ID NO:21 represents the amino acid sequence of mature cynomolgus monkey CD3ε, and the SEQ ID NO:22 represents the amino acid sequence of mature rhesus macaque CD3ε. Thus, as used herein, The term "matured" refers to a protein that does not contain any signal or leader sequences. .

[0038] The homology, length, and cleavage site location of signal peptide sequences vary depending on the protein. It is well known that they are very diverse. Signal peptides can be determined in different ways. For example, SEQ ID NO:13 of the present invention is a SignalP application (http: / / www.cbs.dtu.dk / s Determined according to the service / SignalP / (available at ervices / SignalP / ).

[0039] In certain embodiments, the humanized or chimeric antibody of the present invention is a CD3 epsilon chain, for example It binds to the epsilon chain of human CD3 (SEQ ID NO: 13). In another specific embodiment, this human The modified or chimeric antibody is a metabolite of the N-terminal portion of human CD3ε (epsilon) (SEQ ID NO:13). It binds to epitopes within ano acids 1-27. In such a particular embodiment, the antibody further... Other non-human primate species, such as the crab-eating macaque (Cynomolgus macaque CD3 epsilon SEQ ID NO:21) and / or may cross-react with rhesus monkeys (rhesus monkey CD3 epsilon SEQ ID NO:22). .

[0040] As used herein, the term "cross-reacts" refers to the humanized or chimeric antibody of the present invention. This refers to the ability of antibodies to bind to their targets in different species. Specifically, this The humanized CD3 antibodies exemplified in the examples described in the specification are human CD3 (Example 2) and cynomolgus. It has the ability to bind to rhesus CD3 (Example 2) and rhesus CD3.

[0041] The present invention includes a CDR sequence as defined herein, and further includes a framework region. Although the antibody may have different sequences outside the CDR sequence, it still maintains complete binding affinity compared to the original antibody. The force is maintained. Therefore, the present invention relates to any of the sequences described herein and a certain sequence This also relates to antibodies that contain identical amino acid sequences in variable regions.

[0042] In relation to this invention, the term "sequence identity" refers to the optimal alignment of two sequences. Considering the number of gaps that need to be introduced and the length of each gap, their distribution This refers to the percentage identity between two arrays as a function of the number of identical positions shared by the columns. In other words, % homology = 100 × number of identical positions / total number of positions). Two nucleotide sequences or nucleotides The percentage identity between amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller

[0021] . In addition, the percentage identity between two amino acid sequences This can also be determined using the Needleman and Wunsch algorithm

[22] . Multiple Alignment is preferably performed using (e.g., Vector NTI Advance® software) The Clustal W algorithm

[23] used in version 11.5 (Invitrogen Inc.) Try using it.

[0043] Therefore, in one aspect, the VH region is, a) VH sequence shown in SEQ ID NO:6; b) The VH sequence shown in SEQ ID NO: 8; c) VH sequence shown in SEQ ID NO:7; and d) VH sequence shown in SEQ ID NO:9 For at least one amino acid sequence shown in the VH sequence selected from the group consisting of the following, Both have 90%, at least 95%, at least 97%, or at least 99% identical amino acid sequences. It has a sexual nature.

[0044] In one particular embodiment, the VH region is a) VH sequence shown in SEQ ID NO:6; b) The VH sequence shown in SEQ ID NO: 8; c) VH sequence shown in SEQ ID NO:7; and d) VH sequence shown in SEQ ID NO:9 For at least one amino acid sequence shown in the VH sequence selected from the group consisting of the following, Both have 96% amino acid sequence identity.

[0045] In one embodiment, the VL region is a) VL sequence shown in SEQ ID NO:10; b) VL sequence shown in SEQ ID NO:11; and c) VL sequence shown in SEQ ID NO:12 For at least one amino acid sequence shown in the VL sequence selected from the group consisting of the following, Both have 90%, at least 95%, at least 97%, or at least 99% identical amino acid sequences. It has a sexual nature.

[0046] In one particular embodiment, the VL region is a) VL sequence shown in SEQ ID NO:10; b) VL sequence shown in SEQ ID NO:11; and c) VL sequence shown in SEQ ID NO:12 For at least one amino acid sequence shown in the VL sequence selected from the group consisting of the following, They both have 95% amino acid sequence identity.

[0047] In one embodiment, the VH region is a) VH sequence shown in SEQ ID NO:6; b) The VH sequence shown in SEQ ID NO: 8; c) VH sequence shown in SEQ ID NO:7; and d) VH sequence shown in SEQ ID NO:9 Selected from the group consisting of .

[0048] In one embodiment, the VL region is a) VL sequence shown in SEQ ID NO:10; b) VL sequence shown in SEQ ID NO:11; and c) VL sequence shown in SEQ ID NO:12 Selected from the group consisting of .

[0049] In one embodiment, only either the VH sequence or the VL sequence is one of the sequences disclosed herein. One is 100% identical to the other, and the other is at least one of the sequences disclosed herein. Both have 90%, at least 95%, at least 97%, or at least 99% identical amino acid sequences. It possesses a sequence identity in the form of sex.

[0050] In one particular embodiment, the VH sequence is a) VH sequence shown in SEQ ID NO:6; b) VH sequence shown in SEQ ID NO:7; c) VH sequence shown in SEQ ID NO:8; and d) VH sequence shown in SEQ ID NO:9 For at least one amino acid sequence shown in the VH sequence selected from the group consisting of the following, Both have 97% amino acid sequence identity, and the VL sequence is i) The VL sequence shown in SEQ ID NO:10; ii) The VL sequence shown in SEQ ID NO:11; and iii) VL sequence shown in SEQ ID NO:12 For at least one amino acid sequence shown in the VL sequence selected from the group consisting of the following, They both have 95% amino acid sequence identity.

[0051] In one embodiment, the VH sequence and the VL sequence are selected from the group consisting of the following sequences: a) SEQ ID NO: 6 and 10 respectively; SEQ ID NO: 7 and 10 respectively; SEQ ID NO :8 and 10; SEQ ID NO:9 and 10; SEQ ID NO:6 and 11; SE Q ID NO: 7 and 11; respectively SEQ ID NO: 8 and 11; respectively SEQ ID NO: 9 and 11; SEQ ID NO: 6 and 12 respectively; SEQ ID NO: 7 and 12 respectively; SEQ ID NO: 8 respectively and 12; and for the sequences shown in SEQ ID NO: 9 and 12 respectively, at least 90% of the same Uniform VH and VL sequences; b) SEQ ID NO: 6 and 10 respectively; SEQ ID NO: 7 and 10 respectively; SEQ ID NO :8 and 10; SEQ ID NO:9 and 10; SEQ ID NO:6 and 11; SE Q ID NO: 7 and 11; respectively SEQ ID NO: 8 and 11; respectively SEQ ID NO: 9 and 11; SEQ ID NO: 6 and 12 respectively; SEQ ID NO: 7 and 12 respectively; SEQ ID NO: 8 respectively and 12; and for the sequences shown in SEQ ID NO: 9 and 12 respectively, at least 95% of the same Uniform VH and VL sequences; c) SEQ ID NO: 6 and 10 respectively; SEQ ID NO: 7 and 10 respectively; SEQ ID NO :8 and 10; SEQ ID NO:9 and 10; SEQ ID NO:6 and 11; SE Q ID NO: 7 and 11; respectively SEQ ID NO: 8 and 11; respectively SEQ ID NO: 9 and 11; SEQ ID NO: 6 and 12 respectively; SEQ ID NO: 7 and 12 respectively; SEQ ID NO: 8 respectively For the sequences shown in SEQ ID NO: 9 and 12, respectively, at least 97% of the same Uniform VH and VL sequences; d) SEQ ID NO: 6 and 10 respectively; SEQ ID NO: 7 and 10 respectively; SEQ ID NO :8 and 10; SEQ ID NO:9 and 10; SEQ ID NO:6 and 11; SE Q ID NO: 7 and 11; respectively SEQ ID NO: 8 and 11; respectively SEQ ID NO: 9 and 11; SEQ ID NO: 6 and 12 respectively; SEQ ID NO: 7 and 12 respectively; SEQ ID NO: 8 respectively For the sequences shown in SEQ ID NO: 9 and 12, respectively, at least 99% of the same Uniform VH and VL sequences; e) SEQ ID NO: 6 and 10 respectively; SEQ ID NO: 7 and 10 respectively; SEQ ID NO :8 and 10; SEQ ID NO:9 and 10; SEQ ID NO:6 and 11; SE Q ID NO: 7 and 11; respectively SEQ ID NO: 8 and 11; respectively SEQ ID NO: 9 and 11; SEQ ID NO: 6 and 12 respectively; SEQ ID NO: 7 and 12 respectively; SEQ ID NO: 8 respectively and 12; and for the sequences shown in SEQ ID NO: 9 and 12 respectively, at least 100% of the same Uniform VH and VL sequences; f) VH sequence and SEQ sequence having at least 90% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; g) VH sequence and SEQ sequence having at least 90% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; h) VH sequence and SEQ sequence having at least 90% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; i) A VH sequence and SEQ sequence that have at least 90% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; j) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; k) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; l) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; m) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; n) A VH sequence and SEQ sequence that have at least 97% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; o) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; p) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; q) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; r) VH sequence and SEQ sequence having at least 99% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; s) VH sequence and SEQ sequence having at least 99% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; t) A VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; u) A VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; v) VH sequence and SEQ sequence having at least 100% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; x) VH sequence and SEQ sequence having at least 100% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; y) VH sequence and SEQ sequence having at least 100% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; z) VH sequence and SEQ sequence having at least 100% identity with the sequence shown in SEQ ID NO:6 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; aa) VH sequence and SE sequence that have at least 90% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 95% identity to the sequence shown in Q ID NO: 10, 11, or 12; ab) VH sequences and SE sequences that have at least 90% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 97% identity to the sequence shown in Q ID NO: 10, 11, or 12; ac) VH sequences and SE sequences having at least 90% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 99% identity to the sequence shown in Q ID NO: 10, 11, or 12; ad) VH sequence and SE sequence having at least 90% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 100% identity to the sequence shown in Q ID NO: 10, 11, or 12; ae) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; af) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; ag) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; ah) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; ai) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; aj) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 97% identity with the sequence shown in ak)SEQ ID NO:7 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; al) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; am) VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; an) VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; ao) VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; ap) VH sequence and SEQ sequence having at least 99% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; aq) VH sequence and SE sequence that have at least 100% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 90% identity to the sequence shown in Q ID NO: 10, 11, or 12; VH sequences and SE sequences that have at least 100% identity to the sequence shown in ar)SEQ ID NO:7 A VL sequence having at least 95% identity to the sequence shown in Q ID NO: 10, 11, or 12; as) VH sequence and SE sequence having at least 100% identity with the sequence shown in SEQ ID NO:7 A VL sequence having at least 97% identity to the sequence shown in Q ID NO: 10, 11, or 12; VH sequence and SE sequence having at least 100% identity to the sequence shown in (at)SEQ ID NO:7 A VL sequence having at least 99% identity to the sequence shown in Q ID NO: 10, 11, or 12; ba) VH sequences and SE sequences that have at least 90% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 95% identity to the sequence shown in Q ID NO: 10, 11, or 12; bb) VH sequences and SE sequences that have at least 90% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 97% identity to the sequence shown in Q ID NO: 10, 11, or 12; bc) VH sequences and SE sequences that have at least 90% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 99% identity to the sequence shown in Q ID NO: 10, 11, or 12; bd) VH sequence and SE sequence that have at least 90% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 100% identity to the sequence shown in Q ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 95% identity with the sequence shown in be)SEQ ID NO:8 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; bf) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; bg) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; bh) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; bi) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; bj) SEQ ID NO: A VH sequence and SEQ that have at least 97% identity with the sequence shown. A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; bk) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; bl) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; bm) VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; bn) VH sequence and SEQ sequence having at least 99% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; bo) VH sequence and SEQ sequence having at least 99% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 99% identity with the sequence shown in bp)SEQ ID NO:8 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; bq) VH sequence and SE sequence that have at least 100% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 90% identity to the sequence shown in Q ID NO: 10, 11, or 12; VH sequences and SE sequences that have at least 100% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 95% identity to the sequence shown in Q ID NO: 10, 11, or 12; bs) VH sequences and SE sequences that have at least 100% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 97% identity to the sequence shown in Q ID NO: 10, 11, or 12; bt) VH sequence and SE sequence having at least 100% identity with the sequence shown in SEQ ID NO:8 A VL sequence having at least 99% identity to the sequence shown in Q ID NO: 10, 11, or 12; ca) VH sequence and SEQ sequence having at least 90% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; cb) VH sequence and SEQ sequence having at least 90% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 90% identity with the sequence shown in cc)SEQ ID NO:9 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 90% identity with the sequence shown in cd)SEQ ID NO:9 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 95% identity with the sequence shown in ce)SEQ ID NO:9 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; cf) VH sequences and SEQ sequences that have at least 95% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; cg) VH sequence and SEQ sequence having at least 95% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 95% identity with the sequence shown in ch)SEQ ID NO:9 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; ci) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; cj) VH sequence and SEQ sequence that have at least 97% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 97% identity with the sequence shown in ck)SEQ ID NO:9 A VL sequence having at least 99% identity with the sequence shown in ID NO: 10, 11, or 12; cl) VH sequence and SEQ sequence having at least 97% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in cm)SEQ ID NO:9 A VL sequence having at least 90% identity with the sequence shown in ID NO: 10, 11, or 12; cn) VH sequence and SEQ sequence that have at least 99% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 95% identity with the sequence shown in ID NO: 10, 11, or 12; co) VH sequence and SEQ sequence having at least 99% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 97% identity with the sequence shown in ID NO: 10, 11, or 12; VH sequence and SEQ sequence having at least 99% identity with the sequence shown in cp)SEQ ID NO:9 A VL sequence having at least 100% identity to the sequence shown in ID NO: 10, 11, or 12; cq) VH sequence and SE sequence having at least 100% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 90% identity to the sequence shown in Q ID NO: 10, 11, or 12; cr) VH sequence and SE sequence having at least 100% identity with the sequence shown in SEQ ID NO:9 A VL sequence having at least 95% identity to the sequence shown in Q ID NO: 10, 11, or 12; cs) VH sequence and SE sequence having at least 100% identity to the sequence shown in SEQ ID NO:9 A VL sequence having at least 97% identity to the sequence shown in Q ID NO: 10, 11, or 12; Furthermore VH sequences and SE sequences that have at least 100% identity to the sequence shown in ct)SEQ ID NO:9 A VL sequence having at least 99% identity to the sequence shown in Q ID NO: 10, 11, or 12.

[0052] In one embodiment, the bonding region is a) The VH sequence shown in SEQ ID NO:6 and the VL sequence shown in SEQ ID NO:10; b) VH sequence shown in SEQ ID NO:8 and 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) VH sequence shown in SEQ ID NO:9 and VL sequence shown in SEQ ID NO:12 Includes VH and VL selected from the group consisting of the following.

[0053] In a particular configuration, 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) VH sequence shown in SEQ ID NO:9 and VL sequence shown in SEQ ID NO:10 It includes VH sequences and VL sequences selected from the group consisting of the following.

[0054] The humanized antibody of the present invention possesses homology suitable for use as a human variable framework region. To identify heavy chain and light chain human sequences having the heavy chain and light chain variable region amino acid sequence, The columns are generated by comparing them with a database of human germline variable region sequences. This is possible. A series of humanized heavy and light chain variable regions can be used, for example, to frame a mouse CDR. Transplant into the region (identified as described above) and, if necessary, restore antibody binding efficiency. Residues identified as potentially critically important are restored to their specific mouse sequences. To change (to change one or more human amino acid residues in the framework region to their specific position) This can be designed by (mutating to revert to a non-human amino acid). iTope® and TCED® (

[24] ,

[25] , and

[26] ) and other in-silicone The variant with the lowest incidence of potential T cell epitopes, determined by the application of this technology. You can select an array.

[0055] Furthermore, the humanized antibody of the present invention can also be "deimmunized". The presence of human T cell epitopes within protein sequences, such as those of humanized antibodies, suggests that they Because it has the potential to activate helper T cells, its immunogenicity risk profile Since it may increase the risk, deimmunization might be desirable. Such activation of helper T cells can be avoided by deimmunization. This removes T cell epitopes without significantly reducing antibody binding affinity. This can be achieved by introducing mutations into the amino acid sequence of a humanized antibody.

[0056] Therefore, in one aspect of the present invention, (i) a non-human fully variable heavy chain sequence and / or a fully variable light chain sequence. (ii) A step of comparing the sequence with a database of human germline sequences, (ii) A step of obtaining a humanized sequence by selecting a human germline sequence that has high homology, i) a step to optimize the humanized sequence by reverse mutation if necessary, and (iv) appropriate development To produce a humanized antibody by a method that includes the step of expressing the sequence in the present system. It is possible.

[0057] Therefore, the full-length antibody of the present invention (i) contains a non-human variable heavy chain sequence and a variable light chain sequence. (ii) The process of comparing with a database of germ cell sequences, (ii) the highest phase for non-human sequences (iii) Selecting a human germline sequence of the same sex, (iii) In the selected human germline (iv) A process to obtain a humanized sequence by transplanting a non-human CDR, (v) a step of optimizing the humanized sequence, and (vi) A method comprising the step of expressing a full heavy chain sequence and a full light chain sequence in an appropriate expression system. It can be produced by law. Therefore, the full-length antibody of the present invention is as described in Example 1. It can be produced from either the CDR sequence or the full variable region sequence. The production of antibodies is known to those skilled in the art. Therefore, the full-length antibody of the present invention is produced. The method will be obvious to those skilled in the art.

[0058] In this specification, the term "complete heavy chain sequence" refers to variable heavy chain sequences and constant heavy chain sequences, etc. It refers to a sequence of elements.

[0059] In this specification, the term "complete light chain sequence" refers to variable light chain sequences and constant light chain sequences, etc. It refers to a sequence of elements.

[0060] Revertant mutations can be introduced by standard DNA mutagenesis methods. The standard technique for the initial development is described in

[18] . Alternatively, Quickchange(trademark)Site-Direct You can use a commercially available kit such as the ed Mutagenesis Kit (Stratagene), or use de novo DNA synthesis. Therefore, the desired revertant mutation can be introduced.

[0061] Therefore, in one embodiment, the antibody is a humanized antibody.

[0062] Chimeric antibodies are those in which all of the constant region sequences of a non-human (e.g., mouse) antibody are replaced with those of human origin. It can be produced by substituting in the region sequence. Therefore, chimeric antibodies are complete The non-human variable region sequence is maintained. Therefore, the chimeric antibody of the present invention is suitable for expression systems. In this, non-human variable heavy chain (SEQ ID NO:27), non-human variable light chain sequence (SEQ ID NO:28), We expressed a constant heavy chain sequence and a human constant light chain sequence, thereby creating a full-length chimeric antibody. It can be produced by a method that includes the following steps. Alternative methods can also be used. Since methods for producing chimeric antibodies are known to those skilled in the art, the present invention provides for the production of chimeric antibodies. Those in the industry will understand how the body is produced.

[0063] Therefore, in one embodiment, the antibody is a chimeric antibody.

[0064] In one embodiment, the antibody is a full-length antibody. The term "full-length antibody" as used herein refers to: The heavy and light chain constants corresponding to those typically found in wild-type antibodies of this isotype This refers to an antibody that contains all of the variable domains (e.g., a parent antibody or a mutant antibody).

[0065] In one embodiment, the antibody includes an Fc region comprising first and second immunoglobulin heavy chains.

[0066] As used herein, the term "Fc region" refers to a region that extends from the N-terminus to the C-terminus, with a minimum of This refers to the region including the hinge region, CH2 region, and CH3 region. The Fc region is the N-terminus of the hinge region. The side may further include a CH1 region.

[0067] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Therefore, for example, the hinge region of a human IgG1 antibody is the Eu numbering described in Kabat. This corresponds to amino acids 216-230.

[0068] Unless otherwise stated, or unless contextually inconsistent, the constant region array In this specification, amino acids are defined using the Eu-numbering index (Eu-index of numbering). Numbered according to

[27] , and this is “in the Eu numbering as described in Kabat” They might say, "using Kabat's EU numbering," or "using the EU numbering system."

[0069] As used herein, the terms "CH1 region" or "CH1 domain" refer to immunoglobulins. This refers to the CH1 region of the heavy chain. Therefore, for example, the CH1 region of human IgG1 antibody is Eu numbering system In the system, amino acids 118-215 are used. However, the CH1 region is not included in the other sub-regions described herein. Any of the Ipu types is fine.

[0070] As used herein, the terms "CH2 region" or "CH2 domain" refer to immunoglobulins. This refers to the CH2 region of the heavy chain. Therefore, for example, the CH2 region of human IgG1 antibody is part of the Eu numbering system. The 'm' region corresponds to amino acids 231-340. However, the CH2 region is not a subtype as described herein. Either of the following is fine.

[0071] As used herein, the terms "CH3 region" or "CH3 domain" refer to immunoglobulins. This refers to the CH3 region of the heavy chain. Therefore, for example, the CH3 region of human IgG1 antibody is part of the Eu numbering system. The 'm' region corresponds to amino acids 341-447. However, the CH3 region is not included in the other subtypes described herein. Either of the following is fine.

[0072] In one embodiment, the immunoglobulin heavy chain isotypes are IgG1, IgG2, IgG3, and IgG4. A selection is made from the following groups. The immunoglobulin heavy chain is IgG1m(f) (SEQ ID NO:15), It can be any allotype within each immunoglobulin class. Therefore, a specific In one aspect, the isotype of the immunoglobulin heavy chain is IgG1 or any allotype thereof, e.g., for example IgG1m(f) (SEQ ID NO:15).

[0073] When targeting an antigen CD3 that is part of the T cell receptor (TCR), a T cell-specific cell death mechanism is desirable. Other effector functions, such as complement activation, may not be necessary, so a reduction in effector function is desirable. C1q binding is the first step in the complement cascade, and thus serves as an indicator of the complement-dependent cytotoxicity (CDC) ability of the antibody. If the binding of C1q to the antibody can be avoided, activation of the complement cascade can also be avoided.

[0074] Thus, in one aspect, the antibody comprises an Fc region modified such that the binding of C1q to the antibody is at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% reduced compared to the wild-type antibody, where the C1q binding is determined by ELISA. The term "modified" as used herein refers to an amino acid sequence of an Fc region that is not identical to the amino acid sequence of the wild-type Fc region. That is, for example, to modify the binding site for C1q, the binding site for other effector

[0075] molecules, or the binding to the Fc receptor (FcR), amino acid residues at specific positions in the wild-type Fc region are substituted, deleted, or inserted. Such modifications of the amino acid sequence are prepared by substituting one or more amino acids with conservative amino acids <000091? or by substituting one or more amino acids with alternative amino acids that are physically and / or functionally similar to the amino acids present in the wild-type. ​​​​​ This can be done. Substitutions can also be prepared by substituting with non-conservative amino acids.

[0076] In relation to the present invention, amino acids may be described as either conserved amino acids or non-conserved amino acids. Therefore, amino acids can be classified accordingly. Amino acid residues are alternatives. It can also be classified into classes defined by rational and functional properties. Therefore, The amino acid class can be reflected in one or both of the following tables.

[0077] Conservative class amino acid residues TIFF0007851903000001.tif41128 Alternative physical and functional classification of amino acid residues TIFF0007851903000002.tif74144

[0078] With regard to the present invention, substitution in antibodies such as humanized or chimeric antibodies is Original amino acid - position - substituted amino acid It is shown in the form of:

[0079] Symbols to indicate any amino acid residue, referring to well-known amino acid nomenclature. Use a three-letter or one-letter symbol that includes Xaa and X. Therefore, "L234F" or The notation "Leu234Phe" indicates that the antibody has phenylalanine at amino acid position 234, which is leucine. This means that it includes the substitution of "n".

[0080] Substitution of an amino acid at a given position with any other amino acid is, Original amino acid-position, i.e., "L234" It is mentioned that...

