PD-1-binding molecules and methods use thereof

Abstract

To provide selected anti-PD-1 antibodies capable of binding to both cynomolgus monkey PD-1 and to human PD-1.SOLUTION: The present invention provides an anti-human PD-1-binding monospecific monoclonal antibody that comprises a variable heavy chain domain and a variable light chain domain, wherein the variable heavy chain domain comprises the CDRH1 domain, CDRH2 domain, and CDRH3 domain, and the variable light chain domain comprises the CDRL1 domain, CDRL2 domain, and CDRL3 domain.SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a direct result of U.S. patent application Ser. No. 62 / 198,867 (filed July 30, 2015; pending). U.S. Patent Application No. 62 / 239,559 (filed October 9, 2015; pending); U.S. Patent Application No. 62 / 255,140 (filed November 13, 2015; pending; and U.S. Priority over US Patent Application No. 62 / 322,974 (filed April 15, 2016; pending) No. 6,239,999, filed on Oct. 1, 2003, and claims priority thereto, each of which is incorporated herein by reference in its entirety. is used as a reference.

[0002] Sequence Listing Reference This application may contain one or more sequence listings pursuant to 37 CFR §1.821 et seq. These sequence listings are available on a computer readable medium (file name: 1301_0122PCT _Sequence_Listing_ST25.txt, created on July 1, 2016, (Size: 282,789 bytes), and the file is incorporated herein by reference. is incorporated herein by reference in its entirety.

[0003] The present invention provides selected anti-PD-1 antibodies capable of binding to both cynomolgus monkey PD-1 and human PD-1. D-1 antibodies: PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, P D-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD- 1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mA b PD-1 binding molecules comprising the PD-1 binding domain of 14 or PD-1 mAb 15 The present invention is particularly directed to humanized or chimeric versions of such antibodies. or PD-1-binding fragments of such anti-PD-1 antibodies (particularly immunoconjugates, diabodies, etc.) The present invention particularly relates to PD-1 binding molecules comprising: Targeting epitopes of molecules involved in regulating immune checkpoints present on the surface of immune cells The present invention also relates to such PD-1 binding molecules capable of binding to PD-1. or methods of using such PD-1 binding molecules for stimulation of an immune response. The invention also provides a method for identifying one or more PD-1 binding domains of a selected anti-PD-1 antibody as described above. The PD-1 binding molecule comprising the PD-1 receptor is administered to one or more antibodies effective in stimulating an immune response. in combination with additional molecules and / or one or more additional molecules that specifically bind to the cancer antigen. The present invention relates to combination therapy in which the compound is administered in combination with an additional molecule. [Background technology]

[0004] 2. Description of Related Art I. Cell-Mediated Immune Response The immune system of humans and other mammals serves to provide protection against infection and disease. Such protection is provided by both the humoral and cell-mediated immune responses. The humoral immune response produces antibodies and other biomolecules that can recognize and neutralize foreign targets (antigens). In contrast, cell-mediated immune responses involve T cells activating macrophages, Activation of natural killer (NK) cells and antigen-specific cytotoxic T lymphocytes, and recognition of antigens This is accompanied by the release of various cytokines in response to stimulation (Non-Patent Document 1).

[0005] The ability of T cells to optimally mediate an immune response to an antigen depends on two distinct signaling phases. First, antigen-presenting cells (APCs) interact with each other (Non-patent Documents 2 and 3). Antigens present on the surface of antigen-specific naive CD4 + T Such presentation allows the cells to express specific antigens for the presented antigen. T-cell receptors (T-Cell Receptors) instruct T cells to initiate targeted immune responses The signal is then delivered to the APC via the TCR (transducer receptor). A series of costimulatory and inhibitory signals mediated by interactions between T This triggers cell activation and proliferation, ultimately leading to T cell inhibition. The first signal confers specificity to the immune response, while the second signal determines the nature, strength, and duration of the response. It plays a role in determining the duration.

[0006] The immune system is tightly controlled by costimulatory and co-inhibitory ligands and receptors. The molecule provides a second signal for T cell activation and also limits immunity to self. However, the role of positive and negative signals in maximizing immune responses to infection is unclear. It provides a balanced network (Non-Patent Documents 4 and 5). It maintains self-tolerance and the duration of the immune response. The inhibitory pathways that are important for modulating the level and amplitude of immune responses are collectively referred to as immune checkpoints. Importantly, B7.1 (CD80) and B7.2 (CD86) ligands on antigen-presenting cells and CD4 + The binding between CD28 and CTLA-4 receptors on T lymphocytes (non-patented References 6, 1, 7). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation. Binding of B7.1 or B7.2 to CTLA-4 inhibits this activation (non- CD28 is constitutively expressed on the surface of T cells (Patent Documents 1, 7, and 8). On the other hand, CTLA-4 expression is rapidly upregulated following T cell activation (non-specific CTLA-4 is a high-affinity receptor (Non-patent Document 6), so binding occurs first. Initiating T cell proliferation (through CD28) and then (through early expression of CTLA-4) It inhibits T cell proliferation, thereby attenuating its effect when proliferation is no longer needed.

[0007] Further investigation into the ligands for the CD28 receptor revealed a set of related B7 molecules (" The identification and characterization of the B7 superfamily has been achieved. (Non-patent documents 11, 6, 8, 12, 13, 3, 14, 15, 16, 17). Several members of the family are known: B7.1 (CD80), B7.2 (CD86 ), inducible co-stimulatory factor ligand (ICOS-L), programmed death-1 ligand (PD-L 1; B7-H1), programmed death-2 ligand (PD-L2; B7-DC), B7-H3 , B7-H4 and B7-H6 (Non-patent documents 12, 18).

[0008] II. Programmed Death-1 ("PD-1") Programmed death-1 (PD-1, also known as CD279) mediates a wide range of immune responses. Approximately 31 of the extended CD28 / CTLA-4 family of T cell regulators down-regulates It is a member of the type I membrane protein family with a molecular weight of 100 kD (Non-Patent Document 19, Patent Documents 1 to 9).

[0009] PD-1 is expressed on activated T cells, B cells, and monocytes (Non-Patent Documents 20, 21), as well as and at low levels in natural killer (NK) T cells (Non-patent Documents 22, 23). To manifest.

[0010] The extracellular domain of PD-1 shares 23% identity with the equivalent domain of CTLA-4. It is composed of a single immunoglobulin (Ig) V domain that binds to extracellular I The gV domain is followed by a transmembrane region and an intracellular tail. Two tyrosine-based inhibitory motifs and two immunoreceptor tyrosine-based switch motifs It contains phosphorylation sites, suggesting that PD-1 downregulates TCR signaling. (Non-patent documents 19 and 24).

[0011] PD-1 mediates the inhibition of the immune system by binding to B7-H1 and B7-DC ( Non-patent document 14, patent documents 10 to 17).

[0012] B7-H1 and B7-DC are expressed in the heart, placenta, muscle, fetal liver, spleen, lymph nodes, and breast. Human and mouse tissues, such as the gland, and mouse liver, lung, kidney, pancreatic islet cells, and small intestine In humans, B7-H1 protein expression is , human endothelial cells (Non-patent Documents 25, 26, 27), myocardium (Non-patent Document 28), syncytiotrophoblasts The molecule has also been found in resident mammalian cells in several tissues (Non-Patent Document 29). The phage induces the production of interferon (IFN-γ) or tumor necrosis factor (TNF-γ). Macrophages activated by tumor necrosis factor (TNF)-α It is also expressed by the immune system (Non-Patent Document 30) and in tumors (Non-Patent Document 31).

[0013] The interaction between B7-H1 and PD-1 is a critical downward costimulatory pathway for T and B cells. signal (Non-patent Document 23), and its function as a cell death inducer (Non-patent Documents 19, 32 ) More specifically, low concentrations of PD-1 receptors and B7-H Interaction between 1 and 2 ligands and antigen-specific CD8 + Inhibitory effect of T cell proliferation At high concentrations, interaction with PD-1 has been shown to result in signal transduction; Although it does not inhibit cell proliferation, it clearly reduces the production of several cytokines (Non-Patent Document Reference 6) Resting CD4 and CD8 T cells and already activated CD4 and CD8 T cells T cell proliferation and cytokine production by both naive T cells derived from umbilical cord blood has been shown to be inhibited by a soluble B7-H1-Fc fusion protein (non Patent documents 33, 30, 34, 6).

[0014] The role of B7-H1 and PD-1 in T cell activation and proliferation is unclear. These findings suggest that IL-1 may serve as a therapeutic target for the treatment of inflammation and cancer. The use of anti-PD-1 antibodies for the treatment of infections and tumors and for upregulation of adaptive immune responses has been proposed. (See Patent Documents 18, 19, 20, 21, 22, 23, 24, 25, and 26.) Antibodies that can specifically bind to PD-1 have been reported by Non-Patent Documents 20 and 35. (Patent Documents 27, 28, 29, 30, 31, 32, 33, 34, 35, 27, 25, See also 36, 25, 37, 38, 39, 26, 40, 41, 42, 43, 44).

[0015] However, despite all the advances mentioned above, cancer cells or pathogens More powerfully instructs the body's immune system to attack infected cells, especially at relatively low therapeutic concentrations There continues to be a need for improved compositions that can Although this may be a potent protective mechanism against cancer and disease, tumor microenvironments, such as PD-1 expression, This is because they may be hindered by immunosuppressive mechanisms in the environment. Co-inhibitory molecules expressed by tumor cells, immune cells, and stromal cells mediate T cell responses to cancer cells. Therefore, there is a need for potent PD-1 binding molecules. In particular, they have desirable binding kinetic profiles and are highly selective for PD-1 / PD-L antagonizing the PD-1 / PD-L1 axis by blocking PD-1 interactions (which may contribute to cancer or other diseases and conditions), There continues to be a need for potent PD-1 binding molecules. The present invention provides these and other Target the following goals: [Prior art documents] [Patent documents]

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Immunol 2:261‐268 [Non-Patent Document 31] Dong, H. (2003) “B7‐H1 Pathway And Its Role In The Evasion Of Tumor Immunity,” J. Mol. Med. 81:281‐287 [Non-Patent Document 32] Subudhi, SK et al. (2005) “The Balance Of Immune Responses: Costimulation Verse Coinhibition,” J. Molec. Med. 83:193‐202 [Non-Patent Document 33] Freeman, GJ et al. (2000) “Engagement Of The PD‐1 Immunoinhibitory Receptor By A Novel B7 Family Member Leads To Negative Regulation Of Lymphocyte Activation,” J. Exp. Med. 192:1‐9 [Non-Patent Document 34] Carter, L. et al. (2002) “PD‐1:PD‐L Inhibitory Pathway Affects Both CD4(+) and CD8(+) T‐cells And Is Overcome By IL‐2,” Eur. J. Immunol. 32(3):634‐643 [Non-Patent Document 35] Berger, R. et al. (2008) “Phase I Safety And Pharmacokinetic Study Of CT-011, A Humanized Antibody Interacting With PD-1, In Patients With Advanced Hematologic Malignancies,” Clin. Cancer Res. 14(10):3044-3051 Summary of the Invention [Means for solving the problem]

[0018] The present invention provides selected anti-PD-1 antibodies capable of binding to both cynomolgus monkey PD-1 and human PD-1. D-1 antibodies: PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, P D-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD- 1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mA b PD-1 binding molecules comprising the PD-1 binding domain of 14 or PD-1 mAb 15 The present invention is particularly directed to humanized or chimeric versions of such antibodies. or PD-1-binding fragments of such anti-PD-1 antibodies (particularly immunoconjugates, diabodies, etc.) The present invention particularly relates to PD-1 binding molecules comprising: Targeting epitopes of molecules involved in regulating immune checkpoints present on the surface of immune cells The present invention also relates to such PD-1 binding molecules capable of binding to PD-1. or methods of using such PD-1 binding molecules for stimulation of an immune response. The invention also provides a method for identifying one or more PD-1 binding domains of a selected anti-PD-1 antibody as described above. The PD-1 binding molecule comprising the PD-1 receptor is administered to one or more antibodies effective in stimulating an immune response. in combination with additional molecules and / or one or more additional molecules that specifically bind to the cancer antigen. The present invention relates to combination therapy in which the compound is administered in combination with an additional molecule.

[0019] In particular, the present invention provides three heavy chain CDR domains, i.e., CDR H 1. CDR H 2 and CD R H 3 and three light chain CDR domains, i.e., CDR L 1. CDR L 2 and CDR L 3 and equipped The present invention provides an anti-human PD-1 binding molecule comprising: (A)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 1, each having the amino acid sequence: SEQ ID NO: 7 1, having SEQ ID NO: 72 and SEQ ID NO: 73; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 1, each having the amino acid sequence: SEQ ID NO: 7 6, having SEQ ID NO: 77 and SEQ ID NO: 78; or (B)(1) The above CDR H 1 domain, above CDR H2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 2, each having the amino acid sequence: SEQ ID NO: 8 5, having SEQ ID NO: 86 and SEQ ID NO: 87; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 2, each having the amino acid sequence: SEQ ID NO: 9 0, SEQ ID NO: 91 and SEQ ID NO: 92; or (C)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 3 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 9. 9, having SEQ ID NO: 100 and SEQ ID NO: 101; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM The CDRs are the light chain CDRs of PD-1 mAb 3, each having the amino acid sequence: SEQ ID NO: 1. 04, having SEQ ID NO: 105 and SEQ ID NO: 106; or (D)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 4 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 1. 09, having SEQ ID NO: 110 and SEQ ID NO: 111; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM The CDRs are the light chain CDRs of PD-1 mAb 4, each having the amino acid sequence: SEQ ID NO: 1. 14, having SEQ ID NO: 115 and SEQ ID NO: 116; or (E)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 5 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 1. 19, having SEQ ID NO: 120 and SEQ ID NO: 121; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM The CDRs are the light chain CDRs of PD-1 mAb 5, each having the amino acid sequence: SEQ ID NO: 1. 24, having SEQ ID NO: 125 and SEQ ID NO: 126; or (F)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 6 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 1. 29, having SEQ ID NO: 130 and SEQ ID NO: 131; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM The CDRs are the light chain CDRs of PD-1 mAb 6, each having the amino acid sequence: SEQ ID NO: 1 34, having SEQ ID NO: 135 and SEQ ID NO: 136; or (G)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 7 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 1. 39, having SEQ ID NO: 140 and SEQ ID NO: 141; (2) The above CDR L 1 domain, above CDRL 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 7, each having the amino acid sequence: SEQ ID NO: 1 44, having SEQ ID NO: 145 and SEQ ID NO: 146; or (H)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 8 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 1. 61, having SEQ ID NO: 162 and SEQ ID NO: 163; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 8, each having the amino acid sequence: SEQ ID NO: 1 66, SEQ ID NO: 167 and SEQ ID NO: 168; or (I)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM The heavy chain CDRs of PD-1 mAb 9 are shown in Table 1, each with the amino acid sequence: SEQ ID NO: 1. 71, having SEQ ID NO: 172 and SEQ ID NO: 173; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 9, each having the amino acid sequence: SEQ ID NO: 1 76, SEQ ID NO: 177 and SEQ ID NO: 178; or (J)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 10, with the respective amino acid sequences: SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO: 194; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 10, with the respective amino acid sequences: SEQ ID NO: 197, SEQ ID NO: 198 and SEQ ID NO: 199; or (K)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 11, with the respective amino acid sequences: SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 11, with the respective amino acid sequences: SEQ ID NO: 207, SEQ ID NO: 208 and SEQ ID NO: 209; or (L)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 12, with the respective amino acid sequences: SEQ ID NO: 212, SEQ ID NO: 213 and SEQ ID NO: 214; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 12, with the respective amino acid sequences: SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; or (M)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 13, with the respective amino acid sequences: SEQ ID NO: 222, SEQ ID NO: 223 and SEQ ID NO: 224; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 13, with the respective amino acid sequences: SEQ ID NO: 227, SEQ ID NO: 228 and SEQ ID NO: 229; or (N)(1) Above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 14, with the respective amino acid sequences: SEQ ID NO: 232, SEQ ID NO: 233 and SEQ ID NO: 234; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 14, with the respective amino acid sequences: SEQ ID NO: 237, SEQ ID NO: 238 and SEQ ID NO: 239; or (O)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In are the heavy chain CDRs of PD-1 mAb 15, with the respective amino acid sequences: SEQ ID NO: 242, SEQ ID NO: 243 and SEQ ID NO: 244; (2) The above CDR L 1 domain, above CDR L2 domains and the above CDRs L 3 DOM In are the light chain CDRs of PD-1 mAb 15, with the respective amino acid sequences: SEQ ID NO: 247, SEQ ID NO: 248 and SEQ ID NO: 249; or (P)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In the figure, the heavy chain CDRs of hPD-1 mAb 7(1.2) are shown, each with the amino acid sequence : having SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In the figure, the light chain CDRs of hPD-1 mAb 7(1.2) are shown, each with the amino acid sequence : having SEQ ID NO: 157, SEQ ID NO: 145 and SEQ ID NO: 146; or (Q)(1) The above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In the figure, the heavy chain CDRs of hPD-1 mAb 7(1.3) are shown, each with the amino acid sequence : having SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In the figure, the light chain CDRs of hPD-1 mAb 7(1.3) are shown, each with the amino acid sequence : having SEQ ID NO: 157, SEQ ID NO: 158 and SEQ ID NO: 146; or (R)(1) Above CDR H 1 domain, above CDR H 2 domains and the above CDRs H 3 DOM In the figure, the heavy chain CDRs of hPD-1 mAb 9(2.2) are shown, each with the amino acid sequence : having SEQ ID NO: 183, SEQ ID NO: 172 and SEQ ID NO: 173; (2) The above CDR L 1 domain, above CDR L 2 domains and the above CDRs L 3 DOM In the figure, the light chain CDRs of hPD-1 mAb 9(2.2) are shown, each with the amino acid sequence :SEQ ID NO: 188, SEQ ID NO: 189 and SEQ ID NO: 178.