[0081] The original amino acid and / or substituted amino acid may contain two or more amino acids, but all For modifications that do not involve amino acids, two or more of them can be separated by ",", or " / ". For example, the substitution of leucine with phenylalanine, arginine, lysine, or tryptophan at position 234 can be represented as "Leu234Phe,Arg,Lys,Trp" or "Leu234Phe / Arg / Lys / Trp" or "L234F,R,K,W" or "L234F / R / K / W" or "L→F,R,K or W at position 234". For the present invention, such designations can be used interchangeably, and they have the same meaning and purpose.

[0082] In addition, the term "substitution" includes substitution with any one of the other 19 natural amino acids or with other amino acids, such as non-natural amino acids. For example, the substitution of amino acid L at position 234 includes each of the following substitutions: 234A, 234C, 234D, 234E, 234 F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P

[0083] F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. This is equivalent to the designation 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 to any position mentioned in this specification, and any one of such substitutions is specifically included in this specification. F, 234G, 234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P [[ID=$$]] 、および234Y。なお、これは234Xという指定に等しく、ここではXが元のアミノ酸以外の 任意のアミノ酸を指定している。これらの置換は、L234A、L234Cなど、またはL234A,Cな ど、またはL234A / C / などと指定することもできる。同じことが、本明細書において言及す るありとあらゆる位置に、同様に当てはまり、そのような置換のいずれか一つが具体的に 本明細書に含まれる。

[0084] The antibodies of the present invention may also include deletions of amino acid residues. Such deletions are represented by "del". For example, it includes descriptions like L234del. Therefore, in such an embodiment, the Roy at position 234 The synth is missing from the amino acid sequence.

[0085] The terms “amino acid” and “amino acid residue” are used interchangeably herein. Shut up.

[0086] As used herein, the term "C1q binding" refers to the state in which an antibody is bound to its antigen. This refers to the binding of C1q to the antibody. The term used herein means "bound to the antigen." "Binding" refers to the binding of an antibody to its antigen, whether in vivo or in vitro.

[0087] In reference to the C1q bond, the term "reduce" as used herein refers to the wild-type antibody Compared to C1q binding to the body, C1q binding to antibodies is reduced, minimized, or completely eliminated. This refers to the ability of the antibody of this invention to inhibit [the specified condition].

[0088] With regard to the use of the antibody of the present invention in comparative assays, the term "field" as used herein is used. A "bio-type antibody" refers to an antibody that is identical to the test antibody except that it is not inactive. In this context... The term "inactive" refers to the C1q bond determined in Example 10 (i.e., the C1q bond). Modified Fc region is reduced or absent (as determined by ELISA); in Example 4 Fc-mediated T cell proliferation is determined by (i.e., T cell proliferation is based on peripheral blood mononuclear cells (PBMCs)). Modified Fc regions that are reduced or absent (when measured by a functional assay); and / or The Fc-mediated CD69 expression determined in Example 3 (i.e., Fc-mediated CD69 expression based on PBMC) This refers to modified Fc regions that are reduced or absent (as determined in a functional assay). Therefore, wild-type antibodies contain native amino acids in their immunoglobulin heavy chains. For example, For example, it may modify or reduce the ability of antibodies to interact with C1q, Fc receptors, etc. This antibody contains no mino acid modifications. Therefore, such a wild-type antibody is, for example, C1q. It will remain an activated antibody capable of binding to the wild-type antibody and the antibody of the present invention. This refers to the ability of an antibody to induce effector function, such as making an antibody a bispecific antibody. This may include other amino acid modifications that do not affect the same amino acid modification.

[0089] As used herein, the term "ELISA" refers to a method of identifying substances using antibodies and color changes. The test is enzyme-linked immunosorbent assay ( e nzyme- l inugh i mmuno s orbent a Refers to (ssay). 1 The specific antibody is attached to the plate surface. This allows the protein from the sample to be released. Add the second antibody that binds to the antibody from the sample and examine its binding to the first specific antibody. The second antibody is linked to the enzyme, and in the final step, a substance containing the enzyme's substrate is added. It can be obtained. Subsequent reactions produce a detectable signal (most commonly a change in the substrate's color). This occurs. The concept of ELISA is well known in the art, and various people who implement ELISA The method is considered to be part of the method for evaluating the antibody of the present invention. Therefore, this The explanation should not be interpreted as limiting, because, as described in Example 4, various This is because it allows for morphological ELISA.

[0090] Specifically, the ability of the antibody of the present invention to bind to C1q is (i) the antibody to 96 wells (ii) a step of coating the sheet, (ii) a step of adding 3% serum, (iii) an anti-human C1q antibody is added. (iv) process of coloring the plate, and (v) OD 405nm Includes a step of measuring This can be determined by ELISA. Therefore, in one embodiment, the antibody is C to the antibody. 1q binding is at least 70%, at least 80%, at least 90%, compared to wild-type antibodies. It includes Fc regions modified to reduce by at least 95%, at least 97%, or 100%, Here, C1q binding occurs in (i) the step of coating the antibody onto a 96-well plate, and (ii) 3 (iii) Adding % serum, (iii) Adding anti-human C1q antibody, (iv) Developing the plate Process, and (v)OD 405nm It is determined by an ELISA that includes a step to measure the following: In certain embodiments, the bonding of C1q is evaluated as described in Example 10.

[0091] As used herein, the terms "Fc receptor" or "FcR" refer to receptors found on the surface of certain cells. It refers to the protein that is released. FcRs bind to the Fc region of antibodies. FcRs are the proteins that they recognize. There are several different types that are classified based on the type of antibody. For example, Fcγ (cancer (m) The receptor binds to IgG class antibodies.

[0092] The terms "Fcγ receptor," "Fc gamma receptor," or "FcγR" as used herein This refers to a group of Fc receptors belonging to the immunoglobulin superfamily, and opsonization ( It is the most important Fc receptor for inducing phagocytosis of coated microorganisms. This family includes Several members of the antibody spectrum, such as FcγRI(CD64), exhibit different antibody affinities due to their differing substructures. FcγRIIa(CD32a), FcγRIIb(CD32b), FcγRIIIa(CD16a), FcγRIIIb(CD16b) It is included.

[0093] The effector function mediated by Fc is related to the biological activity of human immunoglobulin G (IgG) molecules. It forms part of the effect. Examples of such effect functions include, for example, various effects on the Fc area. Antibody-dependent cell-mediated cytotoxicity is triggered by the binding of effector molecules. Examples include ADCC and complement-dependent cell-mediated cytotoxicity (CDC). In relation to the present invention, "Fc binding" and "F "Fc receptor binding," "FcR binding," and "binding of the antibody Fc region to FcR" refer to the binding of the Fc receptor (F This refers to the binding of the Fc region to cR or effector molecules. The terms "FcγR binding" and "FcγR" are used. "I binding" refers to the binding of the Fc region to the Fc gamma receptor and Fc gamma receptor I, respectively. When the CD3 antibody binds to T cells, the wild-type Fc region of the CD3 antibody is located on other cells, such as monocytes. It binds to FcR, which leads to nonspecific Fc-mediated activation of T cells. Nonspecific Fc-mediated activation would be undesirable. T cells target (or target specific) It can also be activated by heterotactic T cell activation. Such targeted T cell activation can occur in cancer, etc. It would be highly desirable for the treatment of a certain range of indications. The term "targeted T cell activation" refers to the activation of specific targets such as tumor targets on tumor cells. The first binding region binds to the T cell and the second binding region binds to T cell-specific targets such as CD3. By using the included bispecific antibodies, T cells can be directed towards specific cells such as tumor cells. This refers to the process of binding. Therefore, one binding region binds to CD3 present on the T cell, and The other binding region is a bispecific antibody that binds to, for example, a target-specific antigen on tumor cells. By using this method, it facilitates the targeting of T cells to specific cells such as tumor cells. It is possible. Nonspecific Fc-mediated T cell activation is still considered, so Fc-mediated Such undesirable nonspecific Fc-mediated T cell activation by bridges should be avoided, Such activity can be neutralized by inactivating the Fc region. This prevents interaction between the inactive Fc region and the existing Fc receptor. Humanization of the present invention The antibody was found to be inactive when tested in several different assays. See Examples 3-5. Another test CD3 having amino acid modifications in the Fc region. The antibody huCLB-T3 / 4 was also found to be inactive when tested using a different assay. Please refer to Examples 7-10. As described in the examples, amino acid substitutions L234F, L235E, The humanized CD3 antibody of the present invention, including D265A, exhibits low levels of CD69 expression on T cells (Example 3). , inhibition of Fc-mediated T cell proliferation (Example 4), and nonspecific targeting in the form of bispecific antibodies No death of the antibodies was observed (Example 5). Thus, the humanized antibody of the present invention is a wild-type antibody. Compared to that, it shows superior results in several assays.

[0094] The antibody of the present invention may contain modifications in the Fc region. If the antibody contains such modifications, its antibody The body can produce inactive or deactivated antibodies. The term "inactive" as used herein refers to antibodies that are inactive or deactivated. Being "inactive" or "deactivated" means that it binds to at least one Fcγ receptor. It is not possible to induce Fc-mediated crosslinking of FcR, or the two Fc of individual antibodies The region fails to induce FcR-mediated crosslinking of the target antigen, or it fails to bind to C1q. This refers to the Fc region that cannot be activated. This indicates that the Fc region of a humanized or chimeric CD3 antibody is inactive. Therefore, it is convenient to test using a single-specificity format antibody, but in this way identification The inactive Fc region is used in bispecific or other humanized or chimeric multispecific CD3 antibodies. It can be used for this purpose.

[0095] For the purpose of developing therapeutic antibodies, the interaction of antibodies with the Fc gamma receptor and C1q is being investigated. Several mutants can be constructed to inactivate the c region. Examples of variants are described herein.

[0096] Therefore, in one embodiment, the antibody is at least 50% stronger than the wild-type antibody. At least 60%, at least 70%, at least 80%, at least 90%, at least 99% or it contains an Fc region modified to mediate Fc-mediated T cell proliferation with 100% reduction, and here This T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

[0097] As used herein, the term "reduce" means compared to a control protein such as an antibody. This refers to a reduction in activity or expression in certain cases. In particular, when it comes to T cell proliferation, it means "to reduce". This term refers to reducing T cell proliferation compared to the proliferation of T cells bound to wild-type antibodies. This refers to the ability of the antibody of the present invention to minimize or completely inhibit T cell proliferation. The antibody's capabilities were determined by a PBMC-based functional assay, as described in Examples 4 and 8. This can be evaluated. In one embodiment, the assay is performed on human PBMCs. In another embodiment, The assay is performed on cynomolgus monkey PBMC. In yet another embodiment, the assay is performed on rhesus monkey PB The procedure is performed using microcosmography (MC). Since the antibodies of the present invention are cross-reactive, the procedure is based on PBMC as described herein. The assay will increase T cell proliferation as long as the PBMCs of the species used are within the antibody cross-reactivity spectrum. To demonstrate the reduction of PBMC in any species, such as humans, crab-eating macaques, or rhesus macaques, This can be done using [this method].

[0098] The term "functional assay based on peripheral blood mononuclear cells (PBMCs)" as used in this specification refers to this Functional characteristics of the antibody of the invention, such as its ability to affect T cell proliferation or CD69 expression. An assay used to evaluate the condition in which the only cells present are peripheral blood mononuclear cells. This refers to an assay in which PBMCs are mixed with an antibody in the range of 1 to 1000 ng / mL at a concentration of 5%. (vol / vol) The process involves incubating the proliferating cells in a CO2-moistened incubator at 37°C for 3 days. The process involves adding chemical compounds such as BrdU, which are incorporated into the DNA, and incubating for 5 hours. The process involves pelletizing the cells, drying the cells, and optionally storing the cells at 4°C. The process involves coating cells onto an ELISA plate, and then, at room temperature, using anti-BrdU peroxidase. Incubate for 90 minutes, then add 1 mg / mL of 2,2'-azino-bis(3-ethylbenzothiazoline). The process involves developing color with -6-sulfonic acid for approximately 30 minutes, and then stopping the reaction by adding 100 μL of 2% oxalic acid. The process involves adding [a certain substance] and measuring the absorbance at 405 nm using a suitable microplate reader. T cell proliferation is measured by a method that includes the following steps.

[0099] As used herein, the term "proliferation" refers to cell growth in the context of cell division.

[0100] In this specification, the term "BrdU" refers to 5-bromod-2'-de, a homolog of thymidine. This refers to oxyuridine. When BrdU is added to cell culture for a limited period (e.g., 4 hours), It will be incorporated into the DNA of the proliferating cells. After the cells are fixed, it will be incorporated. BrdU can be detected using ELISA with anti-BrdU peroxidase. Therefore, BrdU inclusion becomes a measure of proliferation.

[0101] In one embodiment, the antibody, compared to the wild-type antibody, reduces Fc-mediated CD69 expression. At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, small It includes an Fc region modified to reduce at least 99% or 100%, and here it is the Fc-mediated CD6 9 expression is measured in a functional assay based on PBMC.

[0102] As used herein, the term "reduce" means compared to a control protein such as an antibody. This refers to a reduction in activity or expression in certain cases. In particular, it relates to the expression level of the T cell activation marker CD69. In this case, the term "reduce" means that both antibody binding regions bind to CD3. Compared to the CD69 expression level when wild-type antibodies are bound to cells, the low CD69 expression level This refers to a reduction. The ability of antibodies to reduce CD69 expression is described in Examples 3 and 7. It can be evaluated by a functional assay based on PBMC. Therefore, in one embodiment, PBMC The antibody in the range of 1-1000 ng / mL was incubated with the antibody in a 5% (vol / vol) CO2-moistened incubator at 37°C for 16 minutes. ~Incubation for 24 hours, washing of cells, mouse anti-human CD28- The process involves staining cells with PE antibody and mouse anti-human CD69-APC antibody, and on CD28-positive cells. CD69 expression is measured by a method that includes a step of determining CD69 expression by flow cytometry. It will be done.

[0103] As used herein, the term "CD69" refers to the human transmembrane gene encoded by the CD69 gene. This refers to the differentiation antigen group 69, which is a type C lectin protein. It affects T lymphocytes and natural killer cells. Activation of (NK) cells induces CD69 expression both in vivo and in vitro, leading to proliferation and other effects. The function of CD69 as a signaling receptor involved in cell activation events is in lymphocytes, for example. For example, in natural killer cells and platelets, and in the induction of specific genes, SIG It functions as a NAL signaling receptor.

[0104] The term "functional assay based on peripheral blood mononuclear cells (PBMCs)" as used in this specification refers to this Functional characteristics of the antibody of the invention, such as its ability to affect T cell proliferation or CD69 expression. An assay used to evaluate the condition in which the only cells present are peripheral blood mononuclear cells. This refers to an assay that involves CD69. The PBMC-based functional assays described in Examples 3, 4, 5, and 7 involve CD69 To evaluate the current state, (i) place the PBMC with the antibody in a 5% (vol / vol) CO2 humidified incubator. (ii) Incubate at 37°C for approximately 16-24 hours, (ii) Wash the cells, (iii) 4°C In this process, cells are stained with mouse anti-human CD28-PE antibody and mouse anti-human CD69-APC antibody. (iv) Includes a step of determining the expression of CD69 on CD28-positive cells by flow cytometry. Therefore, in one embodiment, CD69 expression is determined as described in Examples 3, 4, 5, or 7. It can be determined.

[0105] Thus, the Fc region plays a major role in the interaction between C1q and the Fc gamma receptor. Amino acids can be modified. Examples of amino acid positions that can be modified include: Examples include L234, L235, and P331. Combinations of these, for example, L234F / L235E / P331S, are also possible. This can cause a significant decrease in binding to human CD64, CD32A, CD16, and C1q.

[0106] Therefore, in one embodiment, at least one position corresponding to L234, L235 and P331 Some amino acids can be A, A, and S, respectively ([1],

[28] ). Also, L2 34F amino acid substitutions and L235E amino acid substitutions affect the interaction with the Fc gamma receptor and C1q. This can result in a blocked Fc region (

[29] ~

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

[31] ) Therefore, in one embodiment, the amino acid at the position corresponding to D265 is A This can be the case. Binding to C1q mutates positions D270, K322, P329, and P331. This can be prevented by using D270A or K322A or P329A. By mutating to any of the P331A genes, the antibody can be made CDC-deficient (

[0032] ). Therefore, in one embodiment, at least one corresponding to D270, K322, P329 and P331 The amino acids at each of the positions can be A, A, A, and A, respectively.

[0107] Alternative approaches to minimize interaction between the Fc region and the Fc gamma receptor and C1q This approach involves removing the glycosylation site of the antibody. For example, position N297 is replaced with Q, A By mutating to E, and other factors critical to the IgG-Fc gamma receptor interaction. The glycosylated site is removed. Therefore, in one embodiment, the position corresponding to N297 The mino acid can be G, Q, A, or E (

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

[31] ).

[0108] Alternatively, in human IgG2 and IgG4 subclasses, interactions with C1q and Fc gamma receptors It is thought that its function is inherently impaired, but its interaction with the Fcγ receptor (Fc gamma receptor) Use has also been reported (

[34] ~

[35] ). In both isotypes, these residual interactions Creating mutations that block the action and reduce unnecessary side effects associated with FcR binding is possible. Yes, it is possible. In the case of IgG2, these include L234A and G237A, and in the case of IgG4, they include L235E. Therefore, in one embodiment, the positions corresponding to L234 and G237 in the human IgG double chain The amino acids can be A and A, respectively. In one embodiment, L2 in human IgG quadruplex The amino acid at position 35 can be E.

[0109] To further minimize the interaction between IgG2 antibodies and the Fc gamma receptor and C1q Other approaches include those described in

[36] and

[37] .

[0110] The hinge region of antibodies may also be important in relation to their interaction with the Fc gamma receptor and complement. (

[38] -

[39] ). Therefore, mutations or deletions in the hinge region are antibody It can affect the effects pedal's functionality.

[0111] As used herein, the term "crosslinking" refers to crosslinking of an antibody to an Fc region, thereby crosslinking the FcR-retaining fiber. Indirect bridging of antibody Fab arms (monovalent or bivalent) bound to the target antigen by cells. It refers to the binding to the target antigen on the target antigen-holding cell. The antibody can cross-link with other cells that express FcR.

[0112] As used herein, the term "nonspecific death" refers to the death of T cells or other effector cells. Cell death due to the cytotoxic function of cells, wherein the activity of said cells is independent of tumor target antigens. This refers to death caused by sexualization. Therefore, nonspecific death means that tumor target-holding cells are, for example, By inducing CDC, etc., the tumor is killed by antibodies that bind to the tumor target. Rather, it means that they can be killed, for example, by cytotoxic T cells.

[0113] By modifying one or more of at least five specific amino acid positions in the Fc region The present inventors have shown that an inactivated Fc region can be obtained (Examples 3-5, 7-1). 0).

[0114] Therefore, in one embodiment, the antibody comprises first and second immunoglobulin heavy chains, and the first and In at least one of the second immunoglobulin heavy chains, positions L234, L235, and D in the human IgG1 heavy chain. One or more amino acids at positions 265, N297, and P331 are L, L, and D, respectively. , N, and P are not.

[0115] In one embodiment, in both the first and second heavy chains, positions L234, L235, and D2 in the human IgG monohelic acid are present. One or more amino acids at positions corresponding to 65, N297, and P331 are L, L, D, respectively. Not N, and not P.

[0116] In another embodiment, in at least one of the first and second heavy chains, the position in the human IgG monochain One or more amino acids at positions L234, L235, and D265 are L, L, and D265, respectively. It is not D, but the amino acids at the positions corresponding to N297 and P331 in the human IgG monochain, respectively These are N and P.

[0117] As used herein, the term "position-corresponding amino acid" refers to the amino acid in the human IgG monochain. Refers to amino acid position numbers. Unless otherwise specified, or if contextual inconsistencies arise, Except for the amino acids in the constant region sequence, the amino acids in this specification are numbered according to the Eu numbering index (described in

[0027] ). Therefore, amino acids in other sequences or A segment "corresponding to" a sequence is an amino acid or segment in a sequence, for example, ALIGN, C LustalW and other standard sequence alignment programs are typically used with their default settings. When used, it aligns with the other amino acids or segments and is less than that of the human IgG monochain. It has at least 50%, at least 80%, at least 90%, or at least 95% identity. This involves aligning the sequence or segments within the sequence, thereby producing the amino The method for determining the position in the sequence corresponding to the acid position is considered to be well known in the art. It is possible.

[0118] In the present invention, amino acids can be defined as described above.

[0119] Regarding amino acids in heavy chains, the phrase "the amino acid is not..." or similar expressions are, This means that the amino acid is any amino acid other than the specific amino acid mentioned. This should be understood as follows: For example, the amino acid at the position corresponding to L234 in the human IgG single chain is L. "Not present" means that the amino acid is either a natural amino acid other than L, or a non-natural amino acid. It means that it is possible.

[0120] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acid at position D265 is not D.

[0121] In one embodiment, at least one of the first and second heavy chains contains D265 in the human IgG monohelic acid. The amino acids at the corresponding positions are not D, but rather correspond to positions N297 and P331 in the human IgG monochain. The amino acids at these positions are N and P, respectively.

[0122] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acid at position D265 is either a hydrophobic amino acid or a polar amino acid.

[0123] In this specification, the term "hydrophobic" with respect to amino acid residues refers to A, C, F, G, H, I This refers to amino acid residues selected from the group consisting of L, M, R, T, V, W, and Y. In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acid at the position corresponding to position D265 is A, C, F, G, H, I, L, M, R, T, V, W, or Y. It is selected from a group of amino acids.

[0124] The term "polarity" as used herein with respect to amino acid residues refers to C, D, E, H, K, N, This refers to any amino acid residue selected from the group consisting of Q, R, S, and T. Therefore, it is a single state. In this case, at least one of the first and second heavy chains, at position D265 in the human heavy chain The amino acid at the corresponding position is selected from the group consisting of C, E, H, K, N, Q, R, S, and T. ru.

[0125] In another embodiment, in at least one of the first and second heavy chains, in human IgG monocoque The amino acid at the position corresponding to position D265 is an aliphatic uncharged amino acid, an aromatic amino acid, or It is an acidic amino acid.

[0126] In this specification, the term "aliphatic uncharged" in relation to amino acid residues refers to A, G, I, L This refers to any amino acid residue selected from the group consisting of , and V. Therefore, in one embodiment, In at least one of the first and second heavy chains, relative to position D265 in the human IgG monohelic acid The amino acid at the corresponding position is selected from the group consisting of A, G, I, L, and V.

[0127] The term "aromatic" as used herein with respect to amino acid residues is F, T, and W. This refers to any amino acid residue selected from the group. Therefore, in one embodiment, the first heavy In at least one of the chain and the double chain, the position corresponding to position D265 in the human IgG single chain. The amino acids are selected from the group consisting of F, T, and W.

[0128] As used herein with respect to amino acid residues, the term "acidic" refers to the group consisting of D and E. This refers to any more selected amino acid residues. Therefore, in one embodiment, the first heavy chain and the second In at least one of the heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is The group is selected from the group consisting of D and E.

[0129] In a particular embodiment, at least one of the first and second heavy chains is human IgG monolayer. The amino acid at position D265 in the chain is one of A, E, F, G, I, L, T, V, and W. Selected from the group.

[0130] In one embodiment, in both the first and second heavy chains, at position D265 in the human IgG monohelic acid, The amino acid at the corresponding position is not D.

[0131] In one embodiment, in both the primary and secondary heavy chains, the position corresponding to D265 in the human IgG monohelic acid chain is The amino acid is not D, but rather the amino acids at positions N297 and P331 in the human IgG monochain. The acids are N and P, respectively.

[0132] In one embodiment, in both the first and second heavy chains, at position D265 in the human IgG monohelic acid, The amino acid at the corresponding position is either a hydrophobic amino acid or a polar amino acid.