[0020] The present invention further provides that said molecule is an antibody, in particular said molecule is a chimeric antibody or a humanized antibody. The present invention relates to embodiments of all such anti-human PD-1 binding molecules.

[0021] The present invention further relates to a method for producing a heavy chain variable domain comprising the steps of: 7. The amino acid sequence of SEQ ID NO: 149, SEQ ID NO: 179, SEQ ID NO: 181 or SEQ ID NO: 250 The present invention relates to an embodiment of such an anti-human PD-1 binding molecule having a sequence.

[0022] The present invention further relates to a method for producing a light chain variable domain comprising the steps of: 1, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 184, SEQ ID NO: 186 or SEQ ID NO: 2 The present invention relates to an embodiment of such an anti-human PD-1 binding molecule having a sequence of 51 amino acids.

[0023] The present invention further provides an anti-human PD-1 binding molecule that binds to human PD-1 and a second epitope. For embodiments that are bispecific binding molecules capable of simultaneously binding the second epitope, in particular The peptides are epitopes of molecules involved in regulating immune checkpoints present on the surface of immune cells. (In particular, the second epitope is B7-H3, B7-H4, BTLA, CD 40, CD40L, CD47, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA‐4, galectin‐9, GITR, GITRL, H HLA2, ICOS, ICOSL, KIR, LAG‐3, LIGHT, MHC class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, PD-1, P D-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 or VI STA epitope, and most particularly the second epitope is CD137, CT LA-4, LAG-3, OX40, TIGIT, or TIM-3 epitopes) Concerning form.

[0024] The present invention further provides a method for the preparation of a human PD-1-binding molecule comprising administering to a mammalian subject the present invention ... and in particular, wherein said LAG-3 epitope binding site comprises: Regarding morphology: (A)(1) Amino acid sequences: SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively CDRs of the variable heavy chain of LAG-3 mAb 1, H 1 domain, CDR H 2 domains and and CDR H 3 domains; and (2) Amino acid sequences: SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively CDRs of the variable light chain of LAG-3 mAb 1, L 1 domain, CDR L 2 domains and and CDR L 3 domains; or (B)(1) Amino acid sequences: SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively CDRs of the variable heavy chain of hLAG-3 mAb 1 VH1, H 1 domain, CDRH 2 Domains and CDRs H 3 domains; and (2) Amino acid sequences: SEQ ID NO: 55, SEQ ID NO: 47, and SEQ ID NO: 48, respectively CDRs of the variable light chain of hLAG-3 mAb 1 VL4, L 1 domain, CDR L 2 Domains and CDRs L 3 domains; or (C) (1) Amino acid sequences: SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively CDRs of the variable heavy chain of LAG-3 mAb 6, H 1 domain, CDR H 2 domains and and CDR H 3 domains; and (2) Amino acid sequences: SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively CDRs of the variable light chain of LAG-3 mAb 6, L 1 domain, CDR L 2 domains and and CDR L 3 domains; or (D) (1) hLA having SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively. CDRs of the variable heavy chain of G-3 mAb 6 VH1 H 1 domain, CDR H 2 domain and C DR H 3 domains; and (2) Amino acid sequences: SEQ ID NO: 298, SEQ ID NO: 62, and SEQ ID NO: 63, respectively The CDRs of the variable light chain of LAG-3 mAb 6 L 1 domain, CDR L 2 Domains and CDR L 3 domains.

[0025] The present invention further provides that the molecule is a diabody, and in particular that the diabody comprises two or a covalently bonded complex containing three, four, or five polypeptide chains. The present invention further relates to an embodiment of a human PD-1 binding molecule, wherein the molecule is a trivalent binding molecule. and in particular, the trivalent binding molecule comprises three, four, five, six or more polypeptide chains. The present invention relates to embodiments of such anti-human PD-1 binding molecules that are covalent conjugates comprising The disclosure also provides embodiments of such anti-human PD-1 binding molecules, wherein the molecule comprises an Fc region. The present invention further relates to a method for producing such a molecule comprising administering to a subject a subject the use of an albumin binding domain, in particular a deimmunized albumin. The present disclosure relates to embodiments of such anti-human PD-1 binding molecules comprising the binding domains:

[0026] The present invention further provides a method for producing a polypeptide comprising the molecule of claim 1, wherein the molecule comprises an Fc region, and the Fc region is a variant Fc region. the variant Fc region reduces the affinity of the variant Fc region for FcγR; and and / or one or more amino acid modifications that increase serum half-life, and more particularly , the above modifications are as follows: (1)L234A;L235A; (2) L234A and L235A; (3) M252Y; M252Y and S254T; (4) M252Y and T256E; (5) M252Y, S254T, and T256E; or (6) K288D and H435K; and wherein the numbering is All such anti-human PDs are numbered in the EU index as in bat -1 binding molecule embodiment.

[0027] The present invention further provides a method for inhibiting T cell-mediated immune responses using any of the above-described PD-1 binding molecules. The present invention further relates to embodiments that stimulate the answer. It is intended to be used in the treatment of diseases or conditions associated with a suppressed immune system, particularly cancer or infectious diseases. Regarding the embodiment.

[0028] The present invention particularly relates to such uses in the treatment or diagnosis or prognosis of cancer, said cancer being :Adrenal gland cancer;AIDS-related cancer;Alveolar soft part sarcoma;Astrocytic tumor;Bladder cancer;Bone cancer;Brain and spinal cord cancer ;Metastatic brain tumor;Breast cancer;Carotid bulb tumor;Cervical cancer;Chondrosarcoma;Chordoma;Chromogenic renal cell carcinoma; Clear cell carcinoma; colon cancer; colorectal cancer; cutaneous benign fibrous histiocytoma; desmoplastic small round cell tumor; Ependymoma; Ewing's tumor; extraskeletal myxoid chondrosarcoma; fibroplasia imperfecta ossificans; fibrous dysplasia Gallbladder or bile duct cancer; gastrointestinal cancer; gestational trophoblastic disease; germ cell tumors; head and neck cancer; hepatocellular carcinoma Cancer; Islet cell tumor; Kaposi's sarcoma; Kidney cancer, leukemia, lipoma / benign lipoma, liposarcoma / Malignant rhabdomyosarcoma, liver cancer; lymphoma; lung cancer; medulloblastoma; melanoma; meningioma; multiple endocrine tumors Endocrine tumors; multiple myeloma; myelodysplastic syndrome; neuroblastoma; neuroendocrine tumors; ovarian cancer; pancreatic Pancreatic cancer; papillary thyroid cancer; parathyroid tumor; childhood cancer; peripheral nerve sheath tumor; pheochromocytoma; pituitary tumor prostate cancer; posterior uveal melanoma; rare hematological disorders; renal metastatic cancer; rhabdoid tumor ;rhabdomyosarcoma;sarcoma;skin cancer;soft tissue sarcoma;squamous cell carcinoma;gastric cancer;synovial sarcoma;testicular cancer; of cancer cells selected from the group consisting of thymic carcinoma; thymoma; metastatic thyroid cancer; and uterine cancer cells. Characterized by existence.

[0029] The present invention particularly relates to such uses in the treatment or diagnosis or prognosis of cancer, said cancer being :Colorectal cancer;Hepatocellular carcinoma;Glioma;Kidney cancer;Breast cancer;Multiple myeloma;Bladder cancer;Neuroblastoma sarcoma; non-Hodgkin's lymphoma; non-small cell lung cancer; ovarian cancer: Pancreatic cancer; rectal cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); acute B-lymphoma B-ALL; chronic lymphocytic leukemia (CLL); hairy cell leukemia ( HCL); blastic plasmacytoid dendritic cell neoplasm (BPDCN); mantle cell leukemia (MCL) Non-Hodgkin's lymphomas, including small lymphocytic lymphoma (SLL) and small lymphocytic lymphoma (SLL) lymphomas (NHL); Hodgkin's lymphoma; systemic mastocytosis; or Burkitt lymphoma be.

[0030] The present invention further provides a method for detecting and labeling any of the above-described PD-1 binding molecules, and administering the method to a subject in need thereof. The present invention relates to an embodiment in which the present invention is used for detecting [Brief explanation of the drawings]

[0031] [Figure 1] Figure 1 is a schematic diagram of a representative covalent diabody having two epitope-binding sites composed of two polypeptide chains, each with an E-coil or K-coil Heterodimer-Promoting Domain. Cysteine ​​residues may be present in the linker and / or Heterodimer-Promoting Domain, as shown in Figure 3B. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [Figure 2] 2 is a schematic diagram of a representative covalently linked diabody molecule having two epitope-binding sites consisting of two polypeptide chains, each with a CH2 and CH3 domain, such that the linked chains form all or part of an Fc region. VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. [Figure 3A-3C]Figures 3A-3C are schematic diagrams of a representative tetravalent diabody having four epitope-binding sites composed of two pairs of polypeptide chains (i.e., four total polypeptide chains). One polypeptide in each pair has a CH2 and CH3 domain, such that the linked chains form all or part of an Fc region. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. The two pairs of polypeptide chains can be identical. In embodiments where the VL and VH domains recognize different epitopes (as shown in Figures 3A-3C), the resulting molecule has four epitope-binding sites, is bispecific, and is bivalent for each epitope it binds. In embodiments where the VL and VH domains recognize the same epitope (e.g., using the same VL domain CDRs and the same VH domain CDRs for both chains), the resulting molecule has four epitope-binding sites, is monospecific, and is tetravalent for a single epitope. Alternatively, the two pairs of polypeptides can be different. In embodiments in which the VL and VH domains of each pair of polypeptides recognize different epitopes (as shown in Figure 3C), the resulting molecule has four epitope-binding sites, is tetraspecific, and is monovalent for each epitope it binds. Figure 3A shows an Fc diabody containing peptide Heterodimer-Promoting Domains that contain cysteine ​​residues. Figure 3B shows an Fc Region-containing diabody that contains E-coil and K-coil Heterodimer-Promoting Domains that contain cysteine ​​residues and a linker (with an optional cysteine ​​residue). Figure 3C shows an Fc Region-containing diabody containing antibody CH1 and CL Domains. [Figure 4A-4B] 4A and 4B are schematic diagrams of representative covalently linked diabody molecules having two epitope-binding sites composed of three polypeptide chains. Two of the polypeptide chains have CH2 and CH3 Domains, such that the combined chains form all or part of an Fc Region. The polypeptide chain comprising the VL and VH Domains further comprises a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [Figure 5]Figure 5 is a schematic diagram of a representative covalent diabody having four epitope-binding sites composed of five polypeptide chains. Two of the polypeptide chains have CH2 and CH3 Domains such that the combined chains form an Fc region containing all or part of the Fc region. The polypeptide chains comprising the VL and VH Domains further comprise a Heterodimer-Promoting Domain. VL and VH Domains that recognize the same epitope are shown using the same shading or fill pattern. [Figures 6A-6F] Figures 6A-6F are schematic diagrams of representative Fc region-containing trivalent binding molecules with three epitope-binding sites. Figures 6A and 6B each show the domains of a trivalent binding molecule comprising two diabody-type binding domains and a Fab-type binding domain, with different domain orientations, in which the diabody-type binding domains are N-terminal or C-terminal to the Fc region. The molecules in Figures 6A and 6B comprise four chains. Figures 6C and 6D each show the domains of a trivalent binding molecule comprising two diabody-type binding domains N-terminal to the Fc region and a Fab-type binding domain or an scFv-type binding domain in which the light and heavy chains are linked via a polypeptide spacer. The trivalent binding molecules in Figures 6E and 6F each show the domains of a trivalent binding molecule comprising two diabody-type binding domains C-terminal to the Fc region and linked Fab-type binding domains or an scFv-type binding domain in which the diabody-type binding domains reside. The trivalent binding molecules in Figures 6C-6F each comprise three chains. VL and VH domains that recognize the same epitope are shown using the same shading or filling pattern. [Figures 7A-7D]Figures 7A-7D show the binding of anti-PD-1 antibodies PD-1 mAb 1-15 to human PD-1. Binding curves for binding to shPD-1-His are shown in Figure 7A (PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 4, and PD-1 mAb 9), Figure 7B (PD-1 mAb 5, PD-1 mAb 6, and PD-1 mAb 7), and Figure 7C (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15). Binding curves for binding to shPD-1-human Fc are shown in Figure 7D (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15). [Figures 8A-8C] Figures 8A-8C show the binding of anti-PD-1 antibodies PD-1 mAb 1-15 to cynomolgus monkey PD-1. Binding curves for binding to scynoPD-1-hFc are shown in Figure 8A (PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7), Figure 8B (PD-1 mAb 9), and Figure 8C (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, and PD-1 mAb 15). [Figures 9A-9D]Figures 9A-9D show the ability of anti-PD-1 antibodies PD-1 mAbs 1-15 to block the binding of human PD-L1 to human PD-1. The inhibition curves are shown in Figure 9A (PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 15, and PD-1 mAb A), Figure 9B (PD-1 mAb 4), Figure 9C (PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, and PD-1 mAb A), and Figure 9D (PD-1 mAb 3, PD-1 mAb 8, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14, PD-1 mAb 15, and PD-1 mAb A). [Figures 10A-10B] Figures 10A-10B show the tissue specificity of the anti-human PD-1 antibody PD-1 mAb 7. Figure 10A shows histological staining of normal colon (panels i and vii), liver (panels ii and viii), lung (panels iii and ix), pancreas (panels iv and x), kidney (panels v and xi), and heart (panels vi and xii) tissues. Figure 10A, panels i-vi, show the results of tissues incubated with labeled PD-1 mAb 7 (0.313 μg / mL). Figure 10A, panels vii-xii, show the results of tissues incubated with labeled isotype control mAb (0.314 μg / mL). Figure 10B shows histological staining of skin (panels i and iv), tonsil (panels ii and v), and PD-1-expressing NSO cells (panels iii and vi). Figure 10B, panels i-iii show the results for tissues incubated with labeled PD-1 mAb 7 (0.313 μg / mL). [Figure 11] Figure 11 shows the binding profiles of the humanized anti-human PD-1 antibodies hPD-1 mAb 2, hPD-1 mAb 7(1.1), hPD-1 mAb 7(1.2), hPD-1 mAb 9(1.1), and the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B, which have IgG1 (AA) or IgG4 (P) for binding to cell-surface PD-1. [Figures 12A-12B]Figures 12A-12B show the ability of the humanized anti-PD antibodies hPD-1 mAb 2, hPD-1 mAb 7(1.1), hPD-1 mAb 7(1.2), hPD-1 mAb 9(1.1), and the reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B with IgG1 (AA) or IgG4 (P) to block the binding of soluble human PD-L1 (Figure 12A) and soluble human PD-L2 (Figure 12B) to cell-surface human PD-1. [Figure 13] Figure 13 shows the ability of humanized anti-PD antibodies hPD-1 mAb 2, hPD-1 mAb 7(1.1), hPD-1 mAb 7(1.2), hPD-1 mAb 9(1.1), and reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B with IgG1 (AA) or IgG4 (P) to antagonize the PD-1 / PD-L1 axis by blocking the PD-1 / PD-L1 interaction and preventing down-regulation of T cell responses in a Jurkat-luc-NFAT / CHO-PD-L1 luciferase reporter assay. [Figure 14] Figure 14 shows that PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 can stimulate cytokine production to levels comparable to or even higher than the reference anti-PD-1 antibodies (PD-1 mAb A and PD-1 mAb B), and that treatment with PD-1 mAb 2, PD-1 mAb 7, PD-1 mAb 9, and PD-1 mAb 15 in combination with LAG-3 mAb 1 provided the greatest enhancement of cytokine release. IFNγ secretion profiles from Staphylococcal enterotoxin B (SEB)-stimulated PBMCs treated with anti-PD-1 and anti-LAG-3 antibodies alone and in combination. [Figures 15A-15B]Figures 15A-15B show the ability of humanized anti-PD antibodies hPD-1 mAb 2, hPD-1 mAb 7 (1.2), hPD-1 mAb 9 (1.1), and reference anti-PD-1 antibodies PD-1 mAb A and PD-1 mAb B with IgG1 (AA) or IgG4 (P) to stimulate cytokine production. Secretion profiles of IFNγ (Figure 15A) and TNFα (Figure 15B) from SEB-stimulated PBMCs treated with the anti-PD-1 antibodies. [Figures 16A-16B] Figures 16A-16B show that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART D, DART E, DART F, DART G, and DART H can stimulate cytokine production to levels comparable to or even higher than those observed upon administration of the combination of an anti-PD-1 mAb and an anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A), and that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART D, DART E, DART F, and DART G provided the greatest enhancement of cytokine release. IFNγ secretion profiles of PBMCs stimulated with a low concentration of SEB (0.2 ng / mL) treated with the PD-1 x LAG-3 bispecific diabody, or the anti-PD-1 and anti-LAG-3 antibodies alone and in combination, are plotted. Results using PBMCs from two representative donors are shown in Figures 16A and 16B. [Figures 17A-17B] Figures 17A-17B show that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART B, and DART C can stimulate cytokine production to levels comparable to or even higher than those observed upon administration of the combination of anti-PD-1 mAb and anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A). IFNγ secretion profiles of PBMCs from two representative donors stimulated with a high concentration of SEB (85 ng / mL) and treated with the PD-1 x LAG-3 bispecific diabody, or the anti-PD-1 and anti-LAG-3 antibodies alone and in combination, are plotted. Results using PBMCs from two representative donors are shown in Figures 17A and 17B. [Figures 18A-18B] Figures 18A-18B show that the PD-1 x LAG-3 bispecific diabody constructs DART A, DART B, and DART C can stimulate cytokine production to levels comparable to or even higher than those observed upon administration of the combination of anti-PD-1 mAb and anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A). IFNγ secretion profiles of PBMCs from two representative donors stimulated with a moderate concentration of SEB (0.5 ng / mL) treated with the PD-1 x LAG-3 bispecific diabody, or the anti-PD-1 and anti-LAG-3 antibodies alone and in combination, are plotted. Results using PBMCs from two representative donors are shown in Figures 18A and 18B. [Figure 19] Figure 19 shows that the PD-1 x LAG-3 bispecific diabody constructs DART D and DART H can stimulate cytokine production to levels comparable to or even higher than those observed upon administration of the anti-PD-1 mAb + anti-LAG-3 mAb combination (PD-1 mAb A + LAG-3 mAb A), with DART D providing the greatest enhancement of cytokine release. IL-2 secretion profiles of PBMCs from a representative donor stimulated with a high concentration of SEB (85 ng / mL) treated with the PD-1 x LAG-3 bispecific diabody, or the anti-PD-1 and anti-LAG-3 antibodies alone and in combination, are plotted. [Figure 20]Figure 20 shows that the PD-1 x LAG-3 bispecific diabody constructs DART B and DART I can stimulate cytokine production to levels comparable to or even higher than those observed upon administration of anti-PD-1 mAb + anti-LAG-3 mAb combinations (PD-1 mAb A + LAG-3 mAb A, hPD-1 mAb 7 (1.2) + hLAG-3 mAb 1 (1.4), hPD-1 mAb 7 (1.2) + hLAG-3 mAb 6 (1.1)). IFNγ secretion profiles of PBMCs from a representative donor stimulated with a moderate concentration of SEB (0.5 ng / mL) treated with the PD-1 x LAG-3 bispecific diabody, or the anti-PD-1 and anti-LAG-3 antibodies alone and in combination, are plotted. [Figures 21A-21D] Figures 21A-21D show that the PD-1 x LAG-3 bispecific diabody DART I can stimulate cytokine production to levels comparable to or even higher than those observed upon administration of the combination of anti-PD-1 mAb and anti-LAG-3 mAb (PD-1 mAb A + LAG-3 mAb A). The IFNγ (Figures 21A and 21C) and IL-2 (Figures 21B and 21D) secretion profiles of tetanus toxoid (5 μg / mL)-stimulated CD4+ memory cells from two representative donors treated with the PD-1 x LAG-3 bispecific diabody DART-I, combined anti-PD-1 and anti-LAG-3 antibodies, or an isotype control are plotted. Day 7 results using CD4+ memory T cells from two representative donors are shown in Figures 21A-B and 21C-D. [Figure 22] Figure 22 shows that the pharmacokinetics of the PD-1 x LAG-3 bispecific molecule, DART I, are comparable to those of the anti-PD-1 antibody, PD-1 mAb A IgG4 (P), in cynomolgus monkeys. The cell line shows the mean serum concentrations of DART I (solid line) and PD-1 mAb A (dashed line). Individual values ​​for male (black) and female (white) monkeys are plotted for DART I (triangles) and PD-1 mAb A (circles). [Figures 23A-23C]Figures 23A-23C show serum antibody concentrations and percentages of bound PD-1 on the surface of CD4+ or CD8+ T cells over time in animals after treatment with different anti-PD-1 antibodies. The percentage of bound PD-1 on the surface of CD4+ or CD8+ T cells after anti-PD-1 mAb treatment is plotted on the right Y-axis, with symbols representing the % of bound PD-1 on T cells for individual animals and the dashed line representing the mean. Serum mAb concentrations are plotted on the left Y-axis, with symbols representing serum levels for individual animals and the solid line representing a nonlinear fit of the data. Each panel represents data for animals (n=1 / sex / group) administered 10 mg / kg of hPD-1 mAb 7(1.2) IgG4(P) (Figure 23A), PD-1 mAb A IgG4(P) (Figure 23B), or PD-1 mAb B IgG4(P) (Figure 23B) by IV infusion on day 1. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides selected anti-PD-1 antibodies capable of binding to both cynomolgus monkey PD-1 and human PD-1. D-1 antibodies: PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3, P D-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, PD- 1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mA b PD-1 binding molecules comprising the PD-1 binding domain of 14 or PD-1 mAb 15 The present invention is particularly directed to humanized or chimeric versions of such antibodies. or PD-1-binding fragments of such anti-PD-1 antibodies (particularly immunoconjugates, diabodies, etc.) The present invention particularly relates to PD-1 binding molecules comprising: Targeting epitopes of molecules involved in regulating immune checkpoints present on the surface of immune cells The present invention also relates to such PD-1 binding molecules capable of binding to PD-1. or methods of using such PD-1 binding molecules for stimulation of an immune response. The invention also provides a method for identifying one or more PD-1 binding domains of a selected anti-PD-1 antibody as described above. The PD-1 binding molecule comprising the PD-1 receptor is administered to one or more antibodies effective in stimulating an immune response. in combination with additional molecules and / or one or more additional molecules that specifically bind to the cancer antigen. The present invention relates to combination therapy in which the compound is administered in combination with an additional molecule.

[0033] I. Antibodies and Their Binding Domains The antibody of the present invention comprises at least one nucleotide sequence located in the variable domain of the immunoglobulin molecule. Antigen recognition sites allow specific targeting of carbohydrates, polynucleotides, lipids, polypeptides, etc. As used herein, the term "Antibody" refers to monoclonal antibodies, polyclonal antibodies, and Specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, lactic Monoclonal antibody, single-chain Fv (scFv), single-chain antibody, Fab fragment, F(ab') fragment, disulfide peptide-binding bispecific Fv (sdFv), intracellular antibodies, and epitopes of any of the above. In particular, antibodies include immunoglobulin molecules and antibody-binding fragments of immunoglobulin molecules. Immunoglobulin molecules include immunologically active fragments, i.e., molecules that contain the antigen-binding site. Any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses In addition to their known uses in diagnostics, antibodies can also be used in therapeutic applications. Antibodies have been shown to be useful as therapeutic agents. They can bind immunospecifically to protein molecules because they contain specific domains on such molecules. This is because there are multiple forms, segments, or forms ("epitopes") of the same molecule. The pitope-bearing molecule can have immunological activity, which results in the production of antibodies in animals. These molecules are called "antigens." There has been a resurgence of interest in the therapeutic potential of antibodies, which are biotechnology-derived drugs. It is one of the leading classes of bodies In The Treatment Of Infectious Diseases,” Singapore Med. J. 50(7):663-66 6) Over 200 antibody-based drugs are approved for use or in development.

[0034] The term "monoclonal antibody" refers to a homogeneous antibody. The monoclonal antibody refers to a group of antibodies that selectively bind to an antigen (naturally occurring antibodies). Monoclonal antibodies are highly specific and are composed of amino acids (which occur naturally or do not occur naturally). The term "monoclonal antibody" refers to an antibody directed against a single epitope (or antigenic site). "Antibodies" include not only complete and full-length monoclonal antibodies, but also fragments thereof. fragments (Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv), mutants thereof, Fusion proteins comprising antibody moieties, humanized monoclonal antibodies, chimeric monoclonal antibodies and immunoglobulins with antigen recognition sites having the required specificity and ability to bind to antigens. The source of the antigen or the method of making the antigen (e.g., (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals) This term refers to immunoglobulins as a whole and is not intended to be a definition of "antibody." The above-mentioned fragments and the like are included. Methods for producing monoclonal antibodies are known in the art. One method that may be adopted is the methodology of Kohler, G. et al. (1975) “Continuous Cultures of Fused Cells Secreting Antibody Of Predefined Specificity,” Nature 256:495-497 Typically, monoclonal antibodies are derived from mouse, rat, or rabbit. The antibodies are produced by inducing an animal to an immunogenic amount of cells containing the desired epitope. Immunogens are produced by immunizing with cells, cell extracts, or protein preparations. It can be a cell, a cultured cell line, a cancer cell, a protein, a peptide, a nucleic acid or a tissue. The cells that may be used for immunization include, but are not limited to, those that can be used as immunogens. The cells may be cultured for a period of time (e.g., at least 24 hours) before use. It may be used as an immunogen alone or in combination with a non-denaturing adjuvant such as Ribi. (Jennings, VM (1995) “Review of Selected Adjuvants Used in Antibody Producti on,” ILAR J. 37(3):119-125). Generally, cells are used as immunogens in their entirety. Intact cells must be maintained in a healthy and preferably viable state. allowing the immunized animal to detect the antigen better than the immunized cells. The use of denaturing or strong adjuvants, such as Freund's adjuvant, can This may cause rupture and is therefore not recommended. The immunogen should be administered once every two weeks or once a week. It may be administered multiple times at periodic intervals, such as once, or may be administered live in the animal (e.g., during tissue engineering). Alternatively, the administration of a steroid to a desired pathogenic epitope may be used to maintain viability. Existing monoclonal antibodies and other equivalent antibodies with immunospecificity are known in the art. In one embodiment, such antibodies can be recombinantly sequenced and produced by any of the following means: The polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest is maintained in the vector in the host cell and subsequently The host cells can then be expanded and frozen for future use. The nucleotide sequences may be used in the monospecific or multispecific (e.g., bispecific, trispecific) Monospecific and tetraspecific) molecules, as well as affinity-optimized, chimeric, humanized and / or Genetic engineering to improve antibody affinity or other characteristics by producing caninized antibodies. The general principle for humanizing an antibody is to It involves replacing the non-human remainder of the antibody with human antibody sequences while retaining the base sequence.

[0035] A natural antibody (such as an IgG antibody) consists of two light chains complexed with two heavy chains. Each light chain Each heavy chain contains a variable domain (VL) and a constant domain (CL). a variable heart muscle (VH), three constant domains (CH1, CH2, and CH3), and a CH1 domain and CH2 domains. The basic structural unit of immunoglobulins (e.g., IgG) is usually a glycoprotein of approximately 150,000 Da. It is a trimer with a light chain and two heavy chains expressed as a protein. The N-terminal ("N-terminal") portion contains approximately 100-110 variable domains that play a major role in antigen recognition. The carboxy-terminal ("C-terminal") portion of each chain defines a constant region, and the light chains contain a single The heavy chain usually has three constant domains and one hinge domain. Therefore, the structure of the light chain of an IgG molecule is n-VL-CL-c, and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c, where H is the hinge domain, (n and c represent the N-terminus and C-terminus of the polypeptide, respectively). The antibody contains multiple complementarity determining regions (CDRs) that contain residues in contact with the epitope, and It consists of a non-CDR segment called a framework segment (FR), and The work segment generally maintains the structure of the CDR loop and determines the position of the CDR. These contacts are made possible by the presence of specific framework residues that also contact the antigen. Thus, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR 2-FR3-CDR3-FR4-c. The first, second and third CDRs of the antibody light chain Polypeptides that are (or can function as) the first, second, and third CDRs are referred to herein as are CDRs L 1 domain, CDR L 2 domains and CDRs L These are called the three domains. Similarly, the first, second and third CDRs of an antibody heavy chain (or the first, second and third CDRs) Each CDR R is a polypeptide that can function as a CDR R. H 1 domain, CD R H 2 domains and CDRs H 3 domains. L 1 domain, CDR L 2 domains, CDR L 3 domains, CDR H 1 domain, CDR H 2 domains and CDRs H The term 3-domain refers to the fact that when incorporated into a protein, the protein Chain and heavy chain or diabody or single chain binding molecule (e.g., scFv, BiTe, etc.) Whether the protein is an antibody having The present invention is directed to polypeptides that enable a protein to bind to a specific epitope. As used herein, the term "epitope-binding fragment" refers to a fragment of a human epitope. A "binding fragment" is a fragment of an antibody that can immunospecifically bind to an epitope. The term "epitope-binding site" refers to a fragment of a " refers to that portion of a molecule that contains an epitope-binding fragment that plays a role in epitope binding. The epitope-binding site is one, two, three, four or five of the CDR domains of such an antibody. may contain all five or all six of these epitopes and be capable of immunospecifically binding to such epitopes. The immunospecificity, affinity or affinity of such antibodies to an epitope different from that of the antibody Preferably, however, the epitope-binding fragments are such that It contains all six of the CDR domains of an antibody. The fragments may be single polypeptide chains (e.g., scFv) or each may be an amino acid. Two or more polypeptide chains having a mono- and carboxy-terminus (e.g., diabodies, F ab fragment, F(ab')2 fragment, etc.