[0133] In this specification, the term "hydrophobic" with respect to amino acid residues refers to A, C, F, G, H, I This refers to amino acid residues selected from the group consisting of L, M, R, T, V, W, and Y. In one embodiment, in both the first and second heavy chains, at position D265 in the human IgG monohelic acid The amino acids at the corresponding positions are A, C, F, G, H, I, L, M, R, T, V, W, and Y. Selected from the group of ano acids.

[0134] The term "polarity" as used herein with respect to amino acid residues refers to C, D, E, H, K, N, This refers to any amino acid residue selected from the group consisting of Q, R, S, and T. Therefore, it is a single state. In this case, in both the first and second heavy chains, the position corresponding to position D265 in the human heavy chain The amino acids are selected from the group consisting of C, E, H, K, N, Q, R, S, and T. Therefore, in both the first and second heavy chains, the position D265 in the human IgG single heavy chain corresponds to A group of amino acids whose positional amino acids consist of A, C, F, G, H, I, L, M, R, T, V, W, and Y. Selected from.

[0135] In one embodiment, both the first and second heavy chains correspond to position D265 in the human heavy chain. The amino acid at the position is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0136] In another embodiment, in both the first and second heavy chains, position D265 in the human IgG monohelic acid chain The amino acid at the corresponding position is an aliphatic uncharged amino acid, an aromatic amino acid, or an acidic amino acid. It is an acid.

[0137] In this specification, the term "aliphatic uncharged" in relation to amino acid residues refers to A, G, I, L This refers to any amino acid residue selected from the group consisting of , and V. Therefore, in one embodiment, In both the first and second heavy chains, the position corresponding to position D265 in the human IgG monohelic acid chain. The amino acids are selected from the group consisting of A, G, I, L, and V.

[0138] The term "aromatic" as used herein with respect to amino acid residues is F, T, and W. This refers to any amino acid residue selected from the group. Therefore, in one embodiment, the first heavy In both the single chain and the double chain, the amino acid at the position corresponding to position D265 in the human IgG single chain. However, it is selected from the group consisting of F, T, and W.

[0139] As used herein with respect to amino acid residues, the term "acidic" refers to the group consisting of D and E. This refers to any more selected amino acid residues. Therefore, in one embodiment, the first heavy chain and the second In both heavy chains, the amino acids at the position corresponding to position D265 in the human IgG1 heavy chain are D and E Selected from the group consisting of .

[0140] In a particular embodiment, the position in the human IgG monocoque in both the first and second heavy chains The amino acid at the position corresponding to D265 is selected from the group consisting of A, E, F, G, I, L, T, V, and W. It will be selected.

[0141] In a further embodiment, at least one of the first and second heavy chains contains human IgG monolayer The amino acid at the position corresponding to N297 in the chain is not nitrogen.

[0142] In one embodiment, at least one of the first and second heavy chains contains N297 in the human IgG monohelic acid. The amino acid at the corresponding position is not N, but the amino acid at the position corresponding to position P331 in the human IgG single chain. The amino acid is P.

[0143] In one embodiment, in both the first and second heavy chains, at position N297 in the human IgG monohelic acid, The amino acid at the corresponding position is not nitrogen (N).

[0144] In one embodiment, in both the primary and secondary heavy chains, the position corresponding to N297 in the human IgG monohelic acid chain is The amino acid is not N, but rather the amino acid at the position corresponding to position P331 in the human IgG monochain is P. .

[0145] In a further embodiment, at least one of the first and second heavy chains contains human IgG monolayer The amino acids at positions L234 and L235 in the chain are not L and L, respectively.

[0146] In one embodiment, at least one of the first and second heavy chains contains L234 in the human IgG monohelic acid. And the amino acids at the positions corresponding to L235 are not L and L, but rather the positions in the human IgG1 heavy chain. The amino acids at positions N297 and P331 are N and P, respectively.

[0147] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids corresponding to positions L234 and L235 are A, C, D, E, F, G, H, I, K, M, N, P, Q, R Selected from the group consisting of S, T, Y, and V.

[0148] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234 and L235 are hydrophobic or polar amino acids. be.

[0149] In this specification, the term "hydrophobic" with respect to amino acid residues refers to A, C, F, G, H, I This refers to amino acid residues selected from the group consisting of L, M, R, T, V, W, and Y. In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234 and L235 are A, C, F, G, H, I, M, R, respectively. Selected from the group consisting of T, V, W, and Y.

[0150] The term "polarity" as used herein with respect to amino acid residues refers to C, D, E, H, K, N, This refers to any amino acid residue selected from the group consisting of Q, R, S, and T. Therefore, it is a single state. In this case, at least one of the first and second heavy chains, position L2 in the human IgG monohelic acid. The amino acids at positions 34 and L235 are C, D, E, H, K, N, Q, R, S, and O, respectively. It is selected from a group of amino acids consisting of and T.

[0151] In a particular embodiment, at least one of the first and second heavy chains is human IgG monolayer. The amino acids at positions L234 and L235 in the chain are A, C, D, E, F, and G, respectively. The group is selected from H, I, K, M, N, Q, R, S, T, V, W, and Y.

[0152] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 and L are not L and L, respectively.

[0153] In one embodiment, in both the primary and secondary heavy chains, L234 and L235 in the human IgG mono-heavy chain are compared The amino acids at the corresponding positions are not L and L respectively, but rather positions N297 and P3 in the human IgG1 heavy chain. The amino acids at positions 31 are N and P, respectively.

[0154] In one embodiment, in both the first and second heavy chains, L234 and in the human IgG monohelic acid chain The amino acid at the position corresponding to L235 is either a hydrophobic amino acid or a polar amino acid.

[0155] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 are A, C, F, G, H, I, M, R, T, V, W, respectively. The group is selected from the group consisting of and Y.

[0156] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 are C, D, E, H, K, N, Q, R, S, and T, respectively. It is selected from a group of amino acids consisting of the following.

[0157] In a particular embodiment, the position in the human IgG monocoque in both the first and second heavy chains The amino acids at positions L234 and L235 are A, C, D, E, F, G, H, I, K, and M, respectively. Selected from the group consisting of N, Q, R, S, T, V, W, and Y.

[0158] In another embodiment, in at least one of the first and second heavy chains, in human IgG monocoque The amino acids at positions L234 and L235 are aliphatic uncharged amino acids, aromatic amino acids. It is an acid or an acidic amino acid.

[0159] In this specification, the term "aliphatic uncharged" in relation to amino acid residues refers to A, G, I, L This refers to any amino acid residue selected from the group consisting of , and V. Therefore, in one embodiment, In at least one of the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 are selected from the group consisting of A, G, I, and V, respectively. It can be done.

[0160] The term "aromatic" as used herein with respect to amino acid residues is F, T, and W. This refers to any amino acid residue selected from the group. Therefore, in one embodiment, the first heavy In at least one of the chain and the double chain, relative to positions L234 and L235 in the human IgG monohelic acid The amino acids at the corresponding positions are selected from the group consisting of F, T, and W.

[0161] As used herein with respect to amino acid residues, the term "acidic" refers to the group consisting of D and E. This refers to any more selected amino acid residue. Therefore, in one embodiment, the first heavy chain and In at least one of the second heavy chains, the positions corresponding to positions L234 and L235 in the human IgG monohelic acid are Each of the amino acids is selected from the group consisting of D and E.

[0162] In a particular embodiment, in at least one of the first heavy chain and the second heavy chain, L234 and The amino acids at the position corresponding to L235 are A, D, E, F, G, I, T, V, and W, respectively. Selected from the group.

[0163] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234 and L235 are F and E, or A and A, respectively. That is the case.

[0164] In one embodiment, at least one of the first and second heavy chains contains L234 in the human IgG monohelic acid. The amino acids at the positions corresponding to L235 are F and E, or A and A, respectively. The amino acids at positions N297 and P331 in the human IgG monochain are, respectively, N and It's BiP.

[0165] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 are F and E, or A and A, respectively.

[0166] In one embodiment, in both the primary and secondary heavy chains, L234 and L235 in the human IgG mono-heavy chain are compared The amino acids at the corresponding positions are F and E, or A and A, respectively, and it is a human IgG monochain. The amino acids at positions N297 and P331 in the molecule are N and P, respectively.

[0167] In a particular embodiment, at least one of the first and second heavy chains is human IgG monolayer. The amino acids at positions L234 and L235 in the chain are F and E, respectively.

[0168] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 are F and E, respectively.

[0169] In one embodiment, at least one of the first heavy chain and the second heavy chain contains at least human Ig The amino acids at positions L234 and L235 in the G1 heavy chain are A and A, respectively.

[0170] In one embodiment, in both the first and second heavy chains, at least the human IgG monohelic acid is present in The amino acids at positions L234 and L235 are A and A, respectively.

[0171] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234, L235, and D265 are L, L, and D respectively. do not have.

[0172] In one embodiment, at least one of the first and second heavy chains contains L234 in the human IgG monohelic acid. The amino acids at the positions corresponding to L235 and D265 are not L, L, and D, respectively, but rather human I The amino acids at positions N297 and P331 in the gG1 heavy chain are N and P, respectively. .

[0173] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids corresponding to positions L234 and L235 are A, C, D, E, F, G, H, I, K, M, N, P, Q, R The amino acid selected from the group consisting of S, T, Y, V, and W, and corresponding to position D265, is A Selected from the group consisting of C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W It can be done.

[0174] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234, L235, and D265 are hydrophobic amino acids or polar amino acids. It is an acid.

[0175] In this specification, the term "hydrophobic" with respect to amino acid residues refers to A, C, F, G, H, I This refers to amino acid residues selected from the group consisting of L, M, R, T, V, W, and Y. In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acid at the position corresponding to position D265 is A, C, F, G, H, I, L, M, R, T, V, W, or Y. Selected from a group of amino acids, and corresponding to positions L234 and L235 in the human IgG1 heavy chain. The amino acids at each position belong to the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y. It will be selected.

[0176] The term "polarity" as used herein with respect to amino acid residues refers to C, D, E, H, K, N, This refers to any amino acid residue selected from the group consisting of Q, R, S, and T. Therefore, it is a single state. In this case, at least one of the first and second heavy chains, position L2 in the human IgG monohelic acid. The amino acids at positions 34 and L235 are C, D, E, H, K, N, Q, R, S, and O, respectively. Selected from the group of amino acids consisting of and T, and corresponding to position D265 in the human heavy chain The amino acids are selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0177] In a particular embodiment, at least one of the first and second heavy chains is human IgG monolayer. The amino acids at positions L234 and L235 in the chain are A, C, D, E, F, and G, respectively. Selected from the group consisting of H, I, K, M, N, Q, R, S, T, V, W, and Y, and human IgG monochain The amino acids corresponding to position D265 are A, C, E, F, G, H, I, K, L, M, N, Q, R, S Selected from the group consisting of T, V, W, and Y.

[0178] In one embodiment, in both the first and second heavy chains, L234, L235 in the human IgG monohelic acid The amino acids at the positions corresponding to D265 are hydrophobic or polar amino acids.

[0179] In one embodiment, in both the first and second heavy chains, at position D265 in the human IgG monohelic acid, The amino acids at the corresponding positions are A, C, F, G, H, I, L, M, R, T, V, W, and Y. Selected from the group of ano acids, and at positions L234 and L235 in the human IgG1 heavy chain. Each of the no acids is selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y. ru.

[0180] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid is The amino acids at the positions corresponding to L235 are C, D, E, H, K, N, Q, R, S, and T, respectively. A selection from the group of amino acids consisting of the amino acids at the position corresponding to position D265 in the human heavy chain. The acid is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0181] In a particular embodiment, the position in the human IgG monocoque in both the first and second heavy chains The amino acids at positions L234 and L235 are A, C, D, E, F, G, H, I, K, and M, respectively. Selected from the group consisting of N, Q, R, S, T, V, W, and Y, and at position D2 in the human IgG monohelic acid. The amino acids corresponding to position 65 are A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W Selected from the group consisting of , and Y.

[0182] In another embodiment, in at least one of the first and second heavy chains, in human IgG monocoque The amino acids at positions L234, L235, and D265 are aliphatic uncharged amino acids, aromatic It is a group amino acid or an acidic amino acid.

[0183] In this specification, the term "aliphatic uncharged" in relation to amino acid residues refers to A, G, I, L This refers to any amino acid residue selected from the group consisting of , and V. Therefore, in one embodiment, In at least one of the first and second heavy chains, relative to position D265 in the human IgG monohelic acid The amino acid at the corresponding position is selected from the group consisting of A, G, I, L, and V, and human IgG1 The amino acids at positions L234 and L235 in the heavy chain are A, G, I, and V, respectively. Selected from the group consisting of .

[0184] The term "aromatic" as used herein with respect to amino acid residues is F, T, and W. This refers to any amino acid residue selected from the group. Therefore, in one embodiment, the first heavy In at least one of the main chain and the double chain, positions L234, L235 and D26 in the human IgG monohelic acid. The amino acids at the positions corresponding to 5 are selected from the group consisting of F, T, and W, respectively.

[0185] As used herein with respect to amino acid residues, the term "acidic" refers to the group consisting of D and E. This refers to any more selected amino acid residue. Therefore, in one embodiment, the first heavy chain and In at least one of the second heavy chains, relative to positions L234, L235, and D265 in the human IgG monohelic acid, The amino acids at the corresponding positions are selected from groups consisting of D and E.

[0186] In a particular embodiment, at least one of the first and second heavy chains is human IgG monolayer. The amino acid at position D265 in the chain is one of A, E, F, G, I, L, T, V, and W. Selected from the group, the amino acids at positions corresponding to L234 and L235 are A, D, and The group is selected from E, F, G, I, T, V, and W.

[0187] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid, The amino acids at the positions corresponding to L235 and D265 are not L, L, and D, respectively.

[0188] In one embodiment, in both the first and second heavy chains, L234, L235, and in the human IgG monochain, The amino acids at the position corresponding to D265 are not L, L, and D, respectively, and are not part of the human IgG monocoque. The amino acids at positions N297 and P331 in the molecule are N and P, respectively.

[0189] In one embodiment, in both the first and second heavy chains, L234, L235 in the human IgG monohelic acid , and the amino acid at the position corresponding to D265 is an aliphatic uncharged amino acid, an aromatic amino acid, or It is an acidic amino acid.

[0190] In one embodiment, in both the first and second heavy chains, at position D265 in the human IgG monohelic acid, The amino acids at the corresponding positions are selected from the group consisting of A, G, I, L, and V, and are human IgG The amino acids at positions L234 and L235 in the single chain are A, G, I, and Selected from the group consisting of V.

[0191] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid, The amino acids at the positions corresponding to L235 and D265 are selected from the group consisting of D and E, respectively. It will be done.

[0192] In a particular embodiment, the position in the human IgG monocoque in both the first and second heavy chains The amino acid at the position corresponding to D265 is selected from the group consisting of A, E, F, G, I, L, T, V, and W. The selected amino acids, corresponding to positions L234 and L235, are A, D, E, F, G, and I, respectively. Selected from the group consisting of T, V, and W.

[0193] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. The answers are A, A, and A.

[0194] In one embodiment, at least one of the first and second heavy chains contains L234 in the human IgG monohelic acid. The amino acids at the positions corresponding to L235 and D265 are F, E, and A, or A, A , and A, and the amino acids at positions N297 and P331 in the human IgG monohelic acid chain. These are N and P, respectively.

[0195] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid, The amino acids at positions corresponding to L235 and D265 are F, E, and A, or A, A, And A.

[0196] In one embodiment, in both the first and second heavy chains, L234, L235, and in the human IgG monochain, The amino acids at the position corresponding to D265 are F, E, and A, or A, A, and A, respectively. Furthermore, the amino acids at positions N297 and P331 in the human IgG mono-heavy chain are, respectively, N This is YobiP.

[0197] In a particular embodiment, at least one of the first and second heavy chains is human IgG monolayer. The amino acids at positions L234, L235, and D265 in the chain are F, E, and It is A.

[0198] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid, The amino acids at positions L235 and D265 are F, E, and A, respectively.

[0199] In one embodiment, at least one of the first and second heavy chains contains human IgG monocoque The amino acids at positions L234, L235, and D265 are A, A, and A, respectively. ru.

[0200] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid, The amino acids at positions L235 and D265 are A, A, and A, respectively.

[0201] In another embodiment, in at least one of the first and second heavy chains, in human IgG monocoque The amino acids at positions L234, L235, D265, N297, and P331 are F and E, respectively. These are A, Q, and S.

[0202] In one embodiment, in both the first and second heavy chains, position L234 in the human IgG monohelic acid, The amino acids at positions corresponding to L235, D265, N297, and P331 are F, E, A, Q, and It is S.

[0203] In a particular embodiment, the antibody of the present invention has a VH sequence shown in SEQ ID NO:8, SEQ ID NO:10 It contains the VL sequence shown, and at least one of the heavy chains is at position L234 in the human IgG1 heavy chain. The amino acids at positions L235 and D265 are F, E, and A, respectively.

[0204] In another embodiment, the antibody of the present invention is the VH sequence shown in SEQ ID NO:8 and the sequence shown in SEQ ID NO:12. It contains a VL sequence, and at least one of the heavy chains is located at positions L234 and L235 in the human IgG1 heavy chain. The amino acids at the positions corresponding to D265 are F, E, and A, respectively.

[0205] In another embodiment, the antibody of the present invention is shown in the VH sequence shown in SEQ ID NO:6 and SEQ ID NO:10. It contains a VL sequence, and at least one of the heavy chains is located at positions L234 and L235 in the human IgG1 heavy chain. The amino acids at the positions corresponding to D265 are F, E, and A, respectively.

[0206] In another embodiment, the antibody of the present invention is shown in the VH sequence shown in SEQ ID NO:6 and SEQ ID NO:12. It contains a VL sequence, and at least one of the heavy chains is located at positions L234 and L235 in the human IgG1 heavy chain. The amino acids at the positions corresponding to D265 are F, E, and A, respectively.

[0207] In another embodiment, the antibody of the present invention is the VH sequence shown in SEQ ID NO:9 and the sequence shown in SEQ ID NO:10. It contains a VL sequence, and at least one of the heavy chains is located at positions L234 and L235 in the human IgG1 heavy chain. The amino acids at the positions corresponding to D265 are F, E, and A, respectively.

[0208] In another embodiment, the antibody of the present invention is shown in the VH sequence shown in SEQ ID NO:9 and SEQ ID NO:12. It contains a VL sequence, and at least one of the heavy chains is located at positions L234 and L235 in the human IgG1 heavy chain. The amino acids at the positions corresponding to D265 are F, E, and A, respectively.

[0209] In one aspect, the antibody of the present invention is a human IgLC2 / IgLC3 constant domain lamina with SEQ ID NO:31. Includes light chains.

[0210] In one aspect, the antibody of the present invention increases the expression level and / or production yield. The light chain (LC) and / or heavy chain (HC) of the sea urchin may be modified. In one embodiment, the present invention Antibodies can be modified in the light chain (LC). Such modifications are known in the art. For example, Zheng, L., Goddard, J.-P., Baumann, U., & Reymond, J.-L. (2004). Exp ression improvement and mechanistic study of the retro-Diels-Alderase catalytic 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3 ), 807-14. doi:10.1016 / j.jmb.2004.06.014 ru.

[0211] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acid at the position corresponding to position T41 is not T.

[0212] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acid at the position corresponding to position T41 is selected from H, I, K, L, Q, R, and V.

[0213] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acid at the position corresponding to position T41 is H, K, or R, for example, K.

[0214] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acid at the position corresponding to position F10 is not F, and the position in the lambda light chain of SEQ ID NO:10 One or more amino acid positions corresponding to T41, K55, and L97 are T, K, and It's not L.

[0215] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acids at positions F10, T41, K55, and L97 are F, T, K, and L, respectively. There isn't one.

[0216] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acid at the position corresponding to position T41 is selected from H, I, K, L, Q, R, or V, for example H , K, and R, for example, selected from K, and corresponding to positions F10, T41, K55, and L97. The amino acids at each position are L, K, N, and H, respectively.

[0217] In one embodiment, the antibody of the present invention comprises a light chain (LC) and positions corresponding to R23 and A35. The amino acids in the positions are not R and A, respectively.

[0218] In one embodiment, the antibody of the present invention comprises a light chain (LC) and an amine at a position corresponding to position R23. The no acids are selected from A, G, H, K, Q, S, and T, for example from A and G, and correspond to A35. The amino acids at the position are changed from I, L, M, P, V, G, F, and W, for example, I, L, M, P, and V Selected from.

[0219] In one embodiment, the antibody of the present invention comprises a light chain (LC) and an amine at a position corresponding to position R23. The no acid is A or G, for example, A, and the amino acid at the position corresponding to position A35 is P.

[0220] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 Amino acids at positions corresponding to positions F10, R23, A35, R47, D71, A82, D83, S86, I87, and F89 The acids are not F, R, A, R, D, A, D, S, I, and F, respectively.

[0221] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acids at the position corresponding to position R23 are selected from A, G, H, K, Q, S, and T, for example, A and G. The amino acids selected from the I, L, M, P, V, G, F, and W are the amino acids at the position corresponding to A35, for example. If selected from I, L, M, P, and positions F10, R47, D71, A82, D83, S86, I87, and F89 The amino acids at the corresponding positions are L, T, G, P, E, A, E, and Y, respectively.

[0222] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acid at the position corresponding to position R23 is A or G, and positions F10, A35, R47, D71, A The amino acids at positions 82, D83, S86, I87, and F89 are L, P, T, G, P, E, respectively. These are A, E, and Y.

[0223] In one aspect, the antibody of the present invention increases the affinity of the antibody by using light chains (LCs) Modifications can occur within the molecule. Modifications that lead to increased antibody affinity are publicly known in the art. It is knowledge.

[0224] In one aspect, the antibody of the present invention reduces the affinity of the antibody by reducing the light chain (LC) It can be modified internally. This is advantageous in some settings and leads to increased effectiveness. This is possible. In particular, the low affinity of the CD3 arm may affect the movement of T cells in circulation and at tumor sites. This can affect motility and therefore lead to better engagement between T cells and tumor cells. See olhOj et al, Molecular Immunology 44 (2007). In particular, this is CD3 antibody This can be useful in a bispecific format where it is used as one of the binding arms. Modifications that lead to a reduction in antibody affinity are known in the art, for example, See Webster et al. Int J Cancer Suppl. 1988;3:13-6.

[0225] In one embodiment, the antibody of the present invention comprises a light chain (LC), (i) Is the amino acid at the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 not F? ,or (ii) Is the amino acid at the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 not K? ,or (iii) The amino acid at the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 is not F. Furthermore, the amino acid at the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 is not K.

[0226] In one embodiment, the antibody of the present invention comprises a constant light chain (LC), (i) Is the amino acid at the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 L? or (ii) Is the amino acid at the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 N? or (iii) The amino acid at the position corresponding to position F10 in the lambda light chain of SEQ ID NO:10 is L. Furthermore, the amino acid at the position corresponding to position K55 in the lambda light chain of SEQ ID NO:10 is nitrogen (N).

[0227] In one embodiment, the antibody of the present invention comprises a light chain (LC), and the lambda light chain of SEQ ID NO:10 The amino acids at positions F10, T41, K55, and L97 are F, T, K, and L, respectively. There is no such modification. Such modifications increase the expression level and decrease affinity. It is useful for both purposes.

[0228] The antibody of the present invention comprises a light chain (LC), with positions F10, T41, and K5 in the lambda light chain of SEQ ID NO: 10. The amino acids at positions 5 and L97 are L, K, N, and H, respectively. Modifications serve both to increase expression levels and to decrease affinity. Tsu.

[0229] In one embodiment, the antibody of the present invention comprises a heavy chain (HC) and a light chain (LC), and SEQ ID NO: 15 The positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A is, the position corresponding to F405 in the human IgG single chain of SEQ ID NO:15 is L, and (i)S The positions corresponding to positions F10, T41, K55, and L97 in the lambda light chain of EQ ID NO:10 are L (ii) is K, N, and H, or corresponds to position T41 in the lambda light chain of SEQ ID NO:10 The position is K.