[0036] The present invention particularly relates to single-chain variable domain fragments ("scFv") of the anti-PD-1 antibodies of the invention, and The single-chain variable domain fragments include short binding peptides and multispecific binding molecules comprising the same. These are produced by combining light chain and / or variable domains using peptides. Bird et al. al. (1988) ("Single-Chain Antigen-Binding Proteins," Science 242:423-426) Approximately 3.5 between the carboxy terminus of one variable domain and the amino terminus of the other variable domain Examples of linking peptides that fill the nm are given. Linkers of other sequences may also be designed and used. Bird et al. (1988), "Single-Chain Antigen-Binding Proteins," Science 242 :423-426). The linker may be used to attach a further agent or attachment to a solid support. It can be modified for function. Single chain variants can be produced recombinantly or synthetically. scF For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, Suitable plasmids containing polynucleotides encoding scFvs can be introduced into yeast, plants, or other organisms. into a suitable host cell, such as a eukaryotic cell, such as an insect or mammalian cell, or a prokaryotic cell, such as E. coli. The polynucleotide encoding the scFv of interest can be introduced into the The resulting scFv can be prepared by conventional procedures such as ligation of the corresponding scFv. It can be isolated using standard protein purification techniques known in the art.

[0037] The present invention particularly relates to humanized variants of the anti-PD-1 antibodies of the present invention, and multispecific antibodies comprising the same. The term "humanized" antibody refers to a chimeric molecule. , typically prepared using recombinant techniques, and containing antigen-binding sites of immunoglobulins from non-human species and the remaining immunoglobulin structures of molecules based on the structure and / or sequence of human immunoglobulins. The anti-human PD-1 antibody of the present invention refers to a chimeric molecule having the antibody PD-1 mAb. b 1, PD-1 mAb 2, PD-1 mAb 3, PD-1 mAb 4, PD- 1 mAb 5, PD-1 mAb 6, PD-1 mAb 7, PD-1 mAb 8 , PD-1 mAb 9, PD-1 mAb 10, PD-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 mAb 14 or PD-1 mA b 15. The variable domains of such antibodies include humanized, chimeric or canine variants thereof. The polynucleotide sequence may be used to generate the derivative and to determine the affinity or other characteristics of the antibody. The general principle for humanizing antibodies is to The non-human remainder of the antibody is replaced with human antibody sequences while retaining the base sequence of the antigen-binding portion of the antibody. There are four general steps to humanize a monoclonal antibody: The steps are as follows: (1) Deleting the light and heavy chain variable domains of the starting antibody; (2) determining the nucleotide and predicted amino acid sequences of the humanized antibody; or designing a caninized antibody, i.e., antibody fragments used during the humanization or caninization process. (3) determining the framework area; (4) the actual humanization or caninization method / technology; and (4) Transfection and expression of humanized antibodies. See, e.g., U.S. Patent No. 4,816,566. No. 7; U.S. Patent No. 5,807,715; U.S. Patent No. 5,866,692; and U.S. Patent No. See Permit No. 6,331,415.

[0038] The antigen binding site may consist of complete variable domains fused to constant domains or in an appropriate framework. The antibody comprises only the complementarity determining regions (CDRs) of such variable domains grafted onto the antibody region. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions. This eliminates the constant regions that are immunogenic in human individuals, but prevents the foreign The possibility of variable domains remains (LoBuglio, AF et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sc i. (USA) 86:4220-4224). Another approach is to provide human-derived constant regions. In addition, the variable domains are modified to resemble the human form as closely as possible. The focus has also been on modifying the variable domains of both the heavy and light chains. The antibody is composed of four framework regions that change in response to the antigen of interest and determine binding capacity. It is known that the nucleotide sequence contains three complementarity-determining regions (CDRs) flanked by complementary regions (FRs), The framework regions are relatively conserved within a given species and are also highly conserved for the CDRs. When preparing a non-human antibody against a specific antigen, By grafting CDRs from a non-human antibody onto FRs present in the human antibody to be modified. The variable domains can be "reshaped" or "humanized" by using this approach for a variety of antibodies. The application of chi is described in Sato, K. et al. (1993) “Reshaping A Human Antibody To Inhibit The I nterleukin 6‐Dependent Tumor Cell Growth,” Cancer Res 53:851‐856. Riechmann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323‐327 ; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilyso zyme Activity,” Science 239:1534‐1536; Kettleborough, C. A. et al. (1991) “Hu manization Of A Mouse Monoclonal Antibody By CDR‐Grafting: The Importance Of Fr amework Residues On Loop Conformation,” Protein Engineering 4:773‐3783; Maeda, H. e t al. (1991) “Construction Of Reshaped Human Antibodies With HIV‐Neutralizing Activity,” Human Antibodies Hybridoma 2:124‐134; Gorman, S. D. et al. (1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:418 1‐4185; Tempest, P.R. et al. (1991) “Reshaping A Human Monoclonal Antibody To It should be noted that there seems to be a misspelling in the original text where "amework残基" should probably be "framework residues".Inhibit Human Respiratory Syncytial Virus Infection in vivo,” Bio / Technology 9: 266-271; Co, MS et al. (1991) “Humanized Antibodies For Antiviral Therapy, ” Proc. Natl. Acad. Sci. (USA) 88:2869‐2873; Carter, P. et al. (1992) “Hum anization Of An Anti‐p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al. (1992) "Chimeric And Hum anized Antibodies With Specificity For The CD33 Antigen,” J. Immunol. 148:1149 In some embodiments, the humanized antibody comprises all CDR sequences. Save the column (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody) In other embodiments, humanized antibodies are modified antibodies that differ in sequence from the original antibody. It has one or more CDRs (1, 2, 3, 4, 5 or 6).

[0039] Many "humanized" immunoglobulins have antigen-binding sites derived from non-human immunoglobulins. d) "antibody molecules are described which contain rodent or modified rodent variable domains and , and their associated complementarity determining regions (CDRs) fused to human constant domains. This includes chimeric antibodies (see, e.g., Winter et al. (1991) “Man-made Antibodies,” Nature 349 :293-299; Lobuglio et al. (1989) “Mouse / Human Chimeric Monoclonal Antibody In M an: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989); Shaw et al. (1987) “Characterization Of A Mouse / Human Chimeric Monoclo nal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen,” J. Immunol. 1 38:4534-4538; and Brown et al. (1987) “Tumor-Specific Genetically Engineered Mur ine / Human Chimeric Monoclonal Antibody,” Cancer Res. 47:3577-3583). The references describe the use of human supporting framework regions prior to fusion with the appropriate human antibody constant domains. (FR) describes rodent CDRs (e.g., Riechm ann, L. et al. (1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323 -327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antibodies lysozyme Activity,” Science 239:1534-1536; and Jones et al. (1986) “Replacing T he Complementarity-Determining Regions In A Human Antibody With Those From A Mou (See "Se," Nature 321:522-525). Another reference is the recombinantly veneered The present invention describes rodent CDRs supported by modified rodent framework regions. See, for example, European Patent Publication No. 519,596. These "humanized" molecules are Rodent anti-human antibodies, which limit the duration and effectiveness of therapeutic applications of these portions of Scipient Antibodies are designed to minimize unwanted immunological responses to the molecule. Another method that may be used for this purpose is that of Daugherty et al. (1991) "Polymerase Chain Reaction n Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Mouse Monoclo nal Antibody Directed Against The CD18 Component Of Leukocyte Integrins,” Nucl. Acids Res. 19:2471-2476 and U.S. Pat. No. 6,180,377; U.S. Pat. No. 6,055,055 No. 4,297; U.S. Patent No. 5,997,867; and U.S. Patent No. 5,866,692 It is disclosed by

[0040] II. Fcγ receptors (FcγRs) The CH2 and CH3 domains of the two heavy chains interact to form the Fc region, and this Fc region The region is recognized by cellular Fc receptors, including but not limited to Fcγ receptors (FcγR). As used herein, the term "Fc region (Fc region)" refers to a domain that is recognized by the Fc receptor. The "C-terminal region" is used to define the C-terminal region of the IgG heavy chain. The amino acid sequence of the CH2-CH3 domain of IgG1 is (SEQ ID NO: 1): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X which are numbered by the EU index as in Kabat , X is a lysine (K) or is absent.

[0041] The amino acid sequence of an exemplary CH2-CH3 domain of human IgG2 is (SEQ ID NO:2): 231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X which are numbered by the EU index as in Kabat , X is a lysine (K) or is absent.

[0042] The amino acid sequence of an exemplary CH2-CH3 domain of human IgG3 is (SEQ ID NO:3): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESSGQPENN YNTTPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 440 447 ALHNRFTQKS LSLSPG X which are numbered by the EU index as in Kabat , X is a lysine (K) or is absent.

[0043] The amino acid sequence of an exemplary human IgG4 CH2-CH3 domain is (SEQ ID NO:4): 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X which are numbered by the EU index as in Kabat , X is a lysine (K) or is absent.

[0044] Throughout this specification, the numbering of residues in the constant region of the IgG heavy chain is based on Kabat et al., Se Quences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NH1 , MD (1991) ("Kabat," which is expressly incorporated herein by reference). The EU index numbering system is based on the EU index as in Kabat. (EU index as in Kabat) is the numbering system for human IgG1 EU antibodies. The amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are The amino acid sequences are specified by the position of the amino acid. Kabat has The amino acid consensus sequence for each subgroup is identified, and each amino acid is identified. Residue numbers are assigned and CDRs are identified as defined by Kabat. (Chothia, C. & Lesk, AM((1987) “Canonical structures for the hypervariab le regions of immunoglobulins,” J. Mol. Biol. 196:901-917 CDR H (Note that 1 begins 5 residues earlier.) The program identifies the antibody in question by referencing the conserved amino acids in the Kabat consensus sequence. To identify antibodies not included in the Kabat overview, align them with one of the sequences The above method for assigning residue numbers is standard in the art. The antibody is designed to be comparable in position to the corresponding antibody in different antibodies, including chimeric or humanized variants. For example, the amino acid at position 50 of a human antibody light chain is easily distinguished from the amino acid at position 50 of a mouse antibody light chain. It occupies the position equivalent to the 50th amino acid of

[0045] Polymorphisms can occur at a number of different positions within the antibody constant region (e.g., EU-I as in Kabat). Index numbering, including but not limited to 192nd, 193rd and 214th CH1 positions that are not included; and positions 270, 272, 312, 315, 356, and 35 have been observed in Fc positions, including but not limited to position 8, and therefore There may be slight differences between the sequences shown and those in the prior art. Polymorphic forms of Gm have been well characterized. Currently, 18 Gm allotypes are known. G1m(1,2,3,17) or G1m(a,x,f,z), G2m(23) or G 2m(n), G3m(5,6,10,11,13,14,15,16,21,24,26 ,27,28) or G3m(b1,c3,b3,b0,b3,b4,s,t,g1,c5 ,u,v,g5)(Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefra nc, G. et al., 1979, Hum. Genet.: 50, 199-211). In particular, the antibody of the present invention is Any allotype, isoallotype, or haplotype of the immunoglobulin gene and allotypes, isoallotypes or haploids of the sequences presented herein. Furthermore, depending on the expression system, the C-terminal The C-terminal amino acid residues (shown in bold above) can be removed post-translationally. The group may be any amino acid residue in the PD-1 binding molecules of the invention. Included are PD-1 binding molecules lacking the C-terminal residues of the CH3 domain. Specifically encompassed by the invention are structures containing the C-terminal lysine residue of the CH3 domain. be.

[0046] Activating and inhibitory signals are mediated by ligation of the Fc region to cellular Fc gamma receptors (FcγRs). The ability of such ligations to produce opposite functions This is due to structural differences between different FcγRs. The receptor tyrosine-based inhibitory motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs) are Two distinct domains within the cytoplasmic signaling domain are responsible for the different responses. Recruitment of distinct cytoplasmic enzymes to these structures determines the outcome of FcγR-mediated cellular responses . The ITAM-containing FcγR complex contains FcγRI, FcγRIIA, and FcγRIIIA. Human neutrophils express only FcγRIIB, whereas ITIM-containing complexes contain only FcγRIIB. The FcγRIIA gene is expressed. Clustering aggregates ITAMs with receptor-associated kinases that promote ITAM phosphorylation. ITAM phosphorylation serves as a docking site for Syk kinase. Its activation leads to activation of downstream substrates (e.g., PI3K). Cellular activation can lead to inflammation. The FcγRIIB gene is expressed on B lymphocytes, leading to the release of stimulatory mediators. Its extracellular domain is 96% identical to FcγRIIA, and in IgG complexes, it is distinguishable from FcγRIIA. The presence of ITIM in the cytoplasmic domain of FcγRIIB binds FcγRIIB in a manner that cannot be achieved by FcγRIIB. The above-mentioned inhibitory subclass of γRs is defined. Recently, the molecular basis of this inhibition has been established. Upon co-ligation with activating FcγR, the ITIM in FcγRIIB is phosphorylated and binds to polyphosphorylated FcγR. It attracts the SH2 domain of inositol phosphate 5'-phosphatase (SHIP), which , phosphoinositides released as a result of ITAM-containing FcγR-mediated tyrosine kinase activation hydrolyzes sitol, resulting in intracellular Ca ++ Therefore, FcγRI Cross-linking of IBs dampens the activating response to FcγR ligation and inhibits cellular responsiveness. Thus, B cell activation, B cell proliferation and antibody secretion are disrupted.

[0047] III. Bispecific Antibodies, Multispecific Diabodies and DART® Diabodies D The ability of an antibody to bind to an epitope of an antigen depends on the presence of VL and VH domains of the antibody and The interaction between antibody light chains and antibody heavy chains, particularly their VL and VH domains, depends on the amino acid sequence. Interaction with the H domain forms one of the two epitope binding sites of a natural antibody. Natural antibodies can only bind to one epitope species (i.e., they are monospecific). ), which can bind multiple copies of the species (i.e., exhibit bivalency or multivalency).

[0048] The binding domains of the present invention bind to an epitope in an "immunospecific" manner. When used in the context of more frequently, more rapidly, for longer periods, and / or with higher affinity than the epitope If a molecule reacts or binds to the epitope, it will be referred to as " For example, a virus can bind to a specific epitope in an immunospecific manner. Antibodies that specifically bind to other viral or non-viral epitopes may be immunospecific. binds with higher affinity, avidity, more readily, and / or for a longer period of time; It is an antibody that binds to the viral epitope. The antibody (or portion or epitope) that immunospecifically binds is specific or It is understood that the binding may or may not be preferential. "Unospecific binding" does not necessarily require an exclusive binding ( (However, it can contain) Generally, but not necessarily, references to bonds means "specific" binding. Two molecules are bound together by a specific bond. When a receptor exhibits specificity in binding to a specific ligand, it is called "physiologically specific" (physically specific). These molecules are said to be able to bind to each other in a "siospecific" manner.

[0049] The functionality of an antibody is determined by its ability to bind to two distinct antigens (or different epitopes of the same antigen). and / or by generating multispecific antibody-based molecules that can simultaneously bind to the same have a higher valency (i.e., three or more binding sites) for one epitope and / or antigen This can be enhanced by generating antibody-based molecules that

[0050] A wide range of recombinant bispecific antibodies to provide molecules with higher potency than natural antibodies Formats have been developed (e.g., WO 2008 / 003116; WO 2008 / 003116; International Publication No. 2009 / 132876; International Publication No. 2008 / 003103; International Publication No. 200 7 / 146968; WO 2009 / 018386; WO 2012 / 00 9544; WO 2013 / 070565), most of which contain additional epitopes. Binding fragments (e.g., scFv, VL, VH, etc.) are synthesized from antibody cores (IgA, IgD, IgE, Ig for fusion to or within the antibody core (IgM or IgE), or multiple epitopes To fuse binding fragments (e.g., two Fab fragments or scFvs), a linker peptide is used. An alternative format uses epitope-binding fragments (e.g., scFv, V L, VH, etc.) to form a dimerization domain, such as a CH2-CH3 domain or an alternative polypeptide. To fuse the peptide to the nucleotide sequence, a linker peptide is used (WO 2005 / 0709 66; WO 2006 / 107786A; WO 2006 / 107617A No. 2007 / 046893). Typically, such approaches result in compromises. and trade-offs. See, e.g., WO 2013 / 174873; WO 20 and WO 2010 / 136172, which are directed to the use of linkers. Therefore, it is disclosed that problems may arise in a therapeutic setting, and that the binding of two or more antigens The CL and CH1 domains are switched from their natural positions to allow for binding to the V teaches trispecific antibodies in which the L and VH domains are diversified (WO 2004 / 013666). 008 / 027236; WO 2010 / 108127). The molecules disclosed in the document trade binding specificity for the ability to bind additional antigen species. International Publication No. 2013 / 163427 and International Publication No. 2013 / 119903 are The CH2 domain is modified to contain a fusion protein adduct comprising a binding domain. This document discloses that CH2 plays a minimal role in mediating effector function. It is stated that the only role of the ATP is to inhibit the ATP synthesis. WO 2010028796; and WO 2010 / 028795 disclose Fc a recombinant VL domain in which the VL domain is replaced with additional VL and VH domains to form a trivalent binding molecule. WO 2003 / 025018 and WO 2003 / 025018 disclose antibodies against the IgG1-specific ... No. 012069 discloses recombinant diabodies in which each chain contains an scFv domain. WO 2013 / 006544 describes a method for synthesizing a polypeptide as a single polypeptide chain. After being bound to the ATP-binding domain, the heterodimeric structure is obtained by proteolysis. b molecules. Thus, the molecules disclosed in these documents have no effector function. In some cases, the ability to mediate this is traded in whole or in part for the ability to bind additional antigenic species. International Publication No. 2014 / 022540; International Publication No. 2013 / 003652; International Publication International Publication No. 2012 / 162583; International Publication No. 2012 / 156430; International Publication No. 201 1 / 086091; International Publication No. 2008 / 024188; International Publication No. 2007 / 02 4715; WO 2007 / 075270; WO 1998 / 002463 No. WO 1992 / 022583; and WO 1991 / 003493 adding additional binding domains or functional groups to the antibody or antibody portion (e.g., Adding a diabody to the light chain or adding additional VL and VH to the light and heavy chains of the antibody Adding domains or heterologous fusion proteins to each other or linking multiple Fab domains to each other). Thus, the molecules disclosed in these documents bind the native antibody structure to additional antigen species. It is exchanged for the ability to do so.

[0051] The art further provides a method for identifying peptides that can bind to two or more different epitope species (i.e., bivalent or multivalent). Such natural antibodies are particularly advantageous in that they can exhibit bispecific or multispecificity in addition to specificity. The possibility of generating diabodies distinct from antibodies has been attracting attention (e.g., Holliger et al. (1993) “'Diabodies': Small Bivalent And Bispecific Antibody Fragments,” Pr oc. Natl. Acad. Sci. (USA) 90:6444-6448; U.S. Patent No. 2004 / 0058400 (Hollinger et al.); U.S. Patent No. 2004 / 0220388; WO 02 / 027 No. 81 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. e (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both Th e Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor F or Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672; International Publication No. Issue 02 / 02781 (Mertens et al.); Olafsen, T. et al. (2004) “Covalent Di sulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeli ng For Tumor Targeting Applications,” Protein Eng. Des. Sel. 17(1):21-27; Wu, A et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fus ion Protein Is Mediated Through Variable Domain Exchange,” Protein Engineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And It s Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Bio chem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small R ecombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):5 83-588; Baeuerle, PA et al. (2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. 69(12):4941-4944).

[0052] The design of diabodies relies on antibody derivatives known as single-chain variable domain fragments (scFv). Such molecules are based on the synthesis of light and / or heavy chain variable domains using short peptide sequences. Bird et al. (1988) (“Single-Chain Antigen-B The carboxy terminus of one variable domain and the An example of a connecting peptide that bridges approximately 3.5 nm between the amino termini of one variable domain and the other is described. Linkers of other sequences have also been designed and used (Bird et al. (1988) "Single e-Chain Antigen-Binding Proteins,” Science 242:423-426). The single chain variants can be modified for additional functions such as binding to a solid support or attachment to a rigid support. For synthetic production of scFv, automated methods are available. For recombinant production of scFv, a synthesizer can be used. A suitable plasmid containing the polynucleotide is introduced into a yeast, plant, insect or mammalian The vector can be introduced into a suitable host cell, which can be a eukaryotic cell such as Bacillus subtilis or a prokaryotic cell such as E. coli. The polynucleotide encoding the scFv can be prepared by polynucleotide ligation, etc. The resulting scFv can be prepared by conventional procedures such as those described in the art. It can be isolated using protein purification techniques.

[0053] Provision of non-monospecific diabodies allows co-engagement of multiple cells expressing different epitopes. including, but not limited to, the ability to co-ligate Bispecific diabodies offer significant advantages over antibodies. Thus, bispecific diabodies are useful in therapy and immunodiagnosis. Bispecificity has a wide range of applications, including the design and characterization of diabodies in a variety of applications. This allows for greater flexibility in the design and processing of multimeric antigens, allowing for increased avidity of multimeric antigens, Cross-linking of multiple antigens and directed targeting to specific cell types based on the presence of both target antigens Diabody molecules known in the art are small (up to 50 kDa). Regarding small-sized diabodies, their high valency, low dissociation rate, and rapid release from the circulation Elimination has also shown particular use in the field of tumor imaging (Fitzgerald et al. (1997) “Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pich ia pastoris,” Protein Eng. 10:1221).

[0054] The bispecificity of diabodies allows for the co-ligation of different cells, e.g., cytotoxic T cells and tumor The diabody can then be used to crosslink cells (Staerz et al. (1985) “Hybrid Antibodies Can Target Sites For Attack By T Cells,” Nature 314:628-63 1, and Holliger et al. (1996) “Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody,” Protein Eng. 9:299-305; Marvin et al (2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies,” Acta Phar Alternatively or additionally, bispecific diabodies can be used to bind different Receptors can be co-ligated on the surface of different cells or on a single cell. or receptor co-ligation alters effector function and / or immune cell signaling. Multispecific molecules (e.g., bispecific molecules) containing epitope-binding sites are useful for preparing Diabodies) can be expressed by T lymphocytes, natural killer (NK) cells, antigen-presenting cells, or other Expressed on monocytes, B7-H3 (CD276), B7-H4 (VTCN1), BTL A (CD272), CD3, CD8, CD16, CD27, CD32, CD40, CD4 0L, CD47, CD64, CD70(CD27L), CD80(B7-1), CD86 (B7‐2), CD94(KLRD1), CD137(4‐1BB), CD137L(4 -1BBL), CD226, CTLA-4 (CD152), galectin-9, GITR, GITRL, HHLA2, ICOS (CD278), ICOSL (CD275), Killer activating receptor (KIR), LAG-3 (CD223), LIGHT (TNFSF14, C D258), MHC class I or II, NKG2a, NKG2d, OX40 (CD1 34), OX40L (CD134L), PD1H, PD-1 (CD279), PD-L1 (B7‐H1, CD274), PD‐L2 (B7‐CD, CD273), PVR (NEC L5, CD155), SIRPa, TCR, TIGIT, TIM‐3(HAVCR2), and / or VISTA (PD-1H), both of which are directed against immune cell surface determinants. In particular, cell surface receptors involved in the regulation of immune checkpoints (or their ligands) can be The receptor-directed epitope binding site is involved in the inhibitory signaling of immune checkpoint molecules. By antagonizing or blocking the immune response of a subject, the subject's immune response is stimulated, upregulated, or These are useful in generating bispecific or multispecific binding molecules that enhance immune checkpoints. Molecules involved in the regulation of checkpoints include, but are not limited to, B7-H3, B7 -H4, BTLA, CD40, CD40L, CD47, CD70, CD80, CD86, CD94, CD137, CD137L, CD226, CTLA‐4, galectin‐9, G ITR, GITRL, HHLA2, ICOS, ICOSL, KIR, LAG‐3, LIG HT, MHC class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, PD‐1, PD‐L1, PD‐L2, PVR, SIRPa, TCR, TIGI T, TIM-3 and / or VISTA.

[0055] However, the above-mentioned advantages come at significant costs. The formation of a body requires the successful assembly of two or more distinct and different polypeptides (i.e. The formation of diabodies is due to heterodimerization of different polypeptide chain species. This fact is due to the homodimerization of identical polypeptide chains. This is in contrast to monospecific diabodies, which are formed by providing at least two different polypeptides (i.e., two polypeptide species) for synthesis; and homodimerization of such polypeptides results in inactive molecules. (Takemura, S. et al. (2000) "Construction of a Diabody (Small Recombin ant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588 ) Therefore, the production of such polypeptides prevents covalent bonds between polypeptides of the same species. This must be achieved in a way that prevents homodimerization (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antib (body) Using A Refolding System,” Protein Eng. 13(8):583-588). teach non-covalent linking of such polypeptides (e.g., Olafsen et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjug ation And Radiolabeling For Tumor Targeting Applications,” Prot. Engr. Des. Sel . 17:21-27; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Fun ctional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recomb inant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-58 8; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672 reference).

[0056] However, the art does not provide a method for producing a double bond composed of non-covalently linked polypeptides. Recognizing that specific diabodies are unstable and readily decompose into non-functional monomers, (See, for example, Lu, D. et al. (2005) "A Fully Human Recombinant IgG-Like Bispecif ic Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Gr owth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):1 (See 9665-19672).

[0057] Despite this challenge, the art is now seeing the development of DART® ( D ual A ffi Unity R e‐ T Targeting Reagents (Diabodies) Covalent heterodimeric non-monospecific diabodies with specific binding sites have been successfully developed (e.g., US Published Patent No. 2013‐0295121; U.S. Published Patent No. 2010‐0174053; and U.S. Patent Publication No. 2009-0060910; European Patent Publication No. 2714079; State Patent Publication No. 2601216; European Patent Publication No. 2376109; European Patent Publication No. 21 58221; and WO 2012 / 162068; WO 2012 / 01 8687; WO 2010 / 080538, and Sloan, DD et al. (2015) “Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells,” PLoS P athog. 11(11):e1005233. doi: 10.1371 / journal.ppat.1005233; Al Hussaini, M. et al . (2015) “Targeting CD123 In AML Using A T-Cell Directed Dual-Affinity Re-Targe ting (DART(R)) Platform,” Blood pii: blood-2014-05-575704; Chichili, G.R. et al . (2015) “A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogeno us Leukemia: Preclinical Activity And Safety In Nonhuman Primates,” Sci. Transl . Med. 7(289):289ra82; Moore, P.A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lym phoma,” Blood 117(17):4542-4551; Veri, M.C. et al. (2010) “Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):19 33-1943; Johnson, S. et al. (2010) “Effector Cell Recruitment With Novel Fv-Bas ed Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in viv o B-Cell Depletion,” J. Mol. Biol. 399(3):436-449. The polypeptide comprises two or more covalently conjugated polypeptides, and one or more cysteines. The amino acid residues can form disulfide bonds, which covalently link the two polypeptide chains. This involves processing the polypeptide into each of the selected polypeptide species. The addition of a cysteine ​​residue to the C-terminus of such a structure can help to bridge disulfide bonds between polypeptide chains. It has been found that this allows for the binding of The resulting heterodimer is stabilized.

[0058] The two polypeptides of the simplest bispecific DART® diabody are Each comprises three domains: the first polypeptide (from N-terminus to C-terminus): (i) a first immunoglobulin comprising a binding region of the light chain variable domain (VL1) of the first immunoglobulin; (ii) a binding region for the heavy chain variable domain (VH2) of a second immunoglobulin; and (iii) a cysteine ​​residue (or a cysteine-containing domain) and promote heterodimerization of the diabody with the second polypeptide, thereby forming a heterodimer of the diabody. a heterodimer that serves to covalently link the first and second polypeptides of the avaposide to one another; The second polypeptide comprises a third domain containing a Dimer-Promoting Domain. In the N-terminal to C-terminal direction: (i) a light chain variable domain (VL) of a second immunoglobulin (ii) a first domain comprising a binding region of the heavy chain variable domain of the first immunoglobulin; a second domain comprising a binding region of VH1; and (iii) a cysteine ​​residue (or is a cysteine-containing domain) and the heterodimer-promoting domain of the first polypeptide chain. a complementary polypeptide chain that complexes with the first polypeptide chain to promote heterodimerization with the first polypeptide chain; The second polypeptide contains a third domain, which contains a heterodimer-promoting domain. The cysteine ​​residues of the third domain of the tide chain (or cysteine-containing domain) are Covalent attachment of the second polypeptide chain to the first polypeptide chain of the diabody Such molecules are stable, potent, and have two or more In one embodiment, the first and second polypeptides have the ability to simultaneously bind to the antigen. Each of the third domains of the polypeptides is held together by disulfide bonds. The cysteine ​​residues play a role in binding. A schematic diagram is provided, showing the diabody comprising an E-coil / K-coil heterodimer-promoting domain and A cysteine-containing linker is used for covalent binding. As presented in the drawings, one or both of the polypeptides may further comprise a CH2-CH The diabody polypeptides may have a three-domain sequence, thereby providing a diabody polypeptide having a three-domain sequence between the two diabody polypeptides. The complexation results in the activation of cells (e.g., B lymphocytes, dendritic cells, natural killer cells, macrophages, etc.). Fc region capable of binding to Fc receptors on phages, neutrophils, eosinophils, basophils, and mast cells As will be presented in more detail below, the CH2 of such a polypeptide chain and / or CH3 domains need not be identical in sequence, and advantageously, these two domains It is modified to promote complexation between the polypeptide chains.

[0059] Numerous variants of such molecules have been described (see, e.g., U.S. Patent Publication No. 2015 / 013999). 0175697; U.S. Patent Publication No. 2014 / 0255407; U.S. Patent Publication No. 201 4 / 0099318; U.S. Patent Publication No. 2013 / 0295121; U.S. Patent Publication No. 2 010 / 0174053; and U.S. Patent Publication No. 2009 / 0060910; European Patent Publication Patent No. 2714079; European Patent No. 2601216; European Patent No. 23761 09; European Patent Publication No. 2158221; and International Publication No. 2012 / 162068; (See WO 2012 / 018687; WO 2010 / 080538). These Fc-region-containing DART® diabodies contain two pairs of polypeptide chains. The first polypeptide chain may comprise (in the N-terminal to C-terminal direction): (i) a first immunizing (ii) a first domain comprising a binding region for the light chain variable domain (VL1) of globulin; a second domain comprising the binding region of the heavy chain variable domain (VH2) of a second immunoglobulin; (iii) a cysteine ​​residue (or a cysteine-containing domain) and and promoting heterodimerization of the first polypeptide of the diabody with the second polypeptide of the diabody. a third domain that serves to covalently link the first and second polypeptides to one another; and and (iv) a CH2-CH3 domain. The second polypeptide comprises (N-terminal to C-terminal In the direction of: (i) the binding region of the light chain variable domain (VL2) of the second immunoglobulin (ii) a heavy chain variable domain (VH1) of a first immunoglobulin; a second domain comprising a binding region; and (iii) a cysteine ​​residue (or a cysteine-containing a heterodimer-promoting domain) that promotes heterodimerization between the first polypeptide chain and the second polypeptide chain; The first polypeptide contains a nucleotide domain and a third domain containing a nucleotide domain. The dimers complex with each other to form the Fc region. Figures 3A-3C show the different heterodimer promoters. A schematic diagram of three variants of such diabodies utilizing the axial domain is presented.