[0230] In one embodiment, the antibody of the present invention comprises a heavy chain (HC) of SEQ ID NO:25 and a light chain of SEQ ID NO:32. Includes chain (LC).

[0231] In one embodiment, the antibody of the present invention comprises a heavy chain (HC) of SEQ ID NO:25 and a light chain of SEQ ID NO:33. Includes chain (LC).

[0232] In one aspect, the present invention relates to at least one aspect or state described herein. The antibody's first binding region and the binding to one or more targets different from the first binding region. This relates to a multispecific antibody comprising one or more binding regions. Sex antibodies can be bispecific antibodies.

[0233] Therefore, in one aspect, the present invention is applicable to any aspect or embodiment described herein. The antibody's first binding region and its second binding region, which binds to a different target than the first binding region. This relates to bispecific antibodies that include [specific antibodies].

[0234] The term "multispecific antibody" refers to antibodies that are at least two different, for example, at least three. This typically refers to antibodies that have specificity for epitopes but do not overlap. Such epitopes may be on the same target or on different targets. Also, if the epitope is on a different target, such targets are the same cell. Alternatively, it may be on a cell type or on a different cell or cell type.

[0235] The term "bispecific antibody" refers to an antibody that has at least two different, typically overlapping properties. This refers to antibodies that have specificity for epitopes that are not targeted. They may be on the same target or on different targets. Different epitopes If the target is on the same cell or cell type, They may be on different cells or cell types.

[0236] In one embodiment, the bispecific antibody comprises a primary heavy chain and a secondary heavy chain.

[0237] The embodiments relating to modification of the Fc region and the embodiments relating to specific amino acid substitutions are the present invention. It is thought to be part of the bispecific antibody. Therefore, in one embodiment, the first heavy chain and At least one of the second heavy chains is defined as in any embodiment described herein. This document describes the provision of one or more amino acids modified in such a way as an inactive Fc region. This includes, in one embodiment, both the first and second heavy chains are any of the ones described herein. One or more amino acids modified as defined in the embodiment, for example, an inactive Fc region This includes information regarding the provision of the region. Therefore, bispecific antibodies are as described herein. The Fc region includes an Fc region modified according to any aspect or aspect of the first heavy chain and at least one of the second double chains in any aspect or manner described herein It contains one or more amino acids modified to define as follows.

[0238] Therefore, in one embodiment, the bispecific antibody is a VH sequence and SEQ shown in SEQ ID NO:8. It includes the first binding region containing the VL sequence shown in ID NO:10, where the Fc region is determined by ELISA. The binding of C1q to the antibody is at least 70% and at least 80% compared to the wild-type antibody. , qualifies to reduce by at least 90%, at least 95%, at least 97%, or 100%. It is being done.

[0239] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and SEQ ID NO:12 It includes a first binding region containing the VL sequence shown, where the Fc region is the antibody determined by ELISA. The binding of C1q to the body is at least 70%, at least 80%, compared to wild-type antibodies. It is also qualified to reduce by 90%, at least 95%, at least 97%, or 100%. .

[0240] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:10 It includes a first binding region containing the VL sequence shown, where the Fc region is the antibody determined by ELISA. The binding of C1q to the body is at least 70%, at least 80%, compared to wild-type antibodies. It is also qualified to reduce by 90%, at least 95%, at least 97%, or 100%. .

[0241] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:12 It includes a first binding region containing the VL sequence shown, where the Fc region is the antibody determined by ELISA. The binding of C1q to the body is at least 70%, at least 80%, compared to wild-type antibodies. It is also qualified to reduce by 90%, at least 95%, at least 97%, or 100%. .

[0242] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:10 It includes a first binding region containing the VL sequence shown, where the Fc region is the antibody determined by ELISA. The binding of C1q to the body is at least 70%, at least 80%, compared to wild-type antibodies. It is also qualified to reduce by 90%, at least 95%, at least 97%, or 100%. .

[0243] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:12 It includes a first binding region containing the VL sequence shown, where the Fc region is the antibody determined by ELISA. The binding of C1q to the body is at least 70%, at least 80%, compared to wild-type antibodies. It is also qualified to reduce by 90%, at least 95%, at least 97%, or 100%. .

[0244] In another embodiment, a bispecific antibody is used with respect to the VH sequence shown in SEQ ID NO:8 and SEQ ID NO:10. The antibody comprises a first binding region containing the VL sequence shown, and the antibody is at least 5 times stronger than the wild-type antibody. 0%, at least 60%, at least 70%, at least 80%, at least 90%, at least 9 The Fc region is modified to mediate Fc-mediated T cell proliferation that is reduced by 9% or 100%. Here, T cell proliferation was measured in a functional assay based on peripheral blood mononuclear cells (PBMCs). ru.

[0245] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and SEQ ID NO:12 The antibody contains a first binding region including the VL sequence shown, and the antibody is less than that of the wild-type antibody. 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least The Fc region is modified to mediate Fc-mediated T cell proliferation, which is reduced by 99% or 100%. Here, T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs). It can be done.

[0246] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:10 The antibody contains a first binding region including the VL sequence shown, and the antibody is less than that of the wild-type antibody. 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least The Fc region is modified to mediate Fc-mediated T cell proliferation, which is reduced by 99% or 100%. Here, T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs). It can be done.

[0247] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:12 The antibody contains a first binding region including the VL sequence shown, and the antibody is less than that of the wild-type antibody. 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least The Fc region is modified to mediate Fc-mediated T cell proliferation, which is reduced by 99% or 100%. Here, T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs). It can be done.

[0248] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:10 The antibody contains a first binding region including the VL sequence shown, and the antibody is less than that of the wild-type antibody. 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least The Fc region is modified to mediate Fc-mediated T cell proliferation, which is reduced by 99% or 100%. Here, T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs). It can be done.

[0249] In one embodiment, a bispecific antibody is used in the VH sequence shown in SEQ ID NO:9 and SEQ ID NO:12. The antibody comprises a first binding region containing the VL sequence shown, and the antibody is at least 5 times stronger than the wild-type antibody. 0%, at least 60%, at least 70%, at least 80%, at least 90%, at least 9 The Fc region is modified to mediate Fc-mediated T cell proliferation that is reduced by 9% or 100%. Here, T cell proliferation was measured in a functional assay based on peripheral blood mononuclear cells (PBMCs). ru.

[0250] In another embodiment, a bispecific antibody is used with respect to the VH sequence shown in SEQ ID NO:8 and SEQ ID NO:10. The antibody contains a first binding region including the VL sequence shown, and the antibody inhibits Fc-mediated CD69 expression compared to a wild-type antibody. Compared to at least 50%, at least 60%, at least 70%, at least 80%, and at least The Fc region has been modified to reduce it by 90%, at least 99%, or even 100%, and here Therefore, Fc-mediated CD69 expression is determined by a functional assay based on PBMCs.

[0251] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and SEQ ID NO:12 The antibody contains a first binding region including the VL sequence shown, and the antibody inhibits Fc-mediated CD69 expression, and the antibody inhibits wild-type antibody Compared to at least 50%, at least 60%, at least 70%, at least 80%, less The Fc region has been modified to reduce both by 90%, at least 99%, or 100%, and In this case, Fc-mediated CD69 expression is determined by a functional assay based on PBMC.

[0252] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:10 The antibody contains a first binding region including the VL sequence shown, and the antibody inhibits Fc-mediated CD69 expression, and the antibody inhibits wild-type antibody Compared to at least 50%, at least 60%, at least 70%, at least 80%, less The Fc region has been modified to reduce both by 90%, at least 99%, or 100%, and In this case, Fc-mediated CD69 expression is determined by a functional assay based on PBMC.

[0253] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:12 The antibody contains a first binding region including the VL sequence shown, and the antibody inhibits Fc-mediated CD69 expression, and the antibody inhibits wild-type antibody Compared to at least 50%, at least 60%, at least 70%, at least 80%, less The Fc region has been modified to reduce both by 90%, at least 99%, or 100%, and In this case, Fc-mediated CD69 expression is determined by a functional assay based on PBMC.

[0254] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:10 The antibody contains a first binding region including the VL sequence shown, and the antibody inhibits Fc-mediated CD69 expression, and the antibody inhibits wild-type antibody Compared to at least 50%, at least 60%, at least 70%, at least 80%, less The Fc region has been modified to reduce both by 90%, at least 99%, or 100%, and In this case, Fc-mediated CD69 expression is determined by a functional assay based on PBMC.

[0255] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:12 The antibody contains a first binding region including the VL sequence shown, and the antibody inhibits Fc-mediated CD69 expression, and the antibody inhibits wild-type antibody Compared to at least 50%, at least 60%, at least 70%, at least 80%, less The Fc region has been modified to reduce both by 90%, at least 99%, or 100%, and In this case, Fc-mediated CD69 expression is determined by a functional assay based on PBMC.

[0256] In a particular embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and SEQ ID N It includes a first binding region containing the VL sequence shown in O:10, and at least one of the first and second heavy chains. In this case, the amino acids at positions L234, L235, and D265 in the human IgG monohelic acid are These are F, E, and A, respectively.

[0257] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and SEQ ID NO:1 It includes a first binding region containing the VL sequence shown in 2, and at least one of the first and second heavy chains In this case, the amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are These are F, E, and A, respectively.

[0258] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:1 It includes a first binding region containing the VL sequence shown in 0, and at least one of the first and second heavy chains In this case, the amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are These are F, E, and A, respectively.

[0259] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and SEQ ID NO:1 It includes a first binding region containing the VL sequence shown in 2, and at least one of the first and second heavy chains In this case, the amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are These are F, E, and A, respectively.

[0260] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:1 It includes a first binding region containing the VL sequence shown in 0, and at least one of the first and second heavy chains In this case, the amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are These are F, E, and A, respectively.

[0261] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and SEQ ID NO:1 It includes a first binding region containing the VL sequence shown in 2, and at least one of the first and second heavy chains In this case, the amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are These are F, E, and A, respectively.

[0262] Examples of bispecific antibody molecules that can be used in the present invention include (i) different antigens (ii) A single antibody having two arms including a binding region, (ii) by an additional peptide linker, for example Therefore, two scFvs linked in tandem have specificity for two different epitopes. (iii) Each light chain and heavy chain has two variable domains linked by short peptide bonds. The dual-variable domain antibody (D VD-Ig(trademark))(

[40] ), (iv) chemically linked bispecific (Fab')2 fragments (v) A tetravalent bispecific antibody having two binding sites for each target antigen. TandAb (registered trademark) is a fusion of two single-chain diabodies that give rise to a physical body. (vi) A flexibody, which is a combination of scFv and diabody that yields a polyvalent molecule. (flexibody), (vii) dimerization and docking domain in protein kinase A The so-called "dock and lock" molecule based on " (Registered Trademark), when applied to Fab, this is two different Fab fragments linked together It is possible to provide a trivalent bispecific binding protein consisting of the same Fab fragment. (viii) For example, a so-called scorpion including two scFv fused at both ends of a human Fab arm. It includes the (Scorpion) molecule and the (ix) diabody.

[0263] In one embodiment, the bispecific antibody of the present invention is a diabody, a crossbody, Alternatively, dual Fab arm exchange can be achieved through controlled Fab arm exchange. Specific antibodies, such as DuoBody® (registered trademark) as described in this invention (for example) (as described in

[41] )

[0264] Examples of different types of bispecific antibodies include (i) complementarity to force heterodimerization. (ii) an IgG-like molecule having a specific CH3 domain, and (ii) both sides of the molecule each having at least two different Recombinant IgG-like bilayer containing an antibody Fab fragment or a portion of a Fab fragment. (iii) Targeting molecule, full-length IgG antibody, additional Fab fragment or Fab fragment (iv) an IgG fusion molecule fused to part of the diabody, a single-stranded Fv molecule, or a stabilized diabody. Fc fusion molecules in which a heavy chain constant domain, Fc region or part thereof is fused, (v) different F The ab fragments are either fused into one, or fused into the heavy chain constant domain, Fc region, or a part thereof. (vi) Different single-stranded Fv molecules, or different diabody Alternatively, different heavy chain antibodies (e.g., domain antibodies, Nanobodies®) may fuse with each other. Either it is fused to the heavy chain constant domain, Fc region, or part thereof, or another protein is fused to it. Alternatively, antibodies based on ScFv and diabody, as well as heavy chain antibodies, fused to a carrier molecule. Examples include domain antibodies and Nanobodies®, but this is not limited to them. Not just that.

[0265] An example of an IgG-like molecule containing a complementary CH3 domain molecule is Triomab(registered trademark) (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 En Engineered Domain (Strand Exchange Engineered Domain) Body (SEEDbody) (EMD) Serono,

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

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

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

[0052] ), mAb-Fv (Xencor,

[53] ), bivalent bispecific antibody (Roche) and DuoBody (Registered Examples include, but are not limited to, the trademark molecule (Genmab A / S,

[41] ).

[0266] Examples of recombinant IgG-like dual-targeting molecules include dual-targeting molecules. Targeting (DT)-Ig (GSK / Domantis), Two-in-one antibody (Genent ech), Cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star,

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

[55] ), κλBody(NovImmune) and CovX-body(registered trademark)(CovX / P Examples include fizers, but they are not the only ones.

[0267] An example of an IgG fusion molecule is Dual Variable Domain (DVD)-Ig (trademark) (Abbott,

[56] ), Dual-domain double-head antibody (Unilever; Sano fi Aventis,

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

[58] ), sc Fv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc.,

[59] ) and TvAb (Roche,

[59] ,

[60] ) is one example, but it is not limited to them.

[0268] Examples of Fc fusion molecules include ScFv / Fc fusions (Academic Institution) and Scorpion (S CORPION) (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affini Dual Affinity Retargeting Technology Fc-DART(trademark) (MacroGenics,

[62] ,

[63] ) and Dual(ScFv)2-Fab(National Examples include the Research Center for Antibody Medicine (China), but these are limited to those. That's not the case.

[0269] Examples of Fab-fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Axis Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (Registered Trademark) (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech) is one example, but it is not the only one.

[0270] Examples of antibodies based on ScFv, antibodies based on diabody, and domain antibodies include dual-layer antibodies. Bispecific T cell Engager (BiTE®) (Micr omet), Tandem Diabody (Tandab) (Affimed), Dual Dual Affinity Retargeting Technology (DART(trademark))(MacroGenics), Single-chain Diabod y) (Academic), TCR-like antibody (AIT, ReceptorLogics), human serum albumin ScFv fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting ) Nanobodies (registered trademark) (Ablynx), dual-targeting heavy chain single-domain One example is the dual-targeting heavy chain only domain antibody, but It is not limited to them.

[0271] Furthermore, any single-specific antibody that satisfies the assay conditions described herein is bispecific. It is thought that this could form the basis of sex antibodies. That is, one of the binding regions binds to CD3. A heavily specific antibody is tested in a functional assay and meets any single specific requirement expressed herein. It may be derived from an isochromic CD3 antibody. Such bispecific antibodies are incorporated herein by reference. It can be obtained by the method described in

[41] .

[0272] Therefore, in a particular embodiment, each of the first and second heavy chains is at least It includes a hinge region, CH2 and CH3 regions, and in the first heavy chain, T366 and L36 in the human IgG monoheavy chain A position corresponding to a position selected from the group consisting of 8, K370, D399, F405, Y407, and K409. At least one of the amino acids is substituted, and in the second heavy chain, T3 in the human IgG single chain Corresponding to a position selected from the group consisting of 66, L368, K370, D399, F405, Y407, and K409. At least one amino acid at the position is substituted, and moreover, the first heavy chain and the second heavy chain are It is not substituted in the same position. In this context, the term "substituted" is particularly... This refers to a situation where an amino acid at a fixed amino acid position is substituted with another natural or unnatural amino acid. Therefore, the "substituted" amino acid at the position corresponding to the position in the human IgG single chain is, This means that the amino acids in that position are different from the natural amino acids in the IgG single chain.

[0273] In one embodiment, the amino acid in the first heavy chain corresponds to the position of K409 in the human IgG monohelic acid. It is not K, L, or M, but any amino acid at the position corresponding to F405 in the human IgG single chain is F. And in the second heavy chain, T366, L368, K370, D399, F405, in the human IgG monohelic acid, At least one amino acid at a position corresponding to a position selected from the group consisting of Y407 is placed It has been replaced.

[0274] In one embodiment, the amino acid in the first heavy chain corresponds to the position of K409 in the human IgG monohelic acid. It is neither K, L, nor M, and in the aforementioned double chain, the position corresponding to F405 in the human IgG single chain. The amino acid in the position is not F, but any amino acid, and the amino acid at the position corresponding to K409 in the human IgG single chain is K. That is the case.

[0275] In one embodiment, the amino acid in the first heavy chain corresponds to the position of F405 in the human IgG monohelic acid. It is neither F, R, nor G, and in the aforementioned second heavy chain, T366, L368, K370, D3 in the human IgG monohelic acid. The amino acid at the position corresponding to the position selected from the group consisting of 99, Y407, and K409 is substituted. It is being done.

[0276] In one embodiment, the amino acid in the first heavy chain corresponds to the position of K409 in the human IgG monohelic acid. It is neither K, L, nor M, and the amino acid at the position corresponding to F405 in the human IgG single chain is not F.

[0277] In yet another embodiment, in the first heavy chain, the position corresponding to F405 in the human IgG single heavy chain The amino acid is L, and in the second heavy chain, the position corresponding to K409 in the human IgG single heavy chain The amino acid is either R or vice versa.

[0278] Therefore, in one embodiment, in the first heavy chain, the position corresponding to K409 in the human IgG single heavy chain The amino acid is R, and in the double chain, the amino acid is at the position corresponding to F405 in the human IgG single chain. The acid is L.

[0279] In yet another embodiment, the humanized or chimeric CD3 antibody of the present invention comprises the primary and secondary heavy chains At least one of them is an inactivation substitution as disclosed in any one of the above embodiments, for example L234F, L It contains one or more of 235E and D265A, and the amino acid at the position corresponding to F405 is F No. In one embodiment, the humanized or chimeric CD3 antibody of the present invention is the primary and secondary heavy chains At least one of them is an inactivation substitution as disclosed in any one of the above embodiments, for example L234F, L It contains one or more of 235E and D265A, and yet another substitution at the K409 position, for example, K409R. In particular, in one embodiment, the humanized or chimeric CD3 antibody of the present invention is used to combine the primary and secondary heavy chains. Both include the deactivation substitutions disclosed in any one of the above embodiments, for example, L234F, L235E, and It contains one or more D265A and a substitution at the F405 position, for example, F405L. In one embodiment, this invention The humanized or chimeric CD3 antibody contains either one of the above embodiments in both the primary and secondary heavy chains. In addition, the following deactivation substitutions are disclosed, for example, one or more of L234F, L235E, and D265A, and K409 It contains yet another substitution at the position, for example, K409R. Such antibodies produce bispecific antibodies. It is useful for manufacturing.

[0280] Therefore, in yet another embodiment, in at least one of the first and second heavy chains, The amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are F and E, respectively. , and A, and in the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG single heavy chain is It is L, and in the double chain, the amino acid at the position corresponding to K409 in the human IgG single chain is R. be.

[0281] In one embodiment, at least one of the first and second heavy chains contains L234 in the human IgG monohelic acid. The amino acids at positions corresponding to L235, D265, N297, and P331 are F, E, A, N, and O, respectively. It is called P, and in the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG single chain is L. It is present, and in the double chain, the amino acid at the position corresponding to K409 in the human IgG single chain is R. .

[0282] In one alternative embodiment, in at least one of the primary and secondary heavy chains, in human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG monochain is R, In the second heavy chain, the amino acid at the position corresponding to F405 in the human IgG single heavy chain is L.

[0283] In one embodiment, at least one of the first and second heavy chains contains L234 in the human IgG monohelic acid. The amino acids at positions corresponding to L235, D265, N297, and P331 are F, E, A, N, and O, respectively. It is called P, and in the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG single chain is R. It is present, and in the double chain, the amino acid at the position corresponding to F405 in the human IgG single chain is L. .

[0284] In another embodiment, in both the primary and secondary heavy chains, positions L234, L235 in the human IgG monochain, The amino acids at the positions corresponding to D265 are F, E, and A, respectively, and in the first heavy chain... Therefore, the amino acid at the position corresponding to F405 in the human IgG single chain is L, and in the double chain, The amino acid R is located at the position corresponding to K409 in the human IgG single chain.

[0285] In one embodiment, in both the first and second heavy chains, L234, L235, D265 in the human IgG monochain, The amino acids at positions N297 and P331 are F, E, A, N, and P, respectively. In the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG single heavy chain is L, and the second In the heavy chain, the amino acid R is located at the position corresponding to K409 in the human IgG1 heavy chain.

[0286] In one alternative embodiment, in both the primary and secondary heavy chains, positions L234 and L2 in the human IgG monohelic acid are present. The amino acids at positions 35 and D265 are F, E, and A, respectively, in the first heavy chain. In human IgG single chain, the amino acid at the position corresponding to K409 is R, and in the double chain... Therefore, the amino acid at the position corresponding to F405 in the human IgG single-chain is L.

[0287] In one embodiment, in both the first and second heavy chains, L234, L235, D265 in the human IgG monochain, The amino acids at positions N297 and P331 are F, E, A, N, and P, respectively. In the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG single chain is R, and the second In the heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L.

[0288] As described herein, T1 is directed to specific target cells such as cancer cells or tumor cells. Cell mobilization is a method to kill target cells. The inventors have found that both heavy chains are specific Bispecific CD3×HER2 antibodies containing the amino acid substitutions L234F, L235E, and D265A were used in Example 5. As described above, we showed that it is possible to induce the death of AU565 cells. T cell-mediated death is It has a dual specificity, targeting CD3 in the first binding domain and another target in the second binding domain. It can be obtained by antibodies. Therefore, in one embodiment, the first binding region is humanized or With respect to the Mela CD3 antibody, according to any embodiment described herein, the second binding region is the first binding region. It binds to a target different from the region. If the antibody is a bispecific antibody, then a small portion of the antibody Both halves, i.e., one of the pair of heavy and light chains of the antibody, are humanized or cyanoacrylate as described herein. It should be understood that this is a Mela antibody. Therefore, half of the bispecific antibodies are CD3. The humanized or chimeric antibody of the present invention binds to one half, and the other half binds to a second target. It can be a combined humanized form, a chimeric form, a completely non-human form, or a completely human form. Therefore, it is a single state. In this case, the antibody includes the first heavy chain and the second heavy chain, the first light chain and the second light chain, and the first heavy chain and The first light chain is a humanized or chimeric form, and is connected by disulfide crosslinks. It forms a first binding region, and the second heavy chain and second light chain are whole human bodies, and disulfide The first bond region is connected by a bridge to form a second bond region, where the first bond region is described herein. According to any of the aspects or embodiments described herein, the second bonding region is bonded to a different target. In one embodiment, the antibody comprises a first heavy chain and a second heavy chain, a first light chain and a second light chain, and the The single chain and the first light chain are humanized or chimeric, and are connected by disulfide bridges. They are connected to form a first binding region, and the second heavy chain and second light chain are humanized or chimeric. and are connected by disulfide bridges to form a second bonding region, where the first bond The bonding region is in accordance with any aspect or aspect described herein, and the second bonding region is the It binds to a CD3 epitope different from the first binding region.

[0289] Therefore, in one embodiment, the bispecific antibody is the VH sequence shown in SEQ ID NO:8 and SEQ ID NO:1 The first binding region includes the VL sequence shown in 0, the VH sequence shown in SEQ ID NO:19 and the sequence shown in SEQ ID NO:20 It includes a second binding region containing a VL sequence, and in at least one of the first and second heavy chains, The amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are, respectively, F These are E and A, and in the first heavy chain, the amino at the position corresponding to position F405 in the human IgG monochain. The acid is L, and in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is It is R.