[0060] Other Fc-region-containing DART® diabodies contain three polypeptide chains. The first polypeptide of such a DART® diabody may be Domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a C Such DART® dyes contain domains containing the H2-CH3 sequence. The second polypeptide of the abody can comprise: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) heterodimerization and co-dimerization with the first polypeptide chain of the diabody. Such DART™ diabodies contain domains that promote binding. The third polypeptide comprises a CH2-CH3 sequence. The first and second polypeptide chains of the diabody are associated together to form an epitope. a VL1 / VH1 binding site capable of binding to a first epitope, and a VL2 / VH3 binding site capable of binding to a second epitope. Such relatively complex DART® molecules also form a VH2 binding site. It has a cysteine-containing domain that functions to form a covalent complex. The first and second polypeptides involve cysteine ​​residues in the third domains of each polypeptide. are linked to each other via disulfide bonds. In particular, the first and third polypeptide chains are They complex with each other to form an Fc region stabilized by disulfide bonds. A-4B present a schematic diagram of such a diabody containing three polypeptide chains.

[0061] Yet other Fc-region-containing DART® diabodies can be used to target up to three different immunoglobulins. The light and heavy chain variable domains of globulin (VL1 / VH1, VL2 / VH2 and VL3 / VH4) It may comprise five polypeptide chains, each of which may comprise a binding region derived from a different polypeptide chain (called H3). For example, the first polypeptide chain of such a diabody may comprise: (i) a VH1-containing domain; (ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The second and fifth polypeptide chains of such diabodies may comprise: (i) VL1 and (ii) a CL-containing domain. the third polypeptide chain of comprises: (i) a VH1-containing domain; (ii) a CH1-containing domain; (iii) a domain containing a CH2-CH3 sequence; (iv) a VL2-containing domain; (v (v) a VH3-containing Domain; and (vi) a Heterodimer-Promoting Domain, The heterodimer-promoting domain promotes the dimerization of the third and fourth chains. The fourth polypeptide of the atrial body comprises: (i) a VL3-containing domain; (ii) a VH2-containing domain; and (iii) heterodimerization with a third polypeptide chain of the diabody. and a domain that promotes covalent bonding. These relatively complex DARTs (Dysregulation of Antibodies and Antibodies) are complexed with each other to form the Fc region. The α-Lysine™ molecule also contains a cysteine-containing domain that functions to form a covalent complex. This allows each polypeptide chain to be bound together by disulfide bonds involving cysteine ​​residues. linked to at least one additional polypeptide chain. Preferably, such domains are , arranged in an N-terminal to C-terminal direction. Figure 5 shows such a DNA fragment containing five polypeptide chains. 1 presents a schematic diagram of the ear body.

[0062] For applications where a tetravalent molecule is desired but an Fc is not required, alternative configurations are known in the art. It is known in the art that VH1, VL2, VH2, and VL2 domains are It is also called "TandAb" and is formed by the homodimerization of two identical chains, each of which has These include, but are not limited to, tetravalent tandem antibodies (see, e.g., U.S. Patent Publication No. 2005 -0079170; U.S. Patent Publication No. 2007-0031436; U.S. Patent Publication No. 201 0-0099853; U.S. Patent Publication No. 2011-020667; U.S. Patent Publication No. 201 3-0189263; European Patent Publication No. 1078004; European Patent Publication No. 237186 6; European Patent Publication No. 2361936; and European Patent Publication No. 1293514; International Patent Publication No. WO 1999 / 057150; WO 2003 / 025018; and WO 2003 / 025018 (See issue 2013 / 013700).

[0063] Recently, two diabody-type binding domains and one non-diabody-type binding domain, As well as trivalent structures incorporating an Fc region have been described (see, e.g., PCT Application No. PCT / US15 / 3 No. 3076, entitled "Tri‐Specific Binding Molecules and Methods of Use Thereof", 20 and PCT Application No. PCT / US15 / 33081, filed May 29, 2015; entitled "Tri -Specific Binding Molecules That Specifically Bind to Multiple Cancer Antigens (See "Chemical and Method of Use Thereof," filed May 29, 2015.) The molecules may be used to generate monospecific, bispecific, or trispecific molecules. F presents a schematic representation of such a trivalent molecule containing three or four polypeptide chains.

[0064] IV. Anti-Human PD-1 Binding Molecules of the Invention Preferred PD-1-binding molecules of the present invention include antibodies, diabodies, BiTEs, etc. These consist of continuous or discontinuous (e.g., conformational) portions (epithelial cells) of human PD-1 (CD279). The PD-1-binding molecules of the present invention are preferably capable of binding to one or more non-human PD-1-binding domains. the ability of a species, particularly a primate species (and in particular a primate species such as a cynomolgus monkey), to bind to the PD-1 molecule Also shown is a representative human PD-1 polypeptide (NCBI sequence NP_005009.2; 0 amino acid residue signal sequence (underlined) and the 268 amino acid residue mature protein , amino acid sequence (SEQ ID NO: 68): MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL It has.

[0065] In certain embodiments, the PD-1-binding molecules of the invention meet any one of the following criteria: characterized by one or more of: (1) It specifically binds to human PD-1 endogenously expressed on the surface of stimulated human T cells; (2) Equilibrium binding constant (K D ) specifically binds to human PD-1 at 40 nM or less; (3) Equilibrium binding constant (K D ) specifically binds to human PD-1 at 5 nM or less; (4) On-speed (K a )1.5×10 4 M -1 minutes -1 The above specifically binds to human PD-1 ; (5) On-speed (K a )90.0×10 4 M -1 minutes -1 The above results show that the antibody specifically binds to human PD-1. ru; (6) Off-speed (K d )7×10 -4 minutes -1 Binds specifically to human PD-1: (7) Off-speed (K d )2×10 -4 minutes -1 Binds specifically to human PD-1: (8) specifically binds to non-human primate PD-1 (e.g., cynomolgus monkey PD-1); (9) Binding / inhibitory activity of PD-1 ligands (PD-L1 / PD-L2) to PD-1 inhibiting (i.e., blocking or interfering with) (10) stimulating an immune response; and / or (11) Stimulates antigen-specific T cell responses through synergistic effects with anti-human LAG-3 antibody .

[0066] As used herein, the term "antigen-specific T cell response" refers to a T cell A specific T-cell response is a response in which T cells respond to an antigen specific for that antigen. It refers to the T cell response resulting from stimulation of T cells by antigen-specific stimulation. Non-limiting examples of responses include proliferation and cytokine production (e.g., TNF-α, IFN-γ The ability of a molecule to stimulate an antigen-specific T cell response can be measured, for example, as described herein. Staphylococcus aureus enterotoxin type B antigen ("SEB")-stimulated PBMCs as described in It can be determined using an assay.

[0067] A preferred PD-1 binding molecule of the present invention is the murine anti-human PD-1 monoclonal antibody "PD-1 D-1 mAb 1”, “PD-1 mAb 2”, “PD-1 mAb 3”, “PD ‐1 mAb 4”, “PD‐1 mAb 5”, “PD‐1 mAb 6”, “PD‐ 1 mAb 7”, “PD-1 mAb 8”, “PD-1 mAb 9”, “PD-1 mAb 10”, “PD-1 mAb 11”, “PD-1 mAb 12”, “PD PD-1 mAb 13, PD-1 mAb 14, or PD-1 mAb 15 and more preferably, such anti-human PD-1 monoclonal antibodies CDRs of the VH domain of a monoclonal antibody Hone, two or all three of the following, and / or or CDR of the VL domain L It has one, two or all three of the following: Preferred anti-human PD-1 binding molecules are bispecific (or multispecific) antibodies, chimeric or human PD-1 binding molecules. Monoclonal antibodies, BiTe, diabodies, etc., and such binding molecules with variant Fc regions Includes.

[0068] The present invention particularly relates to: (A)(1) The three CDRs of the VH domain of PD-1 mAb 1 H ; (2) The three CDRs of the VL domain of PD-1 mAb 1 L ; (3) the three CDRs of the VH domain of PD-1 mAb 1 H , and PD-1 The three CDRs of the VL domain of mAb 1 L ; (4) the VH domain of hPD-1 mAb 1 VH1; (5) the VL domain of hPD-1 mAb 1 VL1; (6) the VH and VL domains of hPD-1 mAb 1; (B)(1) The three CDRs of the VH domain of PD-1 mAb 2 H ; (2) The three CDRs of the VL domain of PD-1 mAb 2 L ; (3) the three CDRs of the VH domain of PD-1 mAb 2 H , and PD-1 The three CDRs of the VL domain of mAb 2 L ; (4) the VH domain of hPD-1 mAb 2 VH1; (5) the VL domain of hPD-1 mAb 2 VL1; (6) the VH and VL domains of hPD-1 mAb 2; (C)(1) The three CDRs of the VH domain of PD-1 mAb 3 H ; (2) The three CDRs of the VL domain of PD-1 mAb 3 L ; (3) the three CDRs of the VH domain of PD-1 mAb 3 H , and PD-1 The three CDRs of the VL domain of mAb 3 L ; (D)(1) The three CDRs of the VH domain of PD-1 mAb 4 H ; (2) Three CDRs of the VL domain of PD-1 mAb 4 L ; (3) The three CDRs of the VH domain of PD-1 mAb 4 H , and PD-1 The three CDRs of the VL domain of mAb 4 L ; (E)(1) The three CDRs of the VH domain of PD-1 mAb 5 H ; (2) The three CDRs of the VL domain of PD-1 mAb 5 L ; (3) The three CDRs of the VH domain of PD-1 mAb 5 H , and PD-1 The three CDRs of the VL domain of mAb 5 L ; (F)(1) The three CDRs of the VH domain of PD-1 mAb 6 H ; (2) Three CDRs of the VL domain of PD-1 mAb 6 L ; (3) The three CDRs of the VH domain of PD-1 mAb 6 H , and PD-1 The three CDRs of the VL domain of mAb 6 L ; (G)(1) The three CDRs of the VH domain of PD-1 mAb 7 H ; (2) PD-1 mAb 7 or hPD-1 mAb 7 VL2 or The three CDRs of the VL domain of hPD-1 mAb 7 VL3 L ; (3) The three CDRs of the VH domain of PD-1 mAb 7H , and PD-1 mAb 7 or hPD-1 mAb 7 VL2 or hPD-1 mAb 7 The three CDRs of the VL domain of VL3 L ; (4) hPD-1 mAb 7 VH1 or hPD-1 mAb 7 VH 2 VH domains; (5) hPD-1 mAb 7 VL1 or hPD-1 mAb 7 VL 2 or the VL domain of hPD-1 mAb 7 VL 3; (6) hPD-1 mAb 7(1.1) or hPD-1 mAb 7(1 .2) or hPD-1 mAb 7(1.3) or hPD-1 mAb 7(2 .1) or hPD-1 mAb 7(2.2) or hPD-1 mAb 7(2 .3) VH and VL domains; (H)(1) The three CDRs of the VH domain of PD-1 mAb 8 H ; (2) Three CDRs of the VL domain of PD-1 mAb 8 L ; (3) The three CDRs of the VH domain of PD-1 mAb 8 H , and PD-1 The three CDRs of the VL domain of mAb 8 L ; (I)(1) VH domain of PD-1 mAb 9 or hPD-1 mAb 9 VH2 The three main CDRs H ; (2) VL domain of PD-1 mAb 9 or hPD-1 mAb 9 VL2 The three main CDRs L ; (3) VH domain of PD-1 mAb 9 or hPD-1 mAb 9 VH2 The three main CDRs H and PD-1 mAb 9 or hPD-1 mAb 9 V The three CDRs of the VL domain of L2L ; (4) hPD-1 mAb 9 VH1 or hPD-1 mAb 9 VH 2 VH domains; (5) hPD-1 mAb 9 VL1 or hPD-1 mAb 9 VL 2 VL domains; (6) hPD-1 mAb 9(1.1) or hPD-1 mAb 9(1 .2) or hPD-1 mAb 9(2.1) or hPD-1 mAb 9(2 .2) VH and VL domains (J)(1) The three CDRs of the VH domain of PD-1 mAb 10 H ; (2) Three CDRs of the VL domain of PD-1 mAb 10 L ; (3) the three CDRs of the VH domain of PD-1 mAb 10 H , and PD-1 The three CDRs of the VL domain of mAb 10 L ; (K)(1) The three CDRs of the VH domain of PD-1 mAb 11 H ; (2) Three CDRs of the VL domain of PD-1 mAb 11 L ; (3) Three CDRs of the VH domain of PD-1 mAb 11 H , and PD-1 The three CDRs of the VL domain of mAb 11 L ; (L)(1) The three CDRs of the VH domain of PD-1 mAb 12 H ; (2) Three CDRs of the VL domain of PD-1 mAb 12 L ; (3) Three CDRs of the VH domain of PD-1 mAb 12 H , and PD-1 The three CDRs of the VL domain of mAb 12 L ; (M)(1) The three CDRs of the VH domain of PD-1 mAb 13H ; (2) Three CDRs of the VL domain of PD-1 mAb 13 L ; (3) Three CDRs of the VH domain of PD-1 mAb 13 H , and PD-1 The three CDRs of the VL domain of mAb 13 L ; (N)(1) The three CDRs of the VH domain of PD-1 mAb 14 H ; (2) Three CDRs of the VL domain of PD-1 mAb 14 L ; (3) Three CDRs of the VH domain of PD-1 mAb 14 H , and PD-1 The three CDRs of the VL domain of mAb 14 L ; (O)(1) The three CDRs of the VH domain of PD-1 mAb 15 H ; (2) Three CDRs of the VL domain of PD-1 mAb 15 L ; (3) the three CDRs of the VH domain of PD-1 mAb 15 H , and PD-1 The three CDRs of the VL domain of mAb 15 L ; (4) the VH domain of hPD-1 mAb 15 VH1; (5) the VL domain of hPD-1 mAb 15 VL1; (6) VH and VL domains of hPD-1 mAb 15; or PD-1 mAb 1, PD-1 mAb 2, PD-1 mAb 3 , PD-1 mAb 4, PD-1 mAb 5, PD-1 mAb 6, PD-1 m Ab 7, PD-1 mAb 8, PD-1 mAb 9, PD-1 mAb 10, P D-1 mAb 11, PD-1 mAb 12, PD-1 mAb 13, PD-1 binds to the same epitope as mAb 14 or PD-1 mAb 15, or PD-1 binding molecules comprising PD-1 binding domains that compete for binding.

[0069] A. Anti-human PD-1 antibody PD-1 mAb 1 1. Mouse anti-human PD-1 antibody PD-1 mAb 1 The amino acid sequence of the VH Domain of PD-1 mAb 1 (SEQ ID NO: 69) is shown below: CDR H residues are underlined). DVQLQESGPG RVKPSQSLSL TCTVTGFSIT NDYAWN WIRQ FPGNKLEWMG HITYSGSTSY NPSLKS RISI TRDTSKNHFF LQLSSVTPED TATYYCAR DY GSGYPYTLDY WGQGTSVTVS S CDRs of PD-1 mAb 1 H 1 (SEQ ID NO: 71): NDYAWN CDRs of PD-1 mAb 1 H 2 (SEQ ID NO: 72): HITYSGSTSYNPSLKS CDRs of PD-1 mAb 1 H 3 (SEQ ID NO: 73): DYGSGYPYTLDY

[0070] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 1 is SEQ ID NO: 70 (CDR H The nucleotides encoding the residues are underlined): cagatccagt gatgtgcagc ttcaggagtc gggacctggc cgggtgaaac cttctcagtc tctgtccctc acctgcactg tcactggctt ctcaatcacc aatgattatg cctggaac tg gatccgacag tttccaggaa acaaactgga gtggatgggc cacataacct acagtggcag cactagctac aacccatctc tcaaa agtcg aatctctatc actcgggaca catccaagaa ccacttcttc ctgcagttga gttctgtgac tcctgaggac acagccacat attactgtgc aaga gattac ggtagtggct acccctatac tttggactac tggggtcaag gtacctcagt caccgtctcc tcc is.