[0290] In one embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a VL sequence shown in SEQ ID NO:10. The first binding region includes the sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30. It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0291] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0292] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0293] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0294] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0295] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0296] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:8 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0297] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0298] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0299] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0300] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0301] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0302] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0303] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0304] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:6 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0305] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0306] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0307] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0308] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:10. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0309] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0310] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region containing human IgG1 in at least one of the first and second heavy chains. The amino acids at positions L234, L235, and D265 in the heavy chain are F, E, and It is A, and in the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L. Furthermore, in the double chain, the amino acid at the position corresponding to position K409 in the human IgG single chain is R.

[0311] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:19 and the VL sequence shown in SEQ ID NO:20. It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0312] In another embodiment, the bispecific antibody is a VH sequence shown in SEQ ID NO:9 and a V sequence shown in SEQ ID NO:12. The first binding region includes the L sequence, the VH sequence shown in SEQ ID NO:29 and the VL sequence shown in SEQ ID NO:30 It includes a second binding region, and in both the first and second heavy chains, the position in the human IgG monocoque The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. In the first heavy chain, the amino acid at the position corresponding to position F405 in the human IgG single chain is L, and the In the double hemisphere, the amino acid R corresponds to the position K409 in the human IgG single hemisphere.

[0313] As used herein, the term "disulfide bridge" refers to a bridge between two cysteine ​​residues. This refers to a covalent bond. In other words, this interaction can also be called a Cys-Cys interaction.

[0314] As used herein, the term "target" refers to the binding site to which the binding region of the antibody of the present invention binds. It refers to a molecule. When used in relation to antibody binding, this term refers to the target of the generated antibody. It includes any antigen.

[0315] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric, and disulf The second heavy chain and second light chain are connected by a cyclobridge to form the first binding region, and the second heavy chain and second light chain form the complete human body. The first bond is connected by a disulfide bridge to form a second bonding region, where the first bond The region is according to any aspect or aspect described herein, and the second bonding region is different Binding to the GET, at least one of the primary and secondary heavy chains, in the position of the human IgG monocoque. The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. .

[0316] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric, and disulf The second heavy chain and second light chain are connected by a cyclobridge to form the first binding region, and the second heavy chain and second light chain form the complete human body. The first bond is connected by a disulfide bridge to form a second bonding region, where the first bond The region is according to any aspect or aspect described herein, and the second bonding region is the first bonding region It binds to a different CD3 epitope, and in at least one of the primary and secondary hemispheres, The amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are F, respectively. , E, and A.

[0317] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric, and disulf The second heavy chain and second light chain are connected by a cyclobridge to form the first binding region, and the second heavy chain and second light chain form the complete human body. The first bond is connected by a disulfide bridge to form a second bonding region, where the first bond The region is according to any aspect or aspect described herein, and the second bonding region is different GET binds to positions L234, L235 in the human IgG monohelic acid, in both the primary and secondary helical chains. The amino acids at the positions corresponding to D265 are F, E, and A, respectively.

[0318] In one particular embodiment, the first heavy chain and the first light chain are humanized or chimeric, and disulf The second heavy chain and second light chain are connected by a cyclobridge to form the first binding region, and the second heavy chain and second light chain form the complete human body. The first bond is connected by a disulfide bridge to form a second bonding region, where the first bond The region is according to any aspect or aspect described herein, and the second bonding region is the first bonding region It binds to a different CD3 epitope and is present in both the primary and secondary heavy chains of human IgG monohepatic cells. The amino acids at positions L234, L235, and D265 are F, E, and A, respectively. be.

[0319] In one aspect, the bispecific antibody of the present invention binds to human HER2 or human CD20. Includes the binding region.

[0320] In one embodiment, the bispecific antibody of the present invention includes a second binding region that binds to human CD20. .

[0321] In one embodiment, the second binding region is WO2 containing ofatumumab (2F2), 11B8, and 7D8. Antibodies disclosed in 004035607 (Genmab A / S); WO2005103081 (Genmab A / S) containing 2C6 Antibodies disclosed in: AME-133; TRU-015; IMMU-106; Ocrelizumab (Gazyva (Registered Trademark) (Registered Trademark)); Tositumomab (Bexxar(Registered Trademark)); and Rituximab (Rituxan(Registered Trademark) It may be derived from full-length monoclonal CD20 antibodies such as (trademark) / MabThera(registered trademark).

[0322] In one embodiment, the second binding region may be derived from a full-length monoclonal CD20 antibody. The antibody does not contain the amino acid residues at position 170 (alanine) or position 172 (proline), or It is not necessary, but it contains the amino acid residues of asparagine at position 163 and position 166. Bind to an epitope on CD20, or as required.

[0323] In one embodiment, the bispecific antibody of the present invention comprises a second binding region that binds to human CD20. The binding region is, (i) VH CDR1 area of ​​SEQ ID NO:34, VH CDR2 area of ​​SEQ ID NO:35, VH CD R3 region, VL CDR1 region of SEQ ID NO:37, VL CDR2 region of DAS, and VL CDR3 region of SEQ ID NO:38 region, (ii) VH CDR1 region of SEQ ID NO:41, VH CDR2 region of SEQ ID NO:42, VH C DR3 region, VL CDR1 region of SEQ ID NO:44, VL CDR2 region of DAS, and VL CDR of SEQ ID NO:45 3 areas, (iii) VH CDR1 region of SEQ ID NO:48, VH CDR2 region of SEQ ID NO:49, VH CDR1 region of SEQ ID NO:50 VL CDR3 area, VL CDR1 area with SEQ ID NO: 51, VL CDR2 area of ​​DAS, and VL CD with SEQ ID NO: 52 R3 region, or (iv) VH CDR1 region of SEQ ID NO:55, VH CDR2 region of SEQ ID NO:56, VH C DR3 region, VL CDR1 region of SEQ ID NO:58, VL CDR2 region of DAS, and VL CDR of SEQ ID NO:59 3 areas Includes.

[0324] In one embodiment, the bispecific antibody of the present invention comprises a second binding region that binds to human CD20. The binding region is, (i) At least 90%, at least 95%, and less than the amino acid sequence shown in SEQ ID NO:29. A VH sequence having at least 97% or 99% amino acid sequence identity, and At least 90%, at least 95%, and at least the amino acid sequence shown in SEQ ID NO:30 VL sequences having 97% or at least 99% amino acid sequence identity, (ii) At least 90%, at least 95%, and less than the amino acid sequence shown in SEQ ID NO:39 A VH sequence having at least 97% or 99% amino acid sequence identity, and For the amino acid sequence shown in SEQ ID NO:40, at least 90%, at least 95%, at VL sequences having 97% or at least 99% amino acid sequence identity, (iii) At least 90% and at least 95% of the amino acid sequence shown in SEQ ID NO:46, A VH sequence having at least 97% or at least 99% amino acid sequence identity, and , relative to the amino acid sequence shown in SEQ ID NO:47, at least 90%, at least 95%, and VL sequences having 97% or at least 99% amino acid sequence identity, (iv) At least 90%, at least 95%, and less than the amino acid sequence shown in SEQ ID NO: 53 A VH sequence having at least 97% or 99% amino acid sequence identity, and For the amino acid sequence shown in SEQ ID NO:54, at least 90%, at least 95%, at VL sequences with 97% or at least 99% amino acid sequence identity Includes.

[0325] In one embodiment, the bispecific antibody of the present invention comprises a second binding region that binds to human CD20. The second binding region is, (i) VH sequence of SEQ ID NO:29 and VL sequence of SEQ ID NO:30 (ii) VH sequence of SEQ ID NO:39 and VL sequence of SEQ ID NO:40 (iii) The VH sequence of SEQ ID NO:46 and the VL sequence of SEQ ID NO:47, or (iv) VH sequence of SEQ ID NO:53 and VL sequence of SEQ ID NO:54 Includes.

[0326] Nucleic acid constructs, expression vectors, and host cells In one aspect, the present invention relates to nucleic acid constructs encoding one or more sequences shown in Table 1. This invention relates to lacto. Therefore, the present invention is SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 The sequences shown in 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 This concerns nucleic acid constructs that code for either one of the two.

[0327] In another aspect, the present invention relates to the sequence of the humanized or chimeric CD3 antibody of the present invention. A nucleic acid construct that generates, an expression vector comprising the nucleic acid construct of the present invention, the By culturing host cells containing the vector and the host cells under appropriate conditions A method for producing antibodies, wherein antibodies are produced and optionally recovered. Humanized CD3 antibodies can also be represented as "huCD3".

[0328] In one embodiment, the present invention relates to (i) the heavy chain sequence of the humanized or chimeric antibody of the present invention. (ii) a nucleic acid sequence that encodes the light chain sequence of the humanized or chimeric antibody of the present invention The expression vector is provided that includes either (iii)(i) and (ii). The current vector is one or more nuclei in any aspect or manner described herein. Contains an acid construct or nucleic acid sequence.

[0329] In one embodiment, the expression vector of the present invention is SEQ ID NO: 1, 2, 3, 4, and 5, and It encodes one or more heavy chain and light chain CDR sequences selected from the group consisting of GTN sequences. It contains nucleic acid sequences.

[0330] In one embodiment, the present invention relates to SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 19, 20, 27, 28, 2 One or more selected from the group consisting of 9 and 30, or any combination thereof. The present invention provides an expression vector containing a nucleic acid sequence encoding the amino acid sequence of the present invention. In another embodiment, The current vector contains a nucleic acid sequence encoding the VH CDR3 amino acid sequence shown in SEQ ID NO:3. In another embodiment, the expression vector is selected from SEQ ID NO: 6, 7, 8, 9, 19, 27, and 29. It contains a nucleic acid sequence that encodes a VH amino acid sequence. In another embodiment, the expression vector is SEQ Nucleic acids encoding VL amino acid sequences selected from ID NO: 10, 11, 12, 20, 28, and 30 Includes a sequence. In another embodiment, the expression vector is a human antibody light chain, a human antibody heavy chain, or the The present invention includes nucleic acid sequences that encode both constant regions. In another embodiment, the present invention includes SEQ ID NO Expression vector containing nucleic acid sequences encoding amino acid sequences 15, 16, 23, 24, 25, and 26 - Provide.

[0331] In a particular embodiment, the expression vector contains one or more variants of the above amino acid sequence. The mutant contains a nucleic acid sequence that modifies up to 25 amino acids, for example, up to 20. For example, up to 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4. , 3, 2, or 1 amino acid modification, for example, deletion or insertion, preferably substitution, for example, retention Having a conservative substitution or a non-conservative substitution, or at least 80 for either of the sequences A percentage of identity, for example, at least 85% identity, or 90% identity, or 95% identity, for example. For example, 96% identity, 97% identity, or 98% identity to any of the aforementioned amino acid sequences. It has identity or 99% identity. The present invention is different from the nucleic acid sequence described above, but is a genetic code Due to the diversity of the codes, the nucleic acid sequence encoding the same amino acid sequence as the antibody of the present invention is also related. For example, even if the nucleic acid sequence changes, it will remain the same as any of the amino acid sequences described herein. This can result in amino acid sequences. Further nucleic acid sequences can be identified based on the genetic code. The method is well known to those skilled in the art.

[0332] In yet another embodiment, the expression vector is an antibody, such as a human antibody, with a light chain, heavy chain, or light It further includes nucleic acid sequences that encode the constant regions of both the chain and the heavy chain.

[0333] The expression vectors described above can be used for recombinant production of the antibody of the present invention.

[0334] In relation to the present invention, expression vectors include chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acids. With any suitable vector (a nucleic acid sequence containing an appropriate set of expression regulatory elements), It is possible. Examples of such vectors include derivatives of SV40, bacterial plasmids, and Phage DNA, baculovirus, yeast plasmid, or combination of plasmid and phage DNA? Examples include vectors that induce this condition, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid encoding a humanized or chimeric CD3 antibody is placed in a naked DNA or RNA vector. For example, linear expression elements (as described in

[64] , for example), compressed nucleic acid vectors (For example, those listed in

[65] and / or

[66] ), pBR322, pUC19 / 18, and This can be a plasmid vector such as pUC118 / 119, a "midge" minimum-size nucleic acid vector (e.g., as described in

[0067] ), or CaP04 - Precipitating nucleic acid vectors such as precipitation constructs The constructs (for example, those described in

[68] ,

[69] ,

[70] , and

[71] ) It is included in [this]. Such nucleic acid vectors and their uses are well known in the art (e.g. See, for example

[72] and

[73] .

[0335] In one embodiment, the vector is suitable for the expression of humanized or chimeric CD3 antibodies in bacterial cells. Examples of such vectors include, for instance, BlueScript (Stratagene) and the pIN vector. Expression vectors such as (

[74] ), pET vectors (Novagen, Madison, Wisconsin) — is one example.

[0336] In addition to or instead of this, the expression vector is a vector suitable for expression in yeast systems. - Alternatively, any vector suitable for expression in yeast can be used. Suitable vectors include, for example, alpha factor, alcohol oxidase, and PGH. Which of these are constitutive or inductive promoters (

[75] and

[76] (There is a review article on this topic.)

[0337] Nucleic acid constructs and / or vectors are polypeptides such as nascent polypeptide chains. Secretory sequences / localizations that can target the pericellular lumen or cell culture medium. This may also include nucleic acid sequences that encode the occupying sequence. Such sequences are known in the art. Yes, this includes secretion leaders or signal peptides well known in the art, and cells. Organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transport) Sequence (chloroplast transport sequence), membrane localization / anchor sequence (e.g., membrane transport termination sequence, GPI) This includes anchor arrays, etc.

[0338] In the expression vector of the present invention, nucleic acids encoding humanized or chimeric CD3 antibodies are available. Contains or combines appropriate promoters, enhancers, and other expression-promoting elements. It may be related. An example of such a factor is a strong expression promoter (e.g., human CMV IE) Promoter / enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoter (Ter), effective poly(A) termination sequence, replication origin for plasmid production in E. coli , antibiotic resistance genes as selectable markers, and / or favorable cloning Examples include sites (e.g., polylinkers). Nucleic acid constructs and / or vectors. This may include not only constitutive promoters but also inductive promoters such as CMV IE (these Those skilled in the art will understand that the term is a descriptive term for the degree of gene expression under certain conditions. (Probably).

[0339] In one embodiment, an expression vector encoding a humanized or chimeric CD3 antibody is used with a viral vector It can be deployed and / or delivered to host cells or host animals by a ter.

[0340] Such expression vectors are being tested for recombinant production of humanized or chimeric CD3 antibodies. It is possible.

[0341] In one aspect, the present invention provides a host cell containing the expression vector of the present invention.

[0342] In one aspect, a humanized or chimeric form of any aspect or embodiment described herein. CD3 antibodies are produced by recombinant eukaryotic host cells, recombinant prokaryotic host cells, or recombinant micro Provided using biological host cells. Therefore, the present invention is humanized as defined herein. Alternatively, recombinant eukaryotic host cells or recombinant prokaryotic host cells that produce chimeric CD3 antibodies or immunoglobulins. We provide chief cells or recombinant microbial host cells. Examples of host cells include yeast and bacterial cells. Examples include cells and mammalian cells, such as CHO or HEK-293 cells. For example, in one embodiment, Host cells have a sequence that encodes the expression of a humanized or chimeric CD3 antibody as described herein. It contains nucleic acid sequences that are stably incorporated into the cellular genome. In another embodiment, the host cell, Non-integrated nuclei containing sequences encoding the expression of humanized or chimeric CD3 antibodies as described in the details. The acid sequence includes, for example, a plasmid, cosmid, phagemid, or linear expression element.

[0343] As used herein, the terms “recombinant host cell” (or simply “host cell”) are derived from the term “recombinant host cell” This refers to cells into which a vector, nucleic acid construct, or nucleic acid sequence has been introduced. Such terms refer not only to the specific target cells, but also to the descendants of such cells. It should be understood that this also refers to variations or environmental influences over generations. Certain modifications can occur due to either of these factors, so such offspring are actually different from the parent cell. They may not be identical, but the term "host cell" as used herein still falls within the scope of the same category. It is contained within the enclosure. Examples of recombinant host cells include CHO cells, HEK-293 cells, PER.C6, NS0 cells, and eukaryotic host cells such as lymphocytes, prokaryotic cells such as Escherichia coli, and plant cells Examples include dwelling cells and other eukaryotic hosts such as fungi.

[0344] In yet another aspect, the present invention produces humanized or chimeric CD3 antibodies of the present invention. Regarding the method for this purpose, this method is a) The process of culturing the host cells of the present invention as described above, and b) A step of recovering and / or purifying the antibody of the present invention from the culture medium. Includes.

[0345] In yet another aspect, nucleotide sequences encoding humanized or chimeric CD3 antibodies The column codes the second part, such as a therapeutic polypeptide. An example therapeutic polypeptide. Further details are provided in other sections of this specification. In one embodiment, the present invention relates to humanized or chimeric CD3 antibodies. Regarding a method for producing a fusion protein, this method is: a) A step of culturing host cells containing an expression vector containing such a nucleotide sequence, call b) Recover the humanized or chimeric CD3 antibody fusion protein from the culture medium and / or purify it. process Includes.

[0346] composition In one aspect, the present invention relates to antibodies in any aspect and form described herein. Alternatively, the present invention provides a composition containing a bispecific antibody.

[0347] In one aspect, the present invention is defined in any one aspect and embodiment described herein. The present invention provides a pharmaceutical composition comprising an antibody or bispecific antibody and a pharmaceutically acceptable carrier. ru.

[0348] Pharmaceutical compositions are pharmaceutically produced according to conventional techniques, for example, the techniques disclosed in

[77] . Formulated using an acceptable carrier or diluent and any other known adjuvants and excipients. It is possible.

[0349] pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, The present invention should be suitable for humanized or chimeric antibodies and selected modes of administration. Pharmacology The suitability of the carrier and other components of the composition is determined with respect to antigen binding, and the selected compounds of the present invention Absence of significant negative effects on the desired biological properties of a substance or pharmaceutical composition (e.g., significant The effect is determined based on the absence of a relative inhibition of 10% or less, or 5% or less. ru.

[0350] The pharmaceutical compositions of the present invention include diluents, fillers, salts, buffers, and detergents (e.g., nonionic detergents). Cleaning agents (e.g., Tween-20 or Tween-80), stabilizers (e.g., sugars or protein free amines) Suitable for inclusion in pharmaceutical compositions as an acid, preservative, tissue fixative, solubilizer, and / or other chemical substances. It may also include other materials.

[0351] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is not toxic to patients. Without achieving the desired therapeutic response for a specific patient, composition, and mode of administration The dosage can be adjusted to obtain an effective amount of active ingredient. Selectable dosage levels. This refers to the activity of various pharmacokinetic factors, such as the specific composition of the present invention or its amide used. , route of administration, administration time, elimination rate of the specific compound used, duration of treatment, use Other drugs, compounds, and / or materials used in combination with the specific composition, and the year of treatment of the patient Age, sex, weight, condition, overall health status, medical history, and other information known in the medical field. It will depend on the factors.

[0352] The pharmaceutical composition can be administered via any appropriate route and in any manner. In vivo and In the art, there are no suitable routes for administering the humanized or chimeric antibodies of the present invention in vitro. This is well known, and those skilled in the art can choose it.

[0353] In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.

[0354] In this specification, the expressions "parenteral administration" and "administered parenterally" refer to enteral administration. This refers to administration methods other than topical administration, usually by injection, including epidermal and intravenous administration. Intramuscular, intraarterial, intrathecal, intrasacral, intraorbital, intracardiac, intradermal, abdominal, intratendinous, transtracheal, subcutaneous Subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injections. This includes injection and injection.

[0355] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion. .

[0356] A pharmaceutically acceptable carrier is physiologically compatible with the humanized or chimeric antibody of the present invention. , all kinds of suitable solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, It contains antioxidants and absorption retarders, among other things.

[0357] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include: water, saline solution, phosphate buffer solution, ethanol, dextrose, polyol (for example) Glycerol, propylene glycol, polyethylene glycol, etc.) and their appropriate A fine mixture of vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and Sesame oil, carboxymethylcellulose colloid solution, tragacanth gum and injectable. Examples include organic esters, such as ethyl oleate, and / or various buffers. Other carriers are well known in the field of pharmacy.

[0358] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile injectable solutions. Alternatively, sterile powders for preparing dispersions on-site may be used. The use of such media and agents is known in the art. Conventional media Or, unless they are incompatible with the active compound, the drugs in the pharmaceutical composition of the present invention The use of the following is possible. When referring to "active compound," it means the humanized or chimeric compound of the present invention. It is thought that this also refers to antibodies.

[0359] Proper fluidity can be achieved, for example, by using a coating such as lecithin, in the case of a dispersion. Maintaining the required particle size, and by using surfactants, can.

[0360] The pharmaceutical composition of the present invention is a pharmaceutically acceptable antioxidant, for example, (1) a water-soluble antioxidant Stoppers, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, and metasulfite. (2) Sodium phosphate, sodium sulfite, etc., oil-soluble antioxidants, for example, palmitate Scorbyl, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) ), lecithin, propyl gallate, alpha-tocopherol, etc., and (3) metal cleavage Root substances, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, It may also contain phosphoric acid.

[0361] The pharmaceutical composition of the present invention contains isotonic agents, such as sugars and polyalcohols, such as mannitol. The composition may also contain sorbitol, glycerol, or sodium chloride.

[0362] The pharmaceutical composition of the present invention comprises one or more adjuvants suitable for the selected route of administration, for example, pharmaceuticals. Preservatives, wetting agents, emulsifiers, dispersants, and other preservatives that can enhance the shelf life or effectiveness of the composition. Alternatively, it may also contain a buffering agent. The humanized or chimeric antibodies of the present invention can be used, for example, in implants. Rapid delivery of compounds, including transdermal patches and controlled-release formulations containing microencapsulated delivery systems. It can be prepared using a carrier that protects against release. Such carriers include gelatin, mo Glyceryl nostearate, glyceryl distearate, biodegradable biocompatible polymer, For example, ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoethyl Sterls and polylactic acid alone, or together with wax, or in the art It may contain other well-known materials. Methods for preparing such preparations are generally known to those skilled in the art. It is knowledge (see, for example,

[78] ).

[0363] In one embodiment, the humanized or chimeric antibody of the present invention ensures proper distribution in vivo. It can be formulated in such a way. Pharmacologically acceptable carriers for parenteral administration include To prepare sterile aqueous solutions or dispersions and sterile injectable solutions or dispersions on the spot. Examples include sterile powders. The use of such media and drugs for pharmaceutically active substances. The use is well known in the art. Conventional media or drugs are those which have active compounds Unless otherwise incompatible, its use in the pharmaceutical composition of the present invention is conceivable. Other activating compounds Substances or therapeutic compounds can also be incorporated into the composition.

[0364] Pharmaceutical compositions for injection are typically kept under sterile conditions and under manufacturing and storage conditions. It must be stable under certain conditions. The composition is a solution, microemulsion, liposome, Alternatively, it can be formulated as another ordered structure suitable for high drug concentrations. The carrier is, for example, Water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene) Lencolic acid, etc. and suitable mixtures thereof, vegetable oils, such as olive oil, and an aqueous or non-aqueous solvent containing an injectable organic ester, such as ethyl oleate. Alternatively, it can be a dispersion medium. Proper fluidity can be achieved through coatings such as lecithin. By using it, in the case of a dispersion system, the required particle size is maintained, and surfactants It can be maintained by using isotonic agents, such as sugars, polyalcohols, etc. The composition includes, for example, glycerol, mannitol, sorbitol, or sodium chloride. It would be preferable to include it in the substance. Long-term absorption of the injectable composition delays absorption. By including active ingredients, such as monostearate and gelatin, in the composition, It can be produced. A sterile, injectable solution requires the necessary amount of the active compound in a suitable solvent. Incorporate with one or a combination of the components listed above, and then sterilize precisely. It can be prepared by filtration. Generally, a dispersion is made from a basic dispersion medium and necessary components. A sterilization medium containing other components, such as those listed above, is used to combine the active compound with the sterilization medium. It is prepared by mixing. In the case of sterile powders for preparing sterile injectable solutions, An example of the manufacturing method is to pre-sterilize and filter the powder of the active ingredient and any desired additional ingredients. These methods include vacuum drying and freeze-drying (freeze-drying) from a solution.