[0071] The amino acid sequence of the VL Domain of PD-1 mAb 1 (SEQ ID NO: 74) is shown below: CDR L Residues are underlined): QIVLTQSPAL MSASPGEKVT MTC SATSIVS YVY WYQQKPG SSPQPWIY LT SNLAS GVPAR FSGSGSGTSY SLTISSMEAE DAATYYC QQW SDNPYT FGGG TKLEIK CDRs of PD-1 mAb 1 L 1 (SEQ ID NO: 76): SATSIVSYVY CDRs of PD-1 mAb 1 L 2 (SEQ ID NO: 77): LTSNLAS CDRs of PD-1 mAb 1 L 3 (SEQ ID NO: 78): QQWSDNPYT

[0072] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 1 is SEQ ID NO: 75 (CDR L The nucleotides encoding the residues are underlined): caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc atgacctgc a gtgccacctc aattgtaagt tacgtttac t ggtaccagca gaagcctgga tcctcccccc aaccctggat ttat ctcaca tccaacctgg cttct ggagt ccctgctcgc ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa gatgctgcca cttattactg c cagcagtgg agtgataacc cgtacacg tt cggagggggg accaagctgg aaataaaa is.

[0073] 2. Anti-human PD-1 antibody PD-1 mAb to form "hPD-1 mAb 1" b Humanization of 1 The above-mentioned murine anti-human PD-1 antibody PD-1 mAb 1 was used to identify the antigenic epitope. and further deimmunized to reduce its antigenicity upon administration to a human recipient. We have demonstrated the ability to humanize anti-human PD-1 antibodies to achieve the therapeutic effect of PD-1. PD-1 mAb 1 VH1” and one humanized VH domain, herein referred to as “h One humanized VL domain, designated “PD-1 mAb 1 VL1,” was obtained. Thus, an antibody comprising the humanized VL domain paired with the humanized VH domain is called "h It is called "PD-1 mAb 1."

[0074] The amino acid sequence of the VH domain of hPD-1 mAb 1 VH1 (SEQ ID NO: 79) is shown below. See below (CDR H Residues are underlined): DVQLQESGPG LVKPSQTLSL TCTVSGFSIS NDYAWN WIRQ PPGKGLEWIG HITYSGSTSY NPSLKS RLTI TRDTSKNQFV LTMTNMDPVD TATYYCAR DY GSGYPYTLDY WGQGTTVTVS S

[0075] An exemplary polynucleotide encoding hPD-1 mAb 1 VH1 has the sequence Number 80 (CDR H The nucleotides encoding the residues are underlined): gacgtacagc tccaggaaag tggcccaggt ctggtgaagc catcccagac actgagcctg acttgcaccg tgagtggctt ctccatctca aatgactacg cctggaat tg gattaggcag cctcccggta aagggctgga gtggatcggc cacatcacat acagcggctc cacatcatat aatcccagtc tgaag agccg tcttaccatt actcgcgaca ctagtaagaa ccagtttgtt ctgaccatga ccaacatgga ccctgtggat actgcaacat actattgtgc tcga gattat ggttctggtt acccttatac actcgactac tggggacagg gaaccactgt gaccgtgagc tcc is.

[0076] The amino acid sequence of the VL domain of hPD-1 mAb 1 VL1 (SEQ ID NO: 81) is shown below. See below (CDR L Residues are underlined): EIVLTQSPAT LSVSPGEKVT ITC SATSIVS YVY WYQQKPG QAPQPLIY LT SNLAS GIPAR FSGSGSGTDF TLTISSLEAE DAATYYC QQW SDNPYT FGGG TKVEIK

[0077] An exemplary polynucleotide encoding hPD-1 mAb 1 VL1 has the sequence Number 82 (CDR L The nucleotides encoding the residues are underlined): gaaatcgttc tgacccagag cccagcaacc ctgtctgtct cccccggaga aaaggtcacc attacttgc t ctgctacttc tatcgtgtcc tacgtgtac t ggtatcagca gaagcccggt caggctcccc agccattgat atat ctgacc agcaacctgg cttct ggtat cccagctcgt ttttccggta gcgggtccgg gactgatttc actttgacta tcagctctct ggaggcagaa gacgccgcca cctattattg t caacagtgg tcagacaatc catacact tt tggcggtggc accaaagtcg aaataaag is.

[0078] B. Anti-human PD-1 antibody PD-1 mAb 2 1. Mouse anti-human PD-1 antibody PD-1 mAb 2 The amino acid sequence of the VH Domain of PD-1 mAb 2 (SEQ ID NO: 83) is shown below: CDR H Residues are underlined): DVQLVESGGG LVQPGGSRKL SCAASGFVFS SFGMH WVRQA PEKGLEWVA Y ISSGSMSISY ADTVKG RFTV TRDNAKNTLF LQMTSLRSED TAIYYCAS LS DYFDY WGQGT TLTVSS CDRs of PD-1 mAb 2 H 1 (SEQ ID NO: 85): SFGMH CDRs of PD-1 mAb 2H 2 (SEQ ID NO: 86): YISSGSMSISYADTVKG CDRs of PD-1 mAb 2 H 3 (SEQ ID NO: 87): LSDYFDY

[0079] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 2 is SEQ ID NO: 84 (CDR H The nucleotides encoding the residues are underlined): gatgtgcagc tcgtggagtc tgggggaggc ttagtgcagc ctggagggtc cgggaaactc tcctgtgcag cctctggatt cgttttcagt agctttggaa tgcac tgggt tcgtcaggct ccagagaagg ggctggagtg ggtcgca tac atcagtagtg gcagtatgag catttcctat gcagacacag tgaagggc CG attcaccgtc accagagaca atgccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgaggac acggccattt attactgtgc atcc ctgagt gactactttg actac tgggg ccaaggcacc actctcacag tctcctcc is.

[0080] The amino acid sequence of the VL Domain of PD-1 mAb 2 (SEQ ID NO: 88) is shown below: CDR L Residues are underlined): DVVMSQTPLS LPVSLGDQAS ISC RSSQSLV HSTGNTYLH W YLQKPGQSPK LLIY RVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV FFC SQTTHVP WTFGGGTKLE IK CDRs of PD-1 mAb 2 L 1 (SEQ ID NO: 90): RSSQSLVHSTGNTYLH CDRs of PD-1 mAb 2 L 2 (SEQ ID NO: 91): RVSNRFS CDRs of PD-1 mAb 2 L 3 (SEQ ID NO: 92): SQTTHVPWT

[0081] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 2 is SEQ ID NO: 89 (CDR L The nucleotides encoding the residues are underlined): gatgttgtga tgtcccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgc a gatctagtca gagccttgtt cacagtactg gaaacaccta tttacat tgg tacctgcaga agccaggcca gtctccaaag ctcctgatct ac agggtttc taaccgattt tct ggggtcc ccgacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agtagagtgg aggctgagga tctgggagtt tttttctgc t ctcaaactac acatgttccg tggacg ttcg gtggaggcac caagctggaa atcaaa is.

[0082] 2. Anti-human PD-1 antibody PD-1 m to form "hPD-1 mAb 2" Humanization of Ab2 The above-mentioned murine anti-human PD-1 antibody PD-1 mAb 2 was used to identify the antigenic epitope. and further deimmunized to reduce its antigenicity upon administration to a human recipient. We have demonstrated the ability to humanize anti-human PD-1 antibodies to achieve the therapeutic effect of PD-1. PD-1 mAb 2 contains one humanized VH domain, designated "VH1" and one humanized VH domain, designated "hH1" herein. One humanized VL domain, designated PD-1 mAb 2 VL1, was obtained. Thus, any antibody comprising the humanized VL domain paired with the humanized VH domain. This is referred to as "hPD-1 mAb 2."

[0083] The amino acid sequence of the VH domain of hPD-1 mAb 2 VH1 (SEQ ID NO: 93) is shown below. See below (CDR H Residues are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFVFS SFGMH WVRQA PGKGLEWVA Y ISSGSMSISY ADTVKG RFTI SRDNAKNTLY LQMNSLRTED TALYYCAS LS DYFDY WGQGT TVTVSS

[0084] An exemplary polynucleotide encoding hPD-1 mAb 2 VH1 has the sequence Number 94 (CDR H The nucleotides encoding the residues are underlined): gaagtgcaat tggttgagag tggtggtggc ctggtgcagc caggtggaag tctgcggttg tcctgtgcag caagcggatt tgtgttcagc tcttttggga tgcat tgggt gcgccaggct cccggcaagg gtctcgagtg ggtagca tac atctccagcg ggtccatgtc tattagttat gccgacacag tgaaaggc ag gtttactatc tcccgtgaca atgcaaaaaa cacactgtac ctgcaaatga atagcctgcg caccgaggac accgccttgt actactgcgc ttcc ctgtct gattacttcg actac tgggg tcagggcaca actgtgacag tttcttcc is.

[0085] The amino acid sequence of the VL domain of hPD-1 mAb 2 VL1 (SEQ ID NO: 95) is shown below. See below (CDR L Residues are underlined): DVVMTQSPLS LPVTLGQPAS ISC RSSQSLV HSTGNTYLH W YLQKPGQSPQ LLIY RVSNRF S GVPDRFSGSGSGTDFTLKI SRVEAEDVGV YYC SQTTHVP WT FGQGTKLE IK

[0086] An exemplary polynucleotide encoding hPD-1 mAb 2 VL1 has the sequence Number 96 (CDR L The nucleotides encoding the residues are underlined): gacgttgtga tgacacagtc accactgagt ctgccagtta ccctgggcca gccagccagt atttcttgt c ggagttcaca gagtctggta cattccacag gaaatacata tctccat tgg tacctgcaaa aaccagggca gagcccccag ctgctgattt at agagtgtc taatcgattt tct ggcgtgc cagatcggtt cagcggcagc gggtctggca ctgatttcac actgaaaatc tctagggtgg aggcagagga cgtaggcgtt tactactgt a gtcagaccac ccatgtaccc tggact tttg gccaaggtac taagctggaa atcaag is.

[0087] C. Mouse anti-human PD-1 antibody PD-1 mAb 3 The amino acid sequence of the VH domain of PD-1 mAb 3 (SEQ ID NO: 97) is shown below: CDR H residues are underlined); QVQLQQSGAE LVRPGASVTL SCKASGYTFT DYVMH WVKQT PVHGLEWIG T IDPETGGTAY NQKFKG KAIL TADKSSNTAY MELRSLTSED SAVYYFTR EK ITTIVEGTYW YFDV WGTGTT VTVSS CDRs of PD-1 mAb 3 H 1 (SEQ ID NO: 99): DYVMH CDRs of PD-1 mAb 3 H 2 (SEQ ID NO: 100): TIDPETGGTAYNQKFKG CDRs of PD-1 mAb 3 H 3 (SEQ ID NO: 101): EKITTIVEGTYWYFDV

[0088] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 3 is , SEQ ID NO: 98 (CDR H The nucleotides encoding the residues are underlined): caggttcaac tgcaacagtc tggggctgag ctggtgaggc ctggggcttc agtgacgctg tcctgcaagg cttcgggcta cacatttact gactatgtaa tgcac tgggt gaagcagaca cctgtgcatg gcctggaatg gattgga act attgatcctg aaactggtgg tactgcctac aatcagaagt tcaagggc aa ggccatactg actgcagaca agtcctccaa cacagcctac atggagctcc gcagcctgac atctgaggac tctgccgtct attactttac aaga gagaag attactacga tagtagaggg gacatactgg tacttcgatg tc tggggcac agggaccacg gtcaccgtct cctca is.

[0089] The amino acid sequence of the VL domain of PD-1 mAb 3 (SEQ ID NO: 102) is shown below: (CDR L Residues are underlined): DVLLTQTPLS LPVSLGDQAS ISC RSSQNIV HSNGDTYLE W YLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHLP YT FGGGTKLE IK CDRs of PD-1 mAb 3 L 1 (SEQ ID NO: 104): RSSQNIVHSNGDTYLE CDRs of PD-1 mAb 3 L 2 (SEQ ID NO: 105): KVSNRFS CDRs of PD-1 mAb 3 L 3 (SEQ ID NO: 106): FQGSHLPYT

[0090] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 3 is , SEQ ID NO: 103 (CDR L Nucleotides encoding the residues are underlined) : gatgttttgc tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgc a gatctagtca gaacattgta catagtaatg gagacaccta tttggaa tgg tacctgcaga aaccaggcca gtctccaaag ctcctgatct at aaagtttc caaccgattt tct ggggtcc cagacaggtt cagtggcagt gggtcaggga cagattttac actcaaaatc agcagagtgg aggctgagga tctgggagtt tattactgc t ttcaaggttc acatcttccg tacacg ttcg gaggggggac caagctggaa ataaaa is.

[0091] D. Mouse anti-human PD-1 antibody PD-1 mAb 4 The amino acid sequence of the VH domain of PD-1 mAb 4 (SEQ ID NO: 107) is shown below: (CDR H Residues are underlined): DVQLVESGGG LVQPGGSRKL SCAASGFVFS SFGMH WVRQA PEKGLEWVA Y ISSGSMSISY ADTVKG RFTV TRDNAKNTLF LQMTSLRSED TAIYYCAS LT DYFDY WGQGT TLTVSS CDRs of PD-1 mAb 4 L 1 (SEQ ID NO: 109): SFGMH CDRs of PD-1 mAb 4 L 2 (SEQ ID NO: 110): YISSGSMSISYADTVKG CDRs of PD-1 mAb 4 L 3 (SEQ ID NO: 111): LTDYFDY

[0092] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 4 is SEQ ID NO: 108 (CDR HThe nucleotides encoding the residues are underlined): gatgtgcagc tcgtggagtc tgggggaggc ttagtgcagc ctggagggtc cgggaaactc tcctgtgcag cctctggatt cgttttcagt agctttggaa tgcac tgggt tcgtcaggct ccagagaagg ggctggagtg ggtcgca tat attagtagtg gcagtatgag tatttcctat gcagacacag tgaagggc CG attcaccgtc accagagaca atgccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgaggac acggccattt attactgtgc atcc ctgact gactactttg actac tgggg ccaaggcacc actctcacag tctcctca is.

[0093] The amino acid sequence of the VL domain of PD-1 mAb 4 (SEQ ID NO: 112) is shown below: (CDR L Residues are underlined): DVVMSQTPLS LPVSLGDQAS ISC RSSQSLV HSTGNTYFH W YLQKPGQSPK LLIY RVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQTTHVP WT FGGGTKLE IK CDRs of PD-1 mAb 4 L 1 (SEQ ID NO: 114): RSSQSLVHSTGNTYFH CDRs of PD-1 mAb 4 L 2 (SEQ ID NO: 115): RVSNRFS CDRs of PD-1 mAb 4 L 3 (SEQ ID NO: 116): SQTTHVPWT

[0094] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 4 is SEQ ID NO: 113 (CDR L The nucleotides encoding the residues are underlined): gatgttgtga tgtcccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcctgc a gatctagtca gagccttgtt cacagtactg gaaacaccta tttccat tgg tacctgcaga agccaggcca gtctccaaag ctcctgatct ac agggtttc taaccgattt tct ggggtcc ccgacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtttatttctgc t ctcaaactac acatgttccg tggacg ttcg gtggaggcac caagctggaa atcaaa is.

[0095] E. Mouse anti-human PD-1 antibody PD-1 mAb 5 The amino acid sequence of the VH domain of PD-1 mAb 5 (SEQ ID NO: 117) is shown below: (CDR H Residues are underlined): QVQLQQPGVE LVRPGASVKL SCKASGYSFT AYWMN WMKQR PGQGLEWIG V IHPSDSETWL NQKFKD KATL TVDKSSSTAY MQLISPTSED SAVYYCAR EH YGSSPFAY WG QGTLVTVSA CDRs of PD-1 mAb 5 H 1 (SEQ ID NO: 119): AYWMN CDRs of PD-1 mAb 5H 2 (SEQ ID NO: 120): VIHPSDSETWLNQKFKD CDRs of PD-1 mAb 5 H 3 (SEQ ID NO: 121): EHYGSSPFAY

[0096] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 5 is SEQ ID NO: 118 (CDR H The nucleotides encoding the residues are underlined): caggtccaac tgcagcagcc tggggttgaa ctggtgaggc ctggagcttc agtgaagctg tcctgcaagg cttctggcta ctccttcacc gcctactgga tgaac tggat gaaacagagg cctggacagag gccttgagtg gattggc gtg attcatcctt ccgatagtga aacttggtta aatcagaagt tcaag gacaa ggccacattg actgtagaca aatcctccag cacagcctac atgcaactca tcagcccgac atctgaggac tctgcggtct attactgtgc aaga gagcac tacggtagta gcccgtttgc ttac tggggc caagggactc tggtcactgt ctctgca is.

[0097] The amino acid sequence of the VL domain of PD-1 mAb 5 (SEQ ID NO: 122) is shown below: (CDR L Residues are underlined): DIVLTQSPAS LAVSLGQRAT ISC RANESVD NYGMSFMN WF QQKPGQPPKL LIY AASNQGS GVPARFSGSG SGTDFSLNIH PMEEDDTAMY FC QQSKEVPY T FGGGTKLEI K CDRs of PD-1 mAb 5 L 1 (SEQ ID NO: 124): RANESVDNYGMSFMN CDRs of PD-1 mAb 5 L 2 (SEQ ID NO: 125): AASNQGS CDRs of PD-1 mAb 5 L 3 (SEQ ID NO: 126): QQSKEVPYT

[0098] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 5 is SEQ ID NO: 123 (CDR L The nucleotides encoding the residues are underlined): gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc atctcctgc a gagccaacga aagtgttgat aattatggca tgagttttat gaac tggttc caacagaaac caggacagcc acccaaactc ctcatctat g ctgcatccaa ccaaggatcc ggggtccctg ccaggtttag tggcagtggg tctgggacag atttcagcct caacatccat cctatggagg aggatgatac tgcaatgtat ttctgt cagc aaagtaagga ggttccgtac acg ttcggag gggggaccaa gctggaaata aaa is.

[0099] F. Mouse anti-human PD-1 antibody PD-1 mAb 6 The amino acid sequence of the VH domain of PD-1 mAb 6 (SEQ ID NO: 127) is shown below: (CDR H Residues are underlined): EVKLVESGGG LVNPGGSLKL SCAASGFTFS SYGMSWVRQT PEKRLEWVA T ISGGGSDTYY PDSVKG RFTI SRDNAKNNLY LQMSSLRSED TALYYCAR QK ATTWFAY WGQ GTLVTVST CDRs of PD-1 mAb 6 H 1 (SEQ ID NO: 129): SYGMS CDRs of PD-1 mAb 6 H 2 (SEQ ID NO: 130): TISGGGSDTYYPDSVKG CDRs of PD-1 mAb 6 H 3 (SEQ ID NO: 131): QKATTWFAY

[0100] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 6 is SEQ ID NO: 128 (CDR H The nucleotides encoding the residues are underlined): gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgca gagcaagtga gagtgtggac aattacggca tgtccttcat gaac tggttt cagcagaagc ctgggcagcc acctaagctg ctcatccac g ccgcctctaa ccgcggatct ggggtgcctt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgc cagc aatctaaaga ggtgccctat act tttggtg gcgggaccaa ggttgagatc aaa is.

[0101] The amino acid sequence of the VL domain of PD-1 mAb 6 (SEQ ID NO: 132) is shown below: (CDRL Residues are underlined): DIVLTQSPAS LAVSLGQRAT ISC RASESVD NYGISFMN WF QQKPGQPPKL LIY PASNQGS GVPARFSGSG SGTDFSLNIH PMEEDDAAMY FC QQSKEVPW T FGGGTKLEI K CDRs of PD-1 mAb 6 L 1 (SEQ ID NO: 134): RASESVDNYGISFMN CDRs of PD-1 mAb 6 L 2 (SEQ ID NO: 135): PASNQGS CDRs of PD-1 mAb 6 L 3 (SEQ ID NO: 136): QQSKEVPWT

[0102] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 6 is SEQ ID NO: 133 (CDR L The nucleotides encoding the residues are underlined): gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc atctcctgc a gagccagcga aagtgttgat aattatggca ttagttttat gaac tggttc caacagaaac caggacagcc acccaaactc ctcatctat c ctgcatccaa ccaaggatcc ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat cctatggagg aggatgatgc tgcaatgtat ttctgt cagc aaagtaagga ggttccgtgg acg ttcggtg gaggcaccaa gctggaaatc aaa is.

[0103] G. Anti-human PD-1 antibody PD-1 mAb 7 1. Mouse anti-human PD-1 antibody PD-1 mAb 7 The amino acid sequence of the VH domain of PD-1 mAb 7 (SEQ ID NO: 137) is shown below: (CDR H Residues are underlined): QVQLQQPGAE LVRPGASVKL SCKASGYSFT SYWMN WVKQR PGQGLEWIG V IHPSDSETWL DQKFKD KATL TVDKSSTTAY MQLISPTSED SAVYYCAR EH YGTSPFAY WG QGTLVTVSS CDRs of PD-1 mAb 7 H 1 (SEQ ID NO: 139): SYWMN CDRs of PD-1 mAb 7 H 2 (SEQ ID NO: 140): VIHPSDSETWLDQKFKD CDRs of PD-1 mAb 7 H 3 (SEQ ID NO: 141): EHYGTSPFAY

[0104] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 7 is SEQ ID NO: 138 (CDR H The nucleotides encoding the residues are underlined): gaggtccaac tgcagcagcc tggggctgaa ctggtgaggc ctggagcttc agtgaagctg tcctgcaagg cttctggcta ctccttcacc agctactgga tgaac tgggt gaagcagagg cctggacaag gccttgagtg gattggc gtg attcatcctt ccgatagtga aacttggtta gatcagaagt tcaaggac aa ggccacattg actgtagaca aatcctccac cacagcctac atgcaactca tcagcccgac atctgaggac tctgcggtct attactgtgc aagg gagcac tacggtacta gcccgtttgc ttac tggggc caagggactc tggtcactgt gtcttcc is.

[0105] The amino acid sequence of the VL domain of PD-1 mAb 7 (SEQ ID NO: 142) is shown below: (CDR L Residues are underlined): DIVLTQSPAS LAVSLGQRAT ISC RANESVD NYGMSFMN WF QQKPGQPPKL LIH AASNQGS GVPARFSGSG FGTDFSLNIH PMEEDDAAMY FC QQSKEVPY T FGGGTKLEI K CDRs of PD-1 mAb 7 L 1 (SEQ ID NO: 144): RANESVDNYGMSFMN CDRs of PD-1 mAb 7 L 2 (SEQ ID NO: 145): AASNQGS CDRs of PD-1 mAb 7 L 3 (SEQ ID NO: 146): QQSKEVPYT

[0106] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 7 is SEQ ID NO: 143 (CDR L The nucleotides encoding the residues are underlined): gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc atctcctgc a gagccaacga aagtgttgat aattatggca tgagttttat gaac tggttc caacagaaac caggacagcc acccaaactc ctcatccat g ctgcatccaa ccaaggatccggggtccctg ccaggtttag tggcagtggg tttgggacag acttcagcct caacatccat cctatggagg aggatgatgc tgcaatgtat ttctgt cagc aaagtaagga ggttccgtac acg ttcggag gggggaccaa gctggaaata aaa is.

[0107] 2. Anti-human PD-1 antibody PD-1 mAb to form "hPD-1 mAb 7" Humanization of Ab 7 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 7 was used to identify the antigenic epitope. and further deimmunized to reduce its antigenicity upon administration to a human recipient. We have demonstrated the ability to humanize anti-human PD-1 antibodies to achieve the therapeutic effect of PD-1. Two antibodies designated "PD-1 mAb 7 VH1" and "hPD-1 mAb 7 VH2" were identified. Two humanized VH domains, referred to here as "PD-1 mAb 7 VL1" and "hPD-1 Three human mAb VL2, hPD-1 mAb VL3, and hPD-1 mAb VL4 were identified. Which of the humanized VL domains was the humanized VH domain? Therefore, the humanized VH domain paired with the humanized VH domain can be combined with any of the above-mentioned humanized VH domains. Any antibody comprising one of the VL domains is referred to as "hPD-1 mAb 7." , a particular combination of humanized VH / VL domains can be identified by a specific VH / VL domain. For example, hPD-1 mAb 7 VH1 and hPD-1 mAb The humanized antibody comprising b7 VL2 is specifically designated "hPD-1 mAb 7(1.2)" It is called.

[0108] The amino acid sequence of the VH domain of hPD-1 mAb 7 VH1 (SEQ ID NO: 147) As shown below (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMN WVRQA PGQGLEWIG V IHPSDSETWL DQKFKD RVTI TVDKSTSTAY MELSSLRSED TAVYYCAR EH YGTSPFAY WG QGTLVTVSS

[0109] An exemplary polynucleotide encoding hPD-1 mAb 7 VH1 has the sequence Number 148 (CDR H The nucleotides encoding the residues are underlined): caagttcaat tggtacagag cggggcagag gtgaagaaac ccggcgccag tgttaaggtg tcctgcaaag ccagcggtta cagctttaca agctattgga tgaat tgggt gcgtcaagca ccagggcagg gtctggaatg gattggg gtg atacatcctt ctgacagcga aacatggttg gaccagaaat ttaaagat CG tgtgacaatt acagtcgata agtccacaag cactgcttac atggaactct ccagcttgcg gtccgaggac accgctgtgt attattgcgc caga gagcac tacggcacat caccttttgc atac tggggc cagggaactc tcgtaaccgt atcctcc is.

[0110] The amino acid sequence of the VH domain of hPD-1 mAb 7 VH2 (SEQ ID NO: 149) As shown below (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMN WVRQA PGQGLEWAG V IHPSDSETWL DQKFKD RVTI TVDKSTSTAY MELSSLRSED TAVYYCAR EH YGTSPFAY WG QGTLVTVSS

[0111] An exemplary polynucleotide encoding hPD-1 mAb 7 VH2 has the sequence Number 150 (CDR H The nucleotides encoding the residues are underlined): caagttcaat tggtacagag cggggcagag gtgaagaaac ccggcgccag tgttaaggtg tcctgcaaag ccagcggtta cagctttaca agctattgga tgaat tgggt gcgtcaagca ccagggcagg gtctggaatg ggctggg gtg atacatcctt ctgacagcga aacatggttg gaccagaaat ttaaagat CG tgtgacaatt acagtcgata agtccacaag cactgcttac atggaactct ccagcttgcg gtccgaggac accgctgtgt attattgcgc caga gagcac tacggcacat caccttttgc atac tggggc cagggaactc tcgtaaccgt atcctcc is.

[0112] The amino acid sequence of the VL domain of hPD-1 mAb 7 VL1 (SEQ ID NO: 151) As shown below (CDR L Residues are underlined): EIVLTQSPAT LSLSPGERAT LSC RANESVD NYGMSFMN WF QQKPGQPPKL LIH AASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FC QQSKEVPY T FGGGTKVEI K

[0113] An exemplary polynucleotide encoding hPD-1 mAb 7 VL1 has the sequence Number 152 (CDR L The nucleotides encoding the residues are underlined): gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgc a gagcaaatga gagtgtggac aattacggca tgtccttcat gaac tggttt cagcagaagc ctgggcagcc acctaagctg ctcatccac g ccgcctctaa ccagggatct ggggtgcctt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgc cagc aatctaaaga ggtgccctat act tttggtg gcgggaccaa ggttgagatc aaa is.

[0114] The amino acid sequence of the VL domain of hPD-1 mAb 7 VL2 (SEQ ID NO: 153) As shown below (CDR L Residues are underlined): EIVLTQSPAT LSLSPGERAT LSC RASESVD NYGMSFMN WF QQKPGQPPKL LIH AASNQGSGVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FC QQSKEVPY T FGGGTKVEI K

[0115] An exemplary polynucleotide encoding hPD-1 mAb 7 VL2 has the sequence Number 154 (CDR L The nucleotides encoding the residues are underlined): gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgc a gagcaagtga gagtgtggac aattacggca tgtccttcat gaac tggttt cagcagaagc ctgggcagcc acctaagctg ctcatccac g ccgcctctaa ccagggatct ggggtgcctt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgc cagc aatctaaaga ggtgccctat act tttggtg gcgggaccaa ggttgagatc aaa is.

[0116] The amino acid sequence of the VL domain of hPD-1 mAb 7 VL3 (SEQ ID NO: 155) As shown below (CDR L Residues are underlined): EIVLTQSPAT LSLSPGERAT LSC RASESVD NYGMSFMN WF QQKPGQPPKL LIH AASNRGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FC QQSKEVPY T FGGGTKVEI K

[0117] An exemplary polynucleotide encoding hPD-1 mAb 7 VL3 has the sequence Number 156 (CDR L The nucleotides encoding the residues are underlined): gaaatcgtac tcacccagtc acctgcaacc ctttctctga gccccggtga acgtgccact ctcagctgc a gagcaagtga gagtgtggac aattacggca tgtccttcat gaac tggttt cagcagaagc ctgggcagcc acctaagctg ctcatccac g ccgcctctaa ccgcggatct ggggtgcctt cacgtttttc tggatcagga agtggcactg acttcaccct tacaatcagc tctctggagc cagaggactt tgccgtctat ttctgc cagc aatctaaaga ggtgccctat act tttggtg gcgggaccaa ggttgagatc aaa is.

[0118] Both the VL domains of hPD-1 mAb 7 VL2 and hPD-1 mAb 7 VL3 Main CDR L 1 contains an asparagine to serine amino acid substitution and has the amino acid sequence :RA S ESVDNYGMSFMN (SEQ ID NO: 157, the substituted serine is underlined) Similar substitutions can be made in the PD-1 mAb 7 CDRs described above. L 1 domain It is believed that this can be achieved.

[0119] Furthermore, the CDRs of the VL domain of hPD-1 mAb 7 VL3 L 2 is from glutamine Contains an amino acid substitution to arginine, and the amino acid sequence is: AASN R GS (SEQ ID NO: 158, substituted (The omitted arginine is underlined.) Similar substitutions were made in the PD-1 mAb 7 CDR L It is believed that it can be incorporated into either of the two domains.

[0120] H. Mouse anti-human PD-1 antibody PD-1 mAb 8 The amino acid sequence of the VH domain of PD-1 mAb 8 (SEQ ID NO: 159) is shown below: (CDR H Residues are underlined): EGQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMN WVKQN HGKSLEWIG D INPKNGDTHY NQKFKG EATL TVDKSSTTAY MELRSLTSED SAVYYCAS DF DY WGQGTTLT VSS CDRs of PD-1 mAb 8 H 1 (SEQ ID NO: 161): DYYMN CDRs of PD-1 mAb 8 H 2 (SEQ ID NO: 162): DINPKNGDTHYNQKFKG CDRs of PD-1 mAb 8 H 3 (SEQ ID NO: 163): DFDY

[0121] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 8 is SEQ ID NO: 160 (CDR H The nucleotides encoding the residues are underlined): gagggccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagata tcctgtaagg cttctggata cacgttcact gactactaca tgaac tgggt gaagcagaac catggaaaga gccttgagtg gattgga gat attaatccta aaaatggtga cactcactac aaccagaagt tcaagggc ga ggccacattg actgtagaca agtcctccac cacagcctac atggagctcc gcagcctgac atctgaggac tctgcagtct attactgtgc gagc gatttt gactac tggg gccaaggcac cactctcaca gtctcctcc

[0122] The amino acid sequence of the VL domain of PD-1 mAb 8 (SEQ ID NO: 164) is shown below: (CDR L Residues are underlined): DVVMTQTPLS LPVGLGDQAS ISC RSSQTLV YSNGNTYLN W FLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQSTHVP FT FGSGTKLE IK CDRs of PD-1 mAb 8 L 1 (SEQ ID NO: 166): RSSQTLVYSNGNTYLN CDRs of PD-1 mAb 8 L 2 (SEQ ID NO: 167): KVSNRFS CDRs of PD-1 mAb 8 L 3 (SEQ ID NO: 168): SQSTHVPFT

[0123] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 8 is SEQ ID NO: 165 (CDR L The nucleotides encoding the residues are underlined): gatgttgtga tgacccaaac tccactctcc ctgcctgtcg gtcttggaga tcaagcctcc atctcttgc a gatctagtca gacccttgta tatagtaatg