[0365] The sterile injectable solution contains the required amount of active ingredient, as needed, one of the ingredients listed above or The mixture is prepared by combining it with a suitable solvent and then performing sterile microfiltration. It is possible. Generally, a dispersion consists of a basic dispersion medium and other necessary components, such as those listed above. It is prepared by incorporating an active compound into a sterilization medium containing components from a substance. For sterile powders used to prepare sterile injectable solutions, an example of the preparation method is the active ingredient and desired Vacuum drying and the application of the powder with any additional components from the pre-sterilized and filtered solution. It is freeze-dried.

[0366] Therapeutic applications In another aspect, the present invention relates to any of the inventions described herein for use as a pharmaceutical product. In the humanized or chimeric antibody or pharmaceutical composition of the present invention as defined in that aspect or aspect To relate to.

[0367] In another aspect, the present invention relates to the use of the following for the treatment of diseases as described herein Humanized or chimeric antibodies or pharmaceutical compositions of the present invention as defined in any aspect or aspect of the present invention Regarding.

[0368] The humanized or chimeric antibody or pharmaceutical composition of the present invention is effective against cytotoxic T cells. It can be used to treat any cancer in which the cytogenic mechanism is desirable. For example, humanization or cytogenicity. Mela antibodies are used to treat or prevent disorders such as cancer, inflammatory disorders, or autoimmune disorders. Therefore, it can be administered to cultured cells in vitro or ex vivo, or, for example, to human subjects in vivo. It can be administered to. The term "subject" as used herein typically refers to human subjects. This refers to humans who respond to chemically modified or chimeric antibodies or pharmaceutical compositions. Examples of subjects include, for example... by modifying target functions or directly or indirectly leading to cell death. We can list human patients with disabilities that can be corrected or improved.

[0369] In another aspect, the present invention relates to a disease such as cancer in which T cell recruitment contributes to treatment or prevention. The present invention provides a method for treating or preventing harm, and the method provides a therapeutically effective amount of humanized Alternatively, the process may include administering a chimeric antibody or pharmaceutical composition to a subject requiring it. This method typically involves a humanized or an effective amount of the substance to treat or prevent the disorder. The drug is administered to target chimeric antibodies.

[0370] In one particular aspect, the present invention is defined in any aspect and aspect described herein. The humanized or chimeric antibody or pharmaceutical composition of the invention is administered to a subject who requires it. This relates to a method for treating cancer, which includes a process.

[0371] In another aspect, the present invention is defined in any aspect or embodiment described herein. Regarding use or method, humanized or chimeric antibodies are CD3 and cancer-specific targets. Or targets that are overexpressed in cancer or are associated with cancer, such as HER2, CD. 19. EpCAM, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-M It is a bispecific antibody that specifically binds to both AA and other antibodies, and the disease is cancer, for example, breast cancer. Prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, stomach 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 ( For example, synovial sarcoma, low-grade or high-grade B-cell lymphoma, chronic lymphocytic leukemia It is a disease or acute lymphoblastic leukemia.

[0372] The effective dosage and administration regimen of humanized or chimeric antibodies depends on the disease being treated or It depends on the circumstances and can be determined by someone skilled in the art.

[0373] A physician with ordinary knowledge in this field can easily obtain an effective amount of the required pharmaceutical composition. It can be determined and prescribed. For example, a physician can determine the humanized form used in a pharmaceutical composition. Alternatively, the dose of the chimeric antibody may be lower than the dose required to achieve the desired therapeutic effect. The dosage can be started and gradually increased until the desired effect is achieved. Generally, the present invention The appropriate dose of the composition is the minimum dose effective in producing a therapeutic effect in a specific drug regimen. This refers to the amount of a certain humanized or chimeric antibody. Such an effective dose is generally determined by the factors mentioned above. They will become dependent.

[0374] For example, the "effective dose" for therapeutic use is determined by its ability to stabilize the progression of the disease. It can be measured. The ability of a compound to inhibit cancer can be measured, for example, in human tumors. This can be evaluated using an animal model system for prediction. Alternatively, this property of the composition can be evaluated in humans. The ability of a compound or chimeric antibody to inhibit cell growth or induce cytotoxicity, It can also be evaluated by examining it using an in vitro assay known to those skilled in the art. Treatment available. Effective therapeutic compounds, i.e., the therapeutic humanized or chimeric antibodies or pharmaceutical combinations of the present invention. The substance may reduce tumor size or otherwise improve symptoms in the subject. Yes, it is possible. Those skilled in the art can determine the size of the subject, the severity of the subject's symptoms, and the selected specific composition or The amount can be determined based on factors such as the route of administration. .

[0375] The exemplary, non-limiting range of the therapeutically effective dose of the humanized or chimeric antibody of the present invention is about 0.001 to 3 0 mg / kg, for example, approximately 0.001 to 20 mg / kg, for example, approximately 0.001 to 10 mg / kg, for example, approximately 0.001 to 5 mg / kg, For example, approximately 0.001 to 2 mg / kg, for example, approximately 0.001 to 1 mg / kg, for example, approximately 0.001, approximately 0.01, approximately 0.1, approximately 1 These are approximately 5, 8, 10, 12, 15, and 18 mg / kg, respectively.

[0376] Administration may be done, for example, intravenously, intramuscularly, intraperitoneally, or subcutaneously, for example, near the target site. It can be administered to the side.

[0377] The above treatment method and drug regimen in use aim to achieve the optimal desired response (e.g., therapeutic response) The dosage is adjusted to give the answer. For example, a single bolus or several doses over time. Divided doses may be administered, or the dose may be proportionally reduced or increased as required by the treatment situation. It is possible.

[0378] In one embodiment, the effectiveness of the treatment is monitored during treatment, for example, at predetermined points in time.

[0379] If desired, the effective daily dose of the pharmaceutical composition may be taken in any number of units at appropriate intervals throughout the day. In terms of dosage form, even if administered in divided doses of 2, 3, 4, 5, 6 or more, Good. In another embodiment, in order to minimize any undesirable side effects, humanization is also or chimeric antibodies or pharmaceutical compositions that have a slow, sustained effect for a long period of time, for example, 24 hours or more. It is administered by injection.

[0380] It is possible to administer the humanized or chimeric antibody of the present invention alone, but humanized or Chimeric antibodies are preferably administered as part of the pharmaceutical composition described above.

[0381] The effective dose of the humanized or chimeric antibody of the present invention is once a week, once every two weeks, or once every three weeks. It can also be administered over a single treatment period. The treatment period may be, for example, 8 weeks, 12 weeks, or This can be limited until clinical progression is established. Alternatively, the humanization or The effective dose of the chimeric antibody can also be administered every two weeks, every three weeks, or every four weeks.

[0382] In one embodiment, a humanized or chimeric antibody is administered at a dose of mg / m². 2 The weekly amount is calculated by injection. It can be administered. Such a dosage is, for example, the above mg / kg dosage according to the following formula. It can be based on: dose (mg / kg) × 70: 1.8. Such administration can be, for example, 1 to 8 times. It can be repeated 3 to 5 times. The administration should be done over a period of 2 to 24 hours, for example, over a period of 2 to 12 hours. This can be done by continuous infusion over time. In one embodiment, to reduce toxic side effects Humanized or chimeric antibodies are administered by slow, continuous infusion over a long period, for example, 24 hours or more. It can be administered in this way.

[0383] In one embodiment, a humanized or chimeric antibody is administered once a week for up to eight weeks, for example, 4 to 6 weeks. It can be administered in installments, calculated as a fixed weekly dose. The regimen may be repeated once or multiple times, for example, after 6 or 12 months, as needed. This is possible. Such a fixed dosage can be calculated, for example, by estimating the body weight as 70 kg, as described above. The dosage can be based on the mg / kg dose of the present invention or The amount of chimeric antibody is determined, for example, by taking a biological sample and determining the amount of humanized or chimeric antibody according to the present invention. By measuring using an anti-idiotype antibody that targets the complication region, the determination or It can be adjusted.

[0384] In one embodiment, maintenance therapy involves, for example, administering a humanized or cyanoacrylate once a week for a period of six months or more. Mela antibodies can be administered.

[0385] It reduces the risk of developing cancer and delays the onset of certain events in cancer progression. And / or if the cancer is in remission, humanized to reduce the risk of recurrence. Alternatively, chimeric antibodies can be administered prophylactically.

[0386] Parenteral compositions are formulated into dosage units to facilitate administration and ensure uniform dosage. This is possible. In this specification, "medication unit form" refers to a unit dose of medication administered to the subject being treated. This refers to a suitable physically discrete unit, each unit calculated to produce the desired therapeutic effect. The present invention contains a predetermined amount of the active compound together with the necessary pharmaceutical carrier. The specifications of the medication unit form include (a) the characteristics specific to the active compound and the specific therapeutic effect to be achieved. Such active compounds are formulated for therapeutic effects and (b) treatment of susceptibility in individuals. This is determined by or directly depends on the constraints inherent in the art at hand. ru.

[0387] It reduces the risk of developing cancer and delays the onset of certain events in cancer progression. And / or if the cancer is in remission, humanized to reduce the risk of recurrence. Alternatively, chimeric antibodies can be administered prophylactically. This is particularly useful in patients where it is difficult to pinpoint the location of a known tumor. It is possible.

[0388] Diagnostic applications The humanized or chimeric antibodies of the present invention include the humanized or chimeric antibodies described herein. The composition can also be used for diagnostic purposes. Therefore, the present invention is described in this specification. This document provides diagnostic methods and diagnostic compositions using humanized or chimeric antibodies as described in the book. Such methods and compositions are pure diagnostic tools, such as for detecting or identifying diseases. It can be used for diagnostic purposes and to monitor the progress of therapeutic treatment, thus monitoring the progression of the disease. To monitor the condition, to evaluate the post-treatment state, and to monitor disease recurrence. It can also be used for purposes such as assessing the risk of developing a disease. It is possible.

[0389] In one aspect, the present invention diagnoses diseases characterized by the involvement or accumulation of CD3-expressing cells. Regarding the method, this method involves the humanized or chimeric antibody of the present invention, the composition of the present invention, or the The invention includes the step of administering the pharmaceutical composition to a subject, and optionally the humanized or chimeric antibody is It is labeled with a detectable active substance.

[0390] In one aspect, the humanized or chimeric antibody of the present invention is, for example, the said humanized or chimeric antibody Cells expressing specific targets of interest to which the body binds exhibit disease or pathogenesis. In diagnosing diseases involved in the process, the target level in a sample taken from the patient and By detecting the level of cells expressing the target of interest on their cell surface, , used in ex vivo. This is, for example, the test sample, optionally together with a control sample, according to the present invention. The humanized or chimeric antibody is brought into contact with the target under conditions that allow the antibody to bind to the target. This can be achieved by the following. Next, complex formation is detected (for example, using ELISA). This is possible. When using a control sample together with a test sample, humanized or chimeric antibodies may be used. Alternatively, the levels of the antibody-target complex are analyzed in both samples, and statistically in the test sample... Significantly higher levels of humanized or chimeric antibodies or antibody-target complexes were observed in the control group. This shows the target present in the test sample at a high level compared to the sample.

[0391] Examples of conventional immunoassays that can use the humanized or chimeric antibodies of the present invention And, ELISA, RIA, FACS assay, plasmon resonance assay, chromatography assay (i) Histoimmunohistochemistry, Western blot, and / or immunoprecipitation, It is not limited to those.

[0392] Therefore, in one embodiment, the present invention relates to a disease characterized by the involvement or accumulation of CD3-expressing cells. Regarding methods for diagnosing a disease, this method may be used in any aspect or manner described herein. The process includes administering an antibody, bispecific antibody, composition, or pharmaceutical composition to a target, and is optional. The antibodies are labeled with detectable labels.

[0393] In one embodiment, the present invention relates to the presence of a target or cells expressing the target in a sample. Regarding a method for detecting presence, this method is The sample is subjected to conditions that allow for the binding of humanized or chimeric antibodies to the target in the sample. The steps of contacting the invention with a humanized or chimeric antibody, and A process to analyze whether a complex has been formed. It includes. The sample is typically a biological sample.

[0394] In one embodiment, the sample is a specific target and / or cells expressing the target. This refers to tissue samples that are known to contain or suspected to contain [these substances]. In situ detection of target expression involves taking a histological specimen from the patient, according to the present invention. This can be achieved by administering humanized or chimeric antibodies to such specimens. Humanized or chimeric antibodies are applied to the specimen by coating or layering them. It can be given by, and then it is detected using appropriate means. Next, the target As well as the presence of target or target-expressing cells, the target or ter in the test tissue. The distribution of GET-expressing cells can also be determined (for example, in relation to the evaluation of cancer cell propagation). It is possible. By using the present invention, a wide variety of histological methods (e.g., staining techniques) can be performed. In any case, the modifications that may be made to achieve such in-situ detection are This will be easily understood by those skilled in the art.

[0395] In the above assay, the bound antibody can be detected by humanizing or shaping the sample. The antibody can be labeled with a detectable substance. Alternatively, the bound (primary) specific antibody can be labeled. Antibodies that are chromogenic or chimeric, labeled with a detectable substance, and are primary specific humanized antibodies. Alternatively, it can be detected by binding to a chimeric antibody. Furthermore, in the above assay... This includes antibodies or bispecific antibodies in any aspect or manner described herein. A diagnostic composition can be used. Therefore, in one aspect, the present invention is... Diagnostic sets containing antibodies or bispecific antibodies in any of the aspects or manners described in this document Regarding finished products.

[0396] The level of the target in the sample is compared to a target standard labeled with a detectable substance. By competitive immunoassays utilizing labeled target-specific humanized or chimeric antibodies It can also be estimated. In this type of assay, the biological sample and the labeled target standard are used. Combining substance and target-specific humanized or chimeric antibodies, unlabeled target-specific Determine the amount of labeled target standard material bound to a humanized or chimeric antibody. The amount of target in the sample is the amount of labeled target conjugated to target-specific humanized or chimeric antibodies. - It is inversely proportional to the amount of the reference material.

[0397] Target-specific humanized or chimeric antibodies used in in vitro diagnostic techniques, Suitable labels for secondary antibodies and / or target reference materials include various enzymes and complementary enzymes. Examples include, but are limited to, deficient molecular groups, fluorescent materials, luminescent materials, and radioactive materials. That's not the case. Examples of suitable enzymes include horseradish peroxidase and alkaline hydroxylase. Examples include sphatase, β-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / bi Otin is one example, and suitable fluorescent substances include umbelliferone, fluorescein, Fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein Examples of luminescent substances include thane, dansilchloride, and phycoerythrin. Luminol is one example, and suitable examples of radioactive materials include 125 I, 131 I, 35 S, and 3 H These are some examples.

[0398] In one aspect, the target-specific humanized or chimeric antibody of the present invention targets tumors and other lesions. It is used for in vivo imaging of effect-expressing tissues. In the case of the in vivo method, for example (Fab') 2. Antibody fragments such as Fab and Fab' fragments have rapid distribution dynamics, This is particularly advantageous.

[0399] In vivo imaging can be performed using any appropriate technique. For example, 99 Tc, 131 I, 111 Target-specific humanization labeled with In or other gamma-ray emitting isotopes or Using chimeric antibodies (e.g., antibodies or fragments), target expression tissues such as tumors The accumulation or distribution of target-specific antibodies in a gamma cytosis camera (e.g.) For example, the Elscint Apex 409ECT device is a typical low-energy, high-resolution collimator. Alternatively, imaging can be performed using a low-energy universal collimator. teeth, 89Zr, 76 Br, 18 Using labeling with F or other positron-emitting radionuclides, positron emission Using tomography (PET), target-specific humanized or chimeric antibodies in tumors. Alternatively, the distribution of antibody fragments can be imaged. Using such techniques... The resulting image can be used, for example, as a biomarker for the presence of cancer / tumor cells. When using a GET, the target organism in the patient, mammal, or tissue It can be used to evaluate the internal distribution. A variation of this technique is a gamma camera. Rather than focusing on technique, one could cite the use of magnetic resonance imaging (MRI) to improve imaging. This can be done. Conventional immunoscintigraphy methods and their principles are, for example,

[79] ,

[80] and It is described in

[81] . Furthermore, in addition to the above, or in lieu of the above, Such images can also serve as the basis for surgical techniques to remove tumors. Furthermore, Such in vivo imaging techniques are not suitable for patients who have tumors (other biomeridians). Although it is confirmed by factors such as the presence of metastases, the tumor is not analyzed using traditional analytical techniques. In situations where determination is not possible, it also enables the identification and locating of tumors. All of these methods are characteristic features of the present invention.

[0400] The in vivo imaging and other diagnostic methods provided by the present invention are for human patients (for example, previously In patients who have never been diagnosed with cancer, or who are in recovery / remission from cancer It is particularly useful for detecting minute metastases.

[0401] In one embodiment, the present invention provides a target-specific humanized or chimeric antibody for testing It is conjugated with a radiopaque substance that promotes emission, and the conjugation is humanized. Chimeric antibodies are administered to the host by injection into the bloodstream, and the host undergoes labeling and humanization. This provides an in vivo imaging method in which the presence and location of chimeric antibodies are assayed. Through the techniques and other diagnostic methods described herein, the present invention relates to a human patient or human Screening for the presence of disease-related cells in biological samples taken from patients, and / or target-specific humanized or chimeric anti- This provides a method for evaluating the distribution of body parts.

[0402] In the case of diagnostic imaging, radioisotopes are directly used to target-specific humanized or chimeric antibodies. They can be bonded either directly or indirectly using intermediate functional groups. Useful intermediate functional groups include Examples include ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example,

[0082] ).

[0403] Diagnostic methods include the use of radioactive isotopes and radiopaque materials, as well as dyes (e.g., biotin- Using streptavidin complexes, contrast agents, fluorescent compounds or molecules, and magnetic resonance Targets conjugated with enhancers (e.g., paramagnetic ions) for imaging (MRI) This can also be done using MRI-specific antibodies (for example, conjugate MRI techniques and MRI enhancers). See

[83] for a description of the preparation of gated antibodies. The detection agent can be selected from active substances and fluorescent compounds used in MRI. To load a GET-specific humanized or chimeric antibody with a radioactive metal or paramagnetic ion, It is then reacted with a reagent that has a long tail, and multiple ions are attached to that tail. It may be necessary to attach several chelate groups. Such a tail is polylysine. , polymers such as polysaccharides, or porphyri known to be useful for this purpose, for example. Polyamines, crown ethers, bisthiosemicarbazones, polyoximes, etc. Other derivatized or derivatives having a pendant group to which a tactile group can be attached It can be a chain that can be chelated. Chelates are target-specific using standard chemistry. It can be coupled to humanized or chimeric antibodies.

[0404] Therefore, the present invention relates to a diagnostic target-specific humanized or chimeric antibody, which Get-specific humanized or chimeric antibodies are used with contrast agents (e.g., for magnetic resonance imaging, etc.). For pewter tomography, or as an ultrasound contrast enhancer, or, for example, gamma-emitting isotopes. Body, beta-emitting isotope, alpha-emitting isotope, Auger electron-emitting isotope, or positron We provide radioactive nuclides that may be released isotopes, conjugated with them.

[0405] In one aspect, the present invention relates to a diagnostic composition comprising the antibody or bispecific antibody of the present invention. To relate to.

[0406] In yet another aspect, the present invention relates to a target antigen or target antigen in a sample A kit for detecting the presence of cells that express the expression, The present invention provides target-specific humanized or chimeric antibodies, and Instructions for use with the kit Regarding the kit that includes this.

[0407] Therefore, in one aspect, the present invention is a) The sample is subjected to conditions that allow for the formation of a complex between the antibody or bispecific antibody and CD3. A step of contacting with a bright antibody or a bispecific antibody, and b) A step to analyze whether a complex has been formed. The present invention provides a kit for detecting the presence of CD3 antigen or CD3-expressing cells in a sample.

[0408] In one embodiment, the present invention provides a container containing a target-specific humanized or chimeric antibody, To detect the binding of target-specific humanized or chimeric antibodies to the target. The kit provides for diagnosing cancer, containing one or more reagents. Examples include fluorescent tags, enzyme tags, or other detectable tags. And, we can also list reagents for secondary or tertiary antibodies or enzymatic reactions, in this case, The enzymatic reaction produces a product that can be visualized. In one embodiment, the present invention is applicable One or more labeled or unlabeled forms of the present invention in a container, target-specific humanized Or chimeric antibodies, incubation reagents for indirect assays, and labels Depending on their properties, such assays may include substrates or derivatizers for detection. A diagnostic kit will be provided. This may include control reagents and instructions for use.

[0409] To detect the presence of a target in a tissue sample or host, label target specific Diagnostic kits for use with target-specific humanized or chimeric antibodies, such as target antibodies. We can also supply such diagnostic kits, as well as those described in other sections of this specification. Kits for therapeutic use typically contain target-specific humanized or chimeric antibodies. These can be consumed in a freeze-dried form in a container, either alone or with target cells or target peptides. It can be supplied with additional antibodies specific to it. Typically, pharmaceutically acceptable Carrier (e.g., inert diluent) and / or its components, e.g., tris, phosphate, or carbonate buffer, stabilizer, preservative, biocide, inactive protein, for example, serum albumin For example, (usually in a separate container for mixing) and additional reagents (also typically in a separate container) (Packaged in a container) is also included. Certain kits include target-specific humanized or chimeric versions. A secondary antibody that has the ability to bind to an antibody, and is typically contained in a separate container. This also includes the second antibody, which is typically conjugated to a label and is the target of the present invention. Formulated by methods similar to those used for specific humanized or chimeric antibodies. By using the methods described in other sections, you can define a subset of cancer / tumor cells. To characterize such cells and associated tumor tissues, target-specific humanization is used. Chimeric antibodies can be used.

[0410] Anti-idiotype antibody In yet another aspect, the present invention relates to the humanization or chimeric properties of the invention described herein. This concerns anti-idiotype antibodies that bind to the body.

[0411] Anti-idiotype (Id) antibodies are generally uniquely determined by the antigen-binding site of the antibody. It is an antibody that recognizes the group. Anti-Id antibodies are from the same species as CD3 monoclonal antibodies and To immunize animals with a specific genotype using monoclonal antibodies that are being prepared to counter Id. Therefore, they can be prepared. Immune-treated animals typically have these idiotypes. By producing antibodies that respond to determinants (anti-Id antibodies), the idiotypsy of immunized antibodies It can recognize and respond to determinants. Such antibodies include, for example, US4,699,88 As described in 0. Such antibodies are a further feature of the present invention.

[0412] Anti-Id antibodies can further induce an immune response in other animals, producing so-called anti-anti-Id antibodies. It can also be used as an "immunogen" for this purpose. Anti-anti-Id antibodies are epitope-wise, It may be identical to the original monoclonal antibody that induced the anti-Id antibody. Therefore, monoclonal By using antibodies against the idiotype determinants of the Nal antibody, the same specificity can be achieved. It is possible to identify other clones that express the anti-Id antibody. The technique, for example, with respect to the CD3-specific antibody of the present invention, is described in other sections of this specification. The technique involves altering (thereby producing anti-Id antibody variants) and / or derivatives. It can be converted. For example, monoclonal anti-Id antibody can be converted into keyhole limpet hemosemia. The process involves coupling the drug to a carrier such as nin (KLH) and using it to immunize BALB / c mice. This can be done. Serum from these mice is typically not identical to the original / parental CD3 antibody. It will likely contain anti-Id antibodies with similar binding properties.

[0413] array [Table 1] TIFF0007851903000004.tif217170TIFF0007851903000005.tif235170TIFF0007851903000006.tif232170TIFF0007851903000007.tif49170The CDR area has been annotated according to the IMGT definition. [Examples]

[0414] Example 1: Production of humanized CD3 antibody and inactivated antibody mutant Humanization of CD3 antibodies Humanization of mouse CD3 antibodies (US8,236,308, referred to as IgG1-CD3 in this specification) is performed by Antitope ( (Cambridge, UK) developed their improved germline humanization (CDR transplantation) technology (EP0629240) This was done using this technique. Using this technique, four different VH chains (SEQ ID NO: 6, 7, 8, and 9) and three different VL chains (SEQ ID NO: 10, 11, and 12) were designed. These four VH By combining this with three VL chains, 12 different antibodies were produced. These humanized variants In this specification, the variant is denoted as huCD3. Therefore, the humanized variant of the present invention, comprising VH and VL, For example, it is written as IgG1-huCD3-H1L1, which means that the specific mutant is an IgG1 isotype, and It is a t-modified CD3, with the VH amino acid sequence named "H1" and defined as SEQ ID NO:6, and "L1 It is named " and means that it contains the VL amino acid sequence defined in SEQ ID NO:10. Therefore, for example, H1 refers to the variable heavy chain region VH1, and L1 refers to the variable light chain region VL1.

[0415] Specifically, in the examples described herein, the mutant IgG1-huCD3-H1L1 (SEQ ID Humanized CD3 containing the VH1 sequence shown in NO:6 and the VL1 sequence shown in SEQ ID NO:10, IgG1-huCD3-H1 L2 (humanized CD3 containing the VH1 sequence shown in SEQ ID NO:6 and the VL2 sequence shown in SEQ ID NO:11), Ig G1-huCD3-H1L3 (Human, containing the VH1 sequence shown in SEQ ID NO:6 and the VL3 sequence shown in SEQ ID NO:12) CD3), IgG1-huCD3-H3L3 (VH3 sequence shown in SEQ ID NO:8 and VL3 sequence shown in SEQ ID NO:12) Humanized CD3 (including IgG1-huCD3-H4L1), VH4 sequence shown in SEQ ID NO:9 and SEQ ID NO:10 Humanized CD3 containing the VL1 sequence shown, IgG1-huCD3-H3L1 (VH3 sequence and SEQ ID NO:8 shown) Humanized CD3) including the VL1 sequence shown in ID NO:10, IgG1-huCD3-H3L3 (VH shown in SEQ ID NO:8) Humanized CD3 containing the 3 sequence and the VL3 sequence shown in SEQ ID NO:12, and IgG1-huCD3-H4L3 (SEQ A humanized CD3 containing the VH4 sequence shown in ID NO:9 and the VL3 sequence shown in SEQ ID NO:12 was prepared. I tested it.

[0416] In some embodiments, the heavy chain and light chain variable region sequences of huCLB-T3 / 4 (SEQ ID N, respectively) are used. Antibodies containing O:17 and 18) were used as control antibodies (Labrijn et al., PNAS 2013, 110:5145- 50) It was also used to examine different combinations of inactivating mutations in the Fc region (implemented (See Examples 8-10). huCBL-T3 / 4 is a humanized mouse CD3 antibody CLB-T3 / 4 (Parren et al., Res Immunol. 1991, 142(9)749-63). Both sequences (SEQ ID NO: 17 and 18) were subjected to appropriate pcD. Cloned into an NA3.3 (Invitrogen) expression vector and simultaneously transferred to HEK293F cells. The antibody was expressed using a control protocol. The resulting control antibody is denoted as IgG1-huCLB-T3 / 4.

[0417] In some examples, the heavy and light chain variable region sequences (corresponding to the VH sequence) of the CD20 antibody 7D8 are used. An antibody containing SEQ ID NO:29 and SEQ ID NO:30 (corresponding to the VL sequence) was used as a positive control. It was used. When used in association with a positive control, it is named "IgG1-CD20".

[0418] These IgG1-CD3 (i.e., chimeric parental CD3 antibodies), IgG1-huCD3 and IgG1-huCLB-T3 / 4 antibodies The body is used in single-specificity and bispecificity formats, and the bispecificity antibody is It was made as described below.

[0419] HER2 antibody In some examples, an antibody against HER2 was used. V of this HER2-specific antibody (antibody 169) The H and VL sequences (SEQ ID NO: 19 and 20, respectively) were previously described (WO2012). / 143524[Genmab];Labrijn et al., PNAS 2013, 110:5145-50). This antibody is a single-character antibody. It was used in both heterosexual and bispecificity formats. This was named "IgG1-HER2". wear.

[0420] b12 antibody In some examples, the gp120-specific antibody b12 (Barbas, CF. J Mol Biol. 1993 A) was used. pr 5;230(3)812-23) was used as a negative control. This was named "IgG1-b12".

[0421] Expression The antibody either has the inactivating mutation described below, or does not have the inactivating mutation, and is obtained by the method described below. This enables the production of bispecific antibodies with mutations in the CH3 domain, such as IgG1,κ or IgG1, λ expressed as: IgG1-HER2-K409R, IgG1-b12-K409R, IgG1-CD3-F405L. 293fectin( By using Invitrogen (USA) basically as instructed by the manufacturer, the heavy and light chains of the antibody A plasmid DNA mixture encoding both was introduced into Freestyle HEK293F cells (Invitrogen, USA). It was transfected transiently.

[0422] Antibody purification The culture supernatant was filtered through a 0.2 μm dead-end filter and then refrozen in a 5 mL MabSelect SuRe column (GE Hea The sample was loaded into an Ith Care (a type of elution solution) and eluted with 0.1M sodium citrate-NaOH (pH 3). The substance was immediately neutralized with 2M Tris-HCl (pH 9), and 12.6 mM NaH2P04, 140 mM NaCl, pH 7.4 (B.Brau). For n), dialysis was performed overnight. Alternatively, following purification, the eluted material was placed in a Hi Prep desalting column. The solution was then rinsed and the antibody was replaced with 12.6 mM NaH2P04, 140 mM NaCl, pH 7.4 (B. Braun) buffer. After analysis or buffer exchange, the sample was sterile filtered through a 0.2 μm dead-end filter. The antibody concentration was determined by SDS-PAGE and measured by absorbance at 280 nm. The purified antibody was stored at 2-8°C.

[0423] Production of bispecific antibodies Bispecific antibodies utilize the DuoBody® platform technology, namely WO2011 / 1479. 86 and Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2) The Fab arms were fabricated in vitro using the 2-MEA-induced Fab arm exchange described in 013, 5:962-973). To enable the production of bispecific antibodies using this method, a single mutation is present in the CH3 domain. That is, one parental IgG1 antibody has the F405L mutation (i.e., IgG1-CD3 antibody), and the other parental IgG1 antibody In the body, we generated IgG1 molecules carrying the K409R mutation (i.e., HER2 or b12 antibody). To produce heterozygous antibodies, these two parent antibodies are prepared so that the final concentration of each antibody is 0.5 mg / mL. , along with 25 mM or 75 mM 2-mercaptoethylamine-HCl (2-MEA), in a total volume of 500 μL of TE The mixture was incubated at 31°C for 5 hours. The reduction reaction was carried out using a PD-10 column equilibrated with 25 mL of PBS. The process stopped when the reducing agent 2-MEA was removed using (GE-healthcare, product number 17-0851-01). Prior to desalting, adjust the volume to 2.5 mL by adding 2 mL of PBS (B. Braun, product number 362314). 0) was added to the sample. Elution was performed with 3.5 mL of PBS. Amicon Ultra centrifuge unit (30kD MWCO) The sample was collected in Millipore (product number UFC803096) and concentrated by centrifugation at 3000 × g for 8 minutes. Adjust the volume to 500 μL with PBS (if necessary), and filter through a 0.2 μm filter (Millex-GV, product number...). The sample was sterile filtered using SLGV004SL. The bispecific product was stored at 2-8°C.

[0424] An alternative method that yields the same bispecific antibody involves 10 steps to produce the bispecific antibody. Two parent antibodies of 0 μg each were mixed together with 75 mM 2-mercaptoethylamine-HCl (2-MEA) and total The sample was incubated in 400 μL of PBS (B. Braun, product number 3623140) at 31°C for 5 hours. The original reaction was performed using an Amicon Ultra 0.5ml centrifuge unit (10kD MWCO, Millipore, product number UFC50109). 6) Centrifuge at 13000×g for 10 minutes, then wash four times with 400μL of PBS to remove the reducing agent 2-MEA. It stopped when it was removed. The filter was turned over and centrifuged at 1000g for 2 minutes to test The sample was collected in a new tube. The volume was adjusted to 200 μL with PBS (if necessary). Final concentration To determine this, the absorbance (A280) of the bispecific product at 280 nm was measured. HPLC cation exchange Conversion chromatography (HPLC-CEX) (described in WO2013 / 060867) was performed to obtain the bispecific product. The quantity was determined. The sample was stored at 2-8°C.

[0425] The bispecific antibodies produced are referred to as "K409R IgG1 backbone" and "F4" in the following description. It is labeled "05L IgG1 backbone".

[0426] Inactivating mutation Several antibody variants were generated that had one or more amino acid substitutions in the Fc region. (Inactive) The chemical Fc region prevents antibodies from interacting with Fc receptors present on blood cells such as monocytes. or prevent the antibody from interacting with C1q and activating the classical complement pathway. We investigated the reduction in Fc activity in antibody variants containing different combinations of amino acid substitutions. Up to five amino acid substitutions were introduced. These included mutations N297Q, L234A, L235A, L234F, and L23 This includes 5E, D265A, and P331S. At one or more of these five amino acid positions... The substitution was introduced into the K409R and / or F405L IgG1 backbone. The following huCLB-T3 / 4 antibodies were used: A mutant of the Fc region was created: N297Q (referring to the N297Q substitution). It was named IgG1-huCLB-T3 / 4-N297Q. ), LFLE (referring to L234F / L235E substitution, named IgG1-huCLB-T3 / 4-LFLE), LALA (L234A This refers to the L235A substitution (named IgG1-huCLB-T3 / 4-LALA), LFLENQ (L234F / L235E / N297Q substitution). This refers to substitutions (named IgG1-huCLB-T3 / 4-LFLENQ) and LFLEDA (L234F / L235E / D265A substitutions). It is named IgG1-huCLB-T3 / 4-LFLEDA), DA (referring to the D265A substitution). IgG1-huCLB-T3 / 4-DA (named as), DAPS (referring to the D265A / P331S substitution; named IgG1-huCLB-T3 / 4-DAPS), DAN Q (referring to the D265A / N297Q substitution, named IgG1-huCLB-T3 / 4-DANQ), LFLEPS (L234F / L235E / This refers to P331S substitution (named IgG1-huCLB-T3 / 4-LFLEPS) and LFLEDANQPS (L234F / L235E This refers to the substitution / D265A / N297Q / P331S (named IgG1-huCLB-T3 / 4-LFLEDANQPS).

[0427] Specifically, in IgG1-huCD3 antibody variants, LFL includes mutations L234F, L235E, and D265A. The combination of three amino acid substitutions called EDA is used in the K409R and F405L IgG1 backbone. By introducing it into the molecule, an antibody with an inactivated Fc region was produced. The resulting inactive Sexually differentiated antibody variants are named with the suffix "-LFLEDA".

[0428] Example 2: Humanized CD3 antibodies against human and cynomolgus monkey T cell lines expressing CD3 and Binding of the inactivating mutant Humanized CD3 (huCD3) antibodies with or without LFLEDA mutations in the Fc region and bispecificity ( bs) Purified mutant of IgG1-huCD3×HER2 molecule (see Example 1), human T cell line Jurkat (claw E6-1, ATCC (Registered Trademark), TIB-152 (Trademark), LGC Standards GmbH, Wesel, Germany ) or cynomolgus monkey T cell line HSC-F (catalog number JCRB1164; Human Science Research) The binding to the Resource Bank (Osaka, Japan) was analyzed by FACS analysis. The antibody variants were In addition to the inactivating LFLEDA mutation, the molecule also contained the F405L or K409R mutation as described in Example 1. .

[0429] cells (1×10 5 Cells / well) in a polystyrene 96-well round-bottom plate (Greiner bio-one In 650101), together with serial dilutions of the antibody preparation (in the range of 5 to 10,000 ng / mL after 3-fold dilution), 10 The samples were incubated in 0 μL of PBS / 0.1% BSA / 0.02% azide at 4°C for 30 minutes.

[0430] After washing the cells twice in PBS / 0.1% BSA / 0.02% azide, the cells were incubated with secondary antibody in 100 μL at 4°C. The mixture was incubated for 30 minutes. All experiments used PBS / 0.1% BSA / 0.02% Aji as the secondary antibody. R-phycoerythrin (PE) conjugate goat anti-human IgG F(ab') diluted 1:100 in the solution. 2(109-116-098, Jackson ImmunoResearch Laboratories, Inc., Welsh, Pennsylvania) (Stoglobes) were used. Next, the cells were washed twice with PBS / 0.1% BSA / 0.02% azide and 150 The samples were resuspended in μL of PBS / 0.1% BSA / 0.02% azide and analyzed using FACS Cantoll (BD Biosciences). GraphPad Prism V5.04 software (GraphPad Software, California, USA) Using N'Diego, we analyzed the couple curve using nonlinear regression (sigmoid dose-response with variable slope). Ta.

[0431] Figure 1A shows the IgG1λ-huCD3 mutant IgG1-huCD3-H1L1 with a wild-type Fc region (each with SEQ ID NO :6 and 10), IgG1-huCD3-H1L2 (SEQ ID NO:6 and 11 respectively), IgG1-huCD3-H1L3 (SEQ ID NO: 6 and 12 respectively), IgG1-huCD3-H3L3 (SEQ ID NO: 8 and 12 respectively) The binding of ) and IgG1-huCD3-H4L1 (SEQ ID NO: 9 and 10, respectively) to Jurkat cells The combination was observed in all mutants, and IgG1-CD3-LFLEDA (inactivated LFLEDA (Parental CD3 antibody described in Example 1 with mutation) and IgG1-huCD3-H3L with inactivating LFLEDA mutation This indicates that the binding ability of 1-LFLEDA was similar to that of a huCD3 mutant with a wild-type Fc region. The binding of IgG1-huCLB-T3 / 4, which was included as a positive control, to Jurkat cells was compared with IgG1-huCD3. The binding was stronger compared to the mutant. No binding was observed to the negative control antibody IgG1-b12.

[0432] Figure 6A shows IgG1-CD3-LFLEDA with an inactivating LFLEDA mutation (implementation with an inactivating LFLEDA mutation). (Parental CD3 antibody as described in Example 1), IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-3hu CD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD The binding of 3-H4L3-LFLEDA to Jurkat cells is similar to the binding ability of huCD3 mutants with a wild-type Fc region. This indicates similarity. The binding of IgG1-huCLB-T3 / 4, which was included as a positive control, is similar to that of Jurka. Against T cells, the antibody was stronger at low antibody concentrations compared to the IgG1-huCD3 mutant, but at high antibody concentrations... They were similar. Overall, the humanized CD3 mutants bound to CD3 as IgG1-CD3 antibodies. The function was maintained. No binding was observed to the negative control antibody IgG1-b12.

[0433] Figure 1B shows the bispecific antibody mutants bsIgG1 CD3×HER2, bsIgG1 CD3×b12-LFLEDA, and bs This also indicates that IgG1 huCD3-H3L1×HER2-LFLEDA binds to Jurkat cells. The maximum binding capacity of a specific antibody is higher than that of a single-specific antibody. (EC) The concentration at 50 was 6 to 10 times higher. Here too, binding to the negative control antibody IgG1-b12 was observed. It wasn't done.

[0434] Figure 6B shows the bispecific inactivated Fc antibody mutants bsIgG1 huCD3-H3L1×HER2-LFLEDA and bsIgG1-h uCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2- LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA are also Ju This indicates that they bind to rkat cells. The maximum binding value of these bispecific antibodies is the same as that of single-specific antibodies. The EC50 concentration of the bispecific antibody was higher than the maximum binding value of the isovalent antibody. The binding value allows more antibodies to accumulate on the cell surface, thus enabling bispecificity. The antibody level is higher. Here too, no binding was observed for the negative control antibody IgG1-b12. .

[0435] Figure 2A shows IgG1-huCD3 mutants IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, and Ig with wild-type Fc region. G1-huCD3-H1L3, IgG1-huCD3-H3L3, and IgG1-huCD3-H4L1, as well as IgG1-CD3-LFLEDA, Ig The binding of G1-huCD3-H3L1-LFLEDA to the cynomolgus monkey T cell line HSC-F was similar. This involves a control antibody huCLB-T3 / 4 that does not cross-react with cynomolgus monkey CD3, and a negative control antibody IgG1- No binding was observed for b12.

[0436] Figure 7A shows the IgG1-huCD3 mutant IgG1-CD3-LFLEDA against the cynomolgus monkey T cell line HSC-F, and IgG1- huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1-huCD3-H1L1-LFLEDA, IgG1-huCD3-H1 The binding of L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-huCD3-H4L3-LFLEDA is similar. This indicates that binding was observed with the negative control antibody IgG1-b12.

[0437] Figure 2B also shows the bispecific antibody mutants bsIgG1 CD3×HER2 and bsIgG1 huCD3-H3L1-LFLEDA, as well as HS. This indicates that they bind to CF cells. The maximum binding value of these bispecific antibodies is single-specific. The EC50 concentration of the bispecific antibody is higher than that of the monospecific anti-CD3 antibody. The EC50 concentration was 10 to 12 times higher than that of the other antibody. Here too, binding was observed for the negative control antibody IgG1-b12. It wasn't done.

[0438] Figure 7B shows the bispecific inactivated Fc antibody mutants bsIgG1 huCD3-H3L1×HER2-LFLEDA and bsIgG1-h uCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2- LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsIgG1-huCD3-H4L3×HER2-LFLEDA are also This indicates that these bispecific antibodies bind to HSC-F cells. The EC50 concentration of the bispecific antibody is higher than that of the isotropic anti-CD3 mutant. The EC50 concentration of the antibody was 3 to 6 times higher. Here too, binding was not observed for the negative control antibody IgG1-b12. It wasn't noticed.

[0439] Example 3: T cell activation by humanized CD3 antibody mutant CD69 expression is an early marker of T cell activation. CD3 antibodies bind to CD3 expressed by T cells. This involves the binding of the antibody's Fc region (e.g., the IgG1 Fc region) to the Fc receptor expressed by immune cells. Therefore, it can mediate cross-linking between T cells and immune cells. This leads to T cell activation and CD69 induction. They can be linked. Antibody variants containing an inactivated Fc region (LFLEDA mutation) bind to the Fc receptor. No. Therefore, it is not expected that inactivated CD3 antibodies will not induce T cell activation and CD69 expression. This is because the inactive Fc region does not bind to immune cells that express the Fc receptor, and therefore This is because it is not possible to bridge the gap between T cells and immune cells.

[0440] Humanized CD3 (huCD3) mutants with an LFLEDA mutation in the Fc region and LFLEDA mutation in the Fc region Initial activation of T cells after incubation with humanized CD3-deficient (huCD3) mutants. To make a determination, CD69 expression on T cells was evaluated by FACS analysis. The LFLEDA mutant was non In addition to the activating mutation, it contains either the F405L mutation or the K409R mutation described in Example 1.

[0441] Leucosep tube (number 227290; Greiner Bio-one, Alfen Aan De, Netherlands) PBMCs are isolated from whole blood or buffy coat by density gradient separation using (Lynne-Rain). The samples were washed with PBS and resuspended in culture medium.

[0442] huCD3 antibody variants, negative control (IgG1-b12) and positive control (IgE-huCD3 and parental IgG1-CD3) A dose-response series of ) was prepared in culture medium (in the range of 0.1 to 1,000 ng / mL at 10-fold dilution), and human or It was added to the wells of a 96-well round-bottom plate containing cynomolgus macaque PBMC. 16-24 hours After incubation, the cells are pelleted by centrifugation, and the supernatant (containing cytokines) is extracted. The cells containing the substance were collected and stored at -20°C. Next, the cells were treated with PBS / 0.1% BSA / 0.02% azide. Wash and cross-react with cynomolgus monkey CD28 and CD69 in mice using anti-human CD28-PE (854.222.0 10; Sanquin, Amsterdam, Netherlands; T cell marker) and mouse anti-human CD69-APC anti Using the body (340560; BD Biosciences, Franklin Lakes, New Jersey), at 4℃ The samples were stained for 30 minutes. Unbound antibodies were removed by washing twice with PBS / 0.1% BSA / 0.02% azide. The cells were removed. The cells were resuspended in 150 μL / well, and CD69 expression on CD28-positive cells was examined using FACS Canto. Measurements were taken using II (BD Biosciences).

[0443] Figure 3 shows IgG1-CD3 with the wild-type IgG1 Fc region (as described in Example 1) and humanized IgG1-Hu CD3 mutants were found in human (Figure 3A) and cynomolgus monkey (Figure 3B) T cells, similar to This indicates that it induced similar levels of CD69 expression. Inactivated (LFLEDA)IgG1-CD3-LFL EDA and IgG1-huCD3-H3L1 mutants induced low levels of CD69 expression in human T cells. In cynomolgus monkey T cells, CD69 expression induced by the inactivated IgG1-huCD3 mutant is not The control antibody IgG1-b12 also showed an improvement in CD69 expression in human T cells and cynomolgus monkey T cells. It did not induce that.

[0444] Figure 8 shows inactivated (LFLEDA) IgG1-huCD3-H3L1-LFLEDA, IgG1-huCD3-H3L3-LFLEDA, IgG1- 3huCD3-H1L1-LFLEDA, IgG1-huCD3-H1L3-LFLEDA, IgG1-huCD3-H4L1-LFLEDA, and IgG1-h The uCD3-H4L3-LFLEDA mutant 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 slight activation observed by the inactive mutant is due to the bivalent binding of the CD3 antibody to C This might be due to cross-linking of the D3 molecule. This explanation is relevant because the antibody is active at the highest concentration where monovalent antibody binding occurs. This is supported by observations that the transformation is reduced. The control antibody IgG1-b12 also showed a reduction in human T cells. CD69 expression was not induced in cynomolgus monkey T cells.

[0445] Figures 8C and 8D show the inactivated bispecific antibody mutants bsIgG1-huCD3-H3L1×HER2-LFLEDA, bs IgG1-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 EDA induces CD69 expression in human (Figure 8C) or cynomolgus monkey (Figure 8D) T cells. This indicates that it does not occur. However, at even higher antibody concentrations, some induction of CD69 expression can be observed. They figured it out.

[0446] Example 4: T cell proliferation induced by humanized CD3 antibody mutants Humanized CD3 (huCD3) antibody variants against the proliferation of human and cynomolgus monkey T cells (Example 1) The effects of the product described above are based on the Cell Proliferation ELISA kit from Roche Applied Science. Fermentation ELISA, BrdU kit, number 11647229001; Roche Applied Science, Germany Evaluated by Heim. The evaluation was conducted according to the manufacturer's instructions.

[0447] Human or cynomolgus monkey PBMCs isolated from whole blood or buffy coat were cultured in 96 wells. Dilution series of IgG1 huCD3 antibody variants in a plate (ranging from 0.1 to 1,000 ng / mL at 10-fold dilutions) Incubated together with IgE-CD3 and IgG1-huCLB-T3 / 4 as positive controls. -b12 was included as a negative control. After incubation with antibody for 3 days, BrdU (Roche App) Add Lied Science (Mannheim, Germany) to the culture medium and incubate the plate for 5 hours. Next, the cells were pelletized by centrifugation, the supernatant was collected, and stored at -20°C. The rates were dried and stored at 4°C until ELISA was performed.

[0448] BrdU incorporation into DNA was performed using ELISA according to the manufacturer's instructions (Roche Applied Science). This was determined by the following: After fixing the cells to a plate, they were conjugated with peroxidase. The plates were incubated with the anti-BrdU antibody at room temperature for 90 minutes. The plates were then fermented in PBST. Wash and detect binding using ABTS buffer (not the TMB solution provided with the kit). The color development was stopped by adding 2% oxalic acid to the well after 30 minutes. Next, EL808 E The OD405nm was measured using a LISA reader.

[0449] Figure 4 shows PBMCs with parental IgG1-CD3 and humanized IgG1-huCD3 mutants possessing the wild-type IgG1 Fc region. When incubated, even at extremely low antibody concentrations, human T cells (Figure 4A) and cynopsy cells are detected. This shows that strong proliferation of izal T cells (Figure 4B) was induced. Inactivation of IgG1-huCD3 antibody. Even when incubated with sexualized LFLEDA mutants, human T cells (Figures 4A and 9A) and crabs... Proliferation of quis monkey T cells (Figures 4B and 9B) was not induced. Therefore, IgG1-huCD3 anti The deactivated mutant of the body exhibits low levels of CD69 expression in human T cells (as shown in Example 3). Although it induced the proliferation of human T cells, these inactivated IgG1-huCD3 mutants did not induce human T cell proliferation. .

[0450] Figures 9C and 9D show the inactivated bispecific antibody mutants bsIgG1-huCD3-H3L1×HER2-LFLEDA, bs IgG1-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 EDA does not induce proliferation of T cells isolated from humans (Figure 9C) and cynomolgus monkeys (Figure 9D). It represents something.

[0451] Example 5: In vitro T cell-mediated cytotoxicity induced by humanized CD3 antibody variants Tumor-specific T-cell cytotoxicity involves binding to CD3 on one arm and to HER2, etc. on the other arm. This can be mediated by bispecific antibodies that bind to tumor-specific targets. If a specific antibody binds to both T cells and tumor cells simultaneously, it can activate T cells and target tumor cells. This would lead to cell damage. In this embodiment, T cell-mediated action against HER2-positive tumor cells Cell damage was evaluated using bispecific antibodies against CD3 (humanized mutant) and HER2.

[0452] Therefore, 10% (vol / vol) heat-inactivated CCS, 1.5 g / L sodium bicarbonate (Lonza), 1 mM pyropropyl alcohol. Sodium phosphate, 4.5 g / L glucose (Sigma), 50 IU / mL penicillin, and 50 μg / mL sodium phosphate. AU565 (human breast cancer) cells were cultured in RPMI1640 supplemented with leptomycin. 5 cell lines were selected. AU565 cells were kept at 37°C in a %(vol / vol)CO2 humidified incubator. After culturing to the final stage, the cells are treated with trypsin, resuspended in culture medium, and then strained. By passing the cells through the filter, a single-cell suspension was obtained. 5 × 10 cells were placed in each well of a 96-well culture plate. 4 cells After seeding, the cells are incubated at 37°C and 5% CO2 for at least 3 hours, and then the cells are transferred to a plate. I glued it in place.

[0453] Human or cynomolgus monkey PBMCs were isolated from whole blood or buffy coat. Wash the MCs in PBS, resuspend them in culture medium, and add them in a 1:1 ratio to AU565 tumor cells in a 96-well plate. Added proportionally. Mouse anti-human CD3-PerCP (BD, number 345766) cross-reacts with cynomolgus monkey CD3. Using antibodies (for T cell staining), FACS analysis was performed to determine the percentage of T cells present in PBMCs. The T cell content in the PBMC population used was typically 50-60%.

[0454] Bispecific antibody variants bsIgG1 CD3×HER2-LFLEDA, bsIgG1 CD3×b12-LFLEDA, bsIgG1 hu CD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LFLEDA, bsIgG1-huCD3-H1L1×HER2-L FLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huCD3-H4L1×HER2-LFLEDA, and bsI Dilution series of gG1-huCD3-H4L3×HER2-LFLEDA (final dilution in the range of 0.001 ng / mL to 10,000 ng / mL) The appropriate concentrations were prepared in the culture medium and added to the plate. IgG1-HER2-LFLEDA and IgG1-b12 were then treated. It was included as a reference. The LFLEDA antibody variant, in addition to the inactivating mutation, has a bispecific format. It contains the F405L mutation or the K409R mutation for preparation (see Example 1). The cells were incubated at 37°C and 5% CO2 for 3 days. As a criterion for 100% tumor cell death, the cells were divided into 1 The plates were incubated with μM staurosporine (number S6942-200, Sigma). The wells were washed twice with BS, and 150 μL of culture medium containing 10% Alamar blue was added to each well. The plate was incubated at 37°C with 5% CO2 for 4 hours. The absorbance at 590 nm was measured. Envision (Perkin Elmer, Waltham, Massachusetts)

[0455] Bispecific CD3×HER2-LFLEDA antibody variant (bsIgG1-huCLB-T3 / 4×HER2-LFLEDA and bsIg G1-CD3×HER2-LFLEDA) is used at low concentrations in human effector cells (Figure 5A) or cynomolgus cells. We induced the death of AU565 cells using effector cells (Figure 5B). The CD3 bispecific control antibody huCLB-T3 / 4×HER2-LFLEDA, which does not exhibit cross-reactivity, uses human PBMCs. Only when used did it induce the death of AU565 cells (Figure 5A). Therefore, cynomolgus monkeys When effector cells were used in the assay, no target cell death was observed. (Figure 5B). Single-specific IgG1-b12 or IgG1-HER2-LFLEDA or bsIgG1-CD3×b12-LFLEDA Incubation with antibodies did not induce nonspecific death of target cells.

[0456] Bispecific antibody mutants bsIgG1 huCD3-H3L1×HER2-LFLEDA, bsIgG1-huCD3-H3L3×HER2-LF LEDA, bsIgG1-huCD3-H1L1×HER2-LFLEDA, bsIgG1-huCD3-H1L3×HER2-LFLEDA, bsIgG1-huC D3-H4L1×HER2-LFLEDA and bsIgG1-huCD3-H4L3×HER2-LFLEDA are Hito Effector components. Killing AU565 cells using cells (Figure 10A) or cynomolgus monkey effector cells (Figure 10B) Induced at low concentrations. Incubation with monospecific IgG1-b12 or IgG1-HER2-LFLEDA antibodies. The treatment did not induce nonspecific target cell death (Figures 10A and B). Thus, Humanized CD3 mutants containing an inactivated Fc region do not induce nonspecific target cell death. This is because it ensures the activation of targeted T cells and thus avoids the activation of non-targeted T cells, thus inactivating them. This indicates that mutants containing the Fc region can be used.

[0457] Example 6: Activation of rhesus monkey T cells by humanized CD3 antibody mutants After incubation with a humanized CD3 (huCD3) antibody mutant containing the wild-type IgG1 Fc region To determine the initial activation of T cells, CD69 expression on rhesus monkey T cells was evaluated. The isolation of gestal PBMCs and the evaluation of CD69 expression by flow cytometry were described in Example 3. That's how it went.

[0458] Figure 11 shows humanized CD3 antibody mutants IgG1-huCD3-H1L1, IgG1-huCD3-H1L2, and IgG1-huCD3-H1L3. IgG1-huCD3-H3L3 and IgG1-huCD3-H4L1 are associated with CD69 expression in rhesus monkey-derived T cells. This indicates that it was induced 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. However, Therefore, the huCD3 mutant of the present invention can be used in experiments using rhesus monkey CD3. The 3 mutants cross-react with rhesus macaque CD3.

[0459] Example 7: T cell activation by a deactivating mutant of huCLB-T3 / 4 Incubated with IgG1-huCLB-T3 / 4 mutants with mutations in the Fc region (see Example 1). To determine the initial activation of subsequent T cells, CD69 expression on T cells was evaluated by FACS analysis. Ta.

[0460] Leucosep tube (number 227290; Greiner Bio-one, Alfen Aan De, Netherlands) PBMCs are isolated from whole blood or buffy coat by density gradient separation using (Lynne-Rain). The samples were washed with PBS and resuspended in culture medium.

[0461] IgG1-huCLB-T3 / 4 mutant, negative control (IgG1-huCLB-T3 / 4-Fab), and positive control (IgE-huCL Prepare a dose-response series of B-T3 / 4) in culture medium (in the range of 1-1,000 ng / mL at 3-fold dilution), and PBMCs It was added to the wells of the 96-well round-bottom plate. After 16-24 hours of incubation. Next, the cells are pelletized by centrifugation, and the supernatant (containing cytokines) is collected. The cells were then stored at -20°C. Next, the cells were washed with PBS / 0.1% BSA / 0.02% azide and treated with mouse anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, Netherlands; T cell marker) and Mau Anti-human CD69-APC antibody (340560; BD Biosciences, Franklinley, New Jersey) The samples were stained using (Kus) at 4°C for 30 minutes. Washed twice with PBS / 0.1% BSA / 0.02% azide. Unbound antibodies were removed by [method]. Cells were resuspended in 150 μL / well, and C [method] was applied to CD28-positive cells. D69 expression was measured using FACS Canto II (BD Biosciences).

[0462] Figure 12A shows IgE-huCLB-T3 / 4, IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA, and IgG1-huCLB-T This indicates that cells incubated with 3 / 4-DAPS showed higher CD69 expression levels. When incubated with IgG1-huCLB-T3 / 4-N297Q, compared to wild-type IgG1-huCLB-T3 / 4 Somewhat lower levels of CD69 expression are induced, and IgG1-huCLB-T3 / 4-LFLE and IgG1-huCLB-T3 / CD69 induction was even lower when incubated with 4-LFLEPS. PBMCs were incubated with IgG1-CD3 Fab, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1-huCLB-T3 / 4-DANQ and CD69 expression on T cells was not achieved even when incubated with IgG1-huCLB-T3 / 4-LFLEDANQPS antibody. It was not triggered at all.

[0463] Figure 12B shows that cells incubated with IgE-huCLB-T3 / 4 and IgG1-huCLB-T3 / 4 show CD This indicates a high expression level of 69. Incubate with IgG1-huCLB-T3 / 4-LALA. This induces CD69 expression levels that are somewhat lower compared to wild-type IgG1-huCLB-T3 / 4, and IgG1- Even when incubated with huCLB-T3 / 4-LFLEDA and IgG1-b12 (negative control), on T cells No CD69 expression was induced at all.

[0464] Example 8: T cell proliferation by inactivating mutants of huCLB-T3 / 4 The effect of the huCLB-T3 / 4 mutant (described in Example 1) on T cell proliferation was investigated by Roche Ap. plied Science Cell Proliferation ELISA Kit (BrdU Kit, No. 11) 647229001; Evaluated by Roche Applied Science (Mannheim, Germany). The evaluation was conducted by the manufacturer. I followed the creator's instructions.

[0465] PBMCs isolated from whole blood or buffy coat were cultured in a 96-well plate using IgG1-CD3 The mutants were incubated with a dilution series (ranging from 0.1 to 1,000 ng / mL). IgE-CD3 and IgG1-CD3 is included as a positive control, and IgG1-b12 (K409R mutation for producing bispecific antibodies) is included. Those with the characteristic were included as negative controls. After incubation with the antibody for 3 days, BrdU(R Add the culture medium (oche Applied Science, Mannheim, Germany) and incubate the plate for 5 hours. The cells were then baited. Next, the cells were pelleted by centrifugation, the supernatant was collected, and stored at -20°C. The plates were dried and stored at 4°C until ELISA was performed.

[0466] The incorporation of BrdU into DNA is described in the manufacturer's instructions (Cell Proliferation ELISA, BrdU Kit). The determination was made by ELISA according to the number 11647229001 (Roche Applied Science). After fixation to the rate, the antibody conjugated with peroxidase and anti-BrdU antibody was pre-treated. The kit was incubated at room temperature (RT) for 90 minutes. The plate was washed with PBST (and together with the kit). Binding was detected using ABTS buffer (not the TMB solution provided). After 30 minutes, the wells were... The color development was stopped by adding 2% oxalic acid. Next, OD405n was read using an EL808 ELISA reader. m was measured.

[0467] Figure 13A shows PBMCs with IgG1-huCLB-T3 / 4, IgG1-huCLB-T3 / 4-DA, and IgG1-huCLB-T3 / 4-DAPS. When incubated together, even at extremely low antibody concentrations, strong T cell proliferation was induced. This represents the incubation with IgG1-huCLB-T3 / 4-N297Q, which is positive for IgE-huCLB-T3 / 4. Dose-dependent proliferation comparable to that of sex controls was induced in PBMCs using IgG1-huCLB-T3 / 4-Fab and IgG1-b12-N2. 97Q, IgG1-huCLB-T3 / 4-LFLE, IgG1-huCLB-T3 / 4-LFLEDA, IgG1-huCLB-T3 / 4-LFLENQ, IgG1- huCLB-T3 / 4-LFLEPS, together with IgG1-huCLB-T3 / 4-DANQ and IgG1-huCLB-T3 / 4-LFLEDANQPS antibodies. Incubation did not induce T cell proliferation.

[0468] Figure 13B shows that when PBMCs are incubated with IgG1-huCLB-T3 / 4, very low antibody concentrations are observed. This indicates that strong T cell proliferation was induced. IgE-huCLB-T3 / 4 (positive control) Incubation with IgG1-huCLB-T3 / 4-LALA induced dose-dependent proliferation of PBMCs. Incubation with IgG1-huCLB-T3 / 4-LFLEDA did not induce T cell proliferation. .

[0469] Based on the results of Examples 7 and 8, the mutant with the lowest degree of activation is considered to be the one shown in Examples 7 and 8. A subset of T was subjected to further analysis.

[0470] Example 9: In vitro T cell-mediated cells induced by the inactivated antibody mutant huCLB-T3 / 4 injury 10% (vol / vol) heat-inactivated CCS, 1.5 g / L sodium bicarbonate (Lonza), 1 mM sodium pyruvate Lilium, 4.5 g / L glucose (Sigma), 50 IU / mL penicillin, and 50 μg / mL streptoma AU565 (human breast cancer) cells were cultured in RPMI1640 supplemented with isin. The cell line was cultured at 5% (vol / vo l) AU565 cells were cultured in a CO2-moistened incubator at 37°C until nearly confluence. The cells were nourished. The cells were treated with trypsin, resuspended in culture medium, and passed through a cell strainer. Then, a single-cell suspension was obtained. 5 × 10⁶ cells were placed in each well of a 96-well culture plate. 4 Seed cells, The material was incubated at 37°C in 5% CO2 for at least 3 hours to adhere it to the plate.

[0471] Peripheral blood mononuclear cells (PBMCs) were collected from the blood of healthy volunteers using a Leucosep 30mL tube. The isolated PBMCs were isolated according to the manufacturer's protocol (Greiner Bio-one). Wash with BS, resuspend in culture medium, and add AU565 tumor cells in a 1:1 ratio to a 96-well plate. Added: FACS using mouse anti-human CD3-PerCP (BD, no. 345766) antibody (for T cell staining). Analysis was conducted to measure the percentage of T cells present in PBMCs. The T cell content was typically 50-60%.

[0472] Different Fc variants, wild type, N297Q, LFLE, LALA, LFLENQ, LFLEDA, DANQ, and LFLEDEN IgG1-b12, IgG1-huCLB-T3 / 4, IgG1-HER2, and bispecific huCLB-T were expressed as QPS. Dilution series of 3 / 4×b12 and huCLB-T3 / 4×HER2 antibodies (range from 0.004 ng / mL to 1000 ng / mL) The final concentration of the solution was prepared in the culture medium and added to the plate. The plate was incubated at 37°C in 5% CO2 for 3 days. The cells were incubated for a period of time. As a criterion for 100% tumor cell death, the cells were treated with 1 μM staurosporine. It was incubated with (number S6942-200, Sigma). After incubation, the supernatant was Removed and stored at -20°C. Washed the plate twice with PBS and treated with 10% Alamar Blue. 150 μL of culture medium was added to each well. The plate was incubated at 37°C in 5% CO2 for 4 hours. The absorbance at 590 nm was measured (Envision, Perkin Elmer, Massachusetts). Waltham, State.

[0473] Two experiments were conducted using PBMCs from different donors. In the first experiment, the Fc mutant N297Q was used. LFLE, LFLENQ, LFLEDA, DANQ, and LFLEDANQPS were tested (Figures 14A-G). Second experiment Next, we tested the Fc mutants LFLEDA and LALA (Figure 15A-C). Both experiments included references. This includes antibodies with wild-type Fc domains. It also includes wild-type monospecific IgG1-huCLB-T3 / 4 or bispecific antibodies. Incubation with isomer huCLB-T3 / 4 × b12 antibody results in nonspecific death of target cells. Induced (Figures 14A-G and 15A-C). Single-specific IgG1-huCLB-T3 / 4 and bsIgG1-huCLB- The T3 / 4×b12 mutants N297Q (Figures 14A-G) and LALA (Figures 15A-C) still exhibit some degree of nonspecificity. It induced target cell death, but to a lesser extent compared to the wild-type antibody tested in the same experiment. The results were low. Other IgG1-huCLB-T3 / 4 or bsIgG1-huCLB-T3 / 4 tested had inactivating mutations. None of the ×b12 antibodies induced nonspecific target cell death (Figure 14A-G). (Called 15A~C).

[0474] All bispecific huCLB-T3 / 4×HER2 antibodies inactivate dose-dependent death of AU565 cells. Compared to a non-mutated wild-type bispecific huCLB-T3 / 4×HER2 antibody, it exhibits at least comparable efficacy. The reaction was induced by force (Figures 14A-G and 15A-C). Maximum mortality occurred at extremely low concentrations.

[0475] In wild-type or inactivated mutants of single-specific b12 or HER2 antibodies, cytotoxicity was induced. (Figures 14A-G and 15A-C) was as expected.

[0476] Example 10: Evaluation of C1q binding to inactivated antibody variants of huCLB-T3 / 4 The interaction between C1q and antibodies bound to target cells is in the classical pathway of complement activation. This is the first step. Since wild-type IgG1 contains an interaction site with C1q, ELISA can be performed We evaluated the interaction of C1q with these inactivated IgG1 variants.

[0477] IgG1-huCLB-T3 / 4, bsIgG1-huCLB-T3 / 4×HER2 and IgG1-CD20 (positive control) and above The dilution series of the inactivated antibody variant described in Example 1 (ranging from 7 to 30,000 ng / mL at 4-fold dilution) ) were placed in a 96-well Microlon ELISA plate (Greiner, Germany) and coated overnight at 4°C. The plate was washed and bred in PBS supplemented with 0.025% Tween 20 and 0.1% gelatin. Locked. Wash the plate between incubations using 3% pooled human blood. Rabbit anti-human C1q(D) solution was incubated with Sanquin (product number M0008) at 37°C for 1 hour, 100 μL / well. AKO (product number A0136, 1 / 4,000) was incubated at room temperature for 1 hour, and 100 μL / ...

Claims

1. A humanized or chimeric antibody that binds to human CD3, comprising a binding region including a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH and VL regions are (a) VH sequence shown in SEQ ID NO: 8 and VL sequence shown in SEQ ID NO: 10, (b) VH sequence shown in SEQ ID NO: 9 and VL sequence shown in SEQ ID NO: 10, (c) VH sequence shown in SEQ ID NO: 6 and VL sequence shown in SEQ ID NO: 11, (d) VH sequence shown in SEQ ID NO: 6 and VL sequence shown in SEQ ID NO: 12, (e) The VH sequence shown in SEQ ID NO: 7 and the VL sequence shown in SEQ ID NO: 10, (f) The VH sequence shown in SEQ ID NO: 7 and the VL sequence shown in SEQ ID NO: 11, (g) VH sequence shown in SEQ ID NO: 7 and VL sequence shown in SEQ ID NO: 12, (h) The VH sequence shown in SEQ ID NO: 8 and the VL sequence shown in SEQ ID NO: 11, (i) The VH sequence shown in SEQ ID NO: 8 and the VL sequence shown in SEQ ID NO: 12, (j) The VH sequence shown in SEQ ID NO: 9 and the VL sequence shown in SEQ ID NO: 11, (k) VH sequence shown in SEQ ID NO: 9 and VL sequence shown in SEQ ID NO: 12, The antibody selected from the group consisting of the following.

2. The aforementioned bonding region, (a) VH sequence shown in SEQ ID NO: 8 and VL sequence shown in SEQ ID NO: 10, (b) VH sequence shown in SEQ ID NO: 9 and VL sequence shown in SEQ ID NO: 10, The antibody according to claim 1, comprising a VH sequence and a VL sequence selected from the group consisting of the following.

3. The antibody according to claim 1 or 2, which is a humanized antibody.

4. The antibody according to claim 1 or 2, which is a chimeric antibody.

5. The antibody according to any one of claims 1 to 4, which is a full-length antibody.

6. The antibody according to any one of claims 1 to 5, comprising an Fc region containing first and second immunoglobulin heavy chains.

7. The antibody according to any one of claims 1 to 6, wherein the first and second heavy chains are isotypes selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The antibody according to any one of claims 1 to 7, comprising an Fc region modified such that the binding of C1q to the antibody is reduced by at least 70% compared to a wild-type antibody, wherein the C1q binding is determined by ELISA.

9. The antibody according to any one of claims 1 to 8, comprising an Fc region modified such that the binding of C1q to the antibody is reduced by at least 95% compared to a wild-type antibody, and the C1q binding is determined by ELISA.

10. The antibody according to any one of claims 1 to 9, comprising an Fc region modified such that the binding of C1q to the antibody is reduced by at least 97% compared to a wild-type antibody, wherein the C1q binding is determined by ELISA.

11. The antibody according to any one of claims 1 to 10, comprising an Fc region modified so that the antibody mediates Fc-mediated T cell proliferation reduced by at least 50% compared to a wild-type antibody, wherein T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

12. The antibody according to any one of claims 1 to 11, comprising an Fc region modified so that the antibody mediates Fc-mediated T cell proliferation reduced by at least 80% compared to a wild-type antibody, wherein T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

13. The antibody according to any one of claims 1 to 12, comprising an Fc region modified so that the antibody mediates Fc-mediated T cell proliferation reduced by at least 90% compared to a wild-type antibody, wherein T cell proliferation is measured in a functional assay based on peripheral blood mononuclear cells (PBMCs).

14. The antibody according to any one of claims 1 to 13, wherein the antibody comprises an Fc region modified to reduce Fc-mediated CD69 expression by at least 50% compared to a wild-type antibody, and the Fc-mediated CD69 expression is measured in a functional assay based on PBMC.

15. The antibody according to any one of claims 1 to 14, wherein the antibody comprises an Fc region modified to reduce Fc-mediated CD69 expression by at least 80% compared to a wild-type antibody, and the Fc-mediated CD69 expression is measured in a functional assay based on PBMC.

16. The antibody according to any one of claims 1 to 15, wherein the antibody comprises an Fc region modified to reduce Fc-mediated CD69 expression by at least 90% compared to a wild-type antibody, and the Fc-mediated CD69 expression is measured in a functional assay based on PBMC.

17. The antibody according to any one of claims 1 to 16, comprising 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 L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are not L, L, D, N, and P, respectively.

18. The antibody according to claim 17, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is not D.

19. The antibody according to claim 17, wherein in at least one of the first and second heavy chains, the amino acid at the position corresponding to position N297 in the human IgG1 heavy chain is not N.

20. The antibody according to claim 17, wherein in at least one of the first and second heavy chains, the amino acids at positions L234 and L235 in the human IgG1 heavy chain are not L and L, respectively.

21. The antibody according to claim 18 or 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 the human IgG1 heavy chain are F and E, or A and A, respectively.

22. The antibody according to claim 21, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

23. The antibody according to claim 21, wherein in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are A and A, respectively.

24. The antibody according to any one of claims 1 to 17, 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 the human IgG1 heavy chain are not L, L, and D, respectively.

25. The antibody according to claim 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 the human IgG1 heavy chain are F, E, and A, or A, A, and A, respectively.

26. The antibody according to claim 25, 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 the human IgG1 heavy chain are F, E, and A, respectively.

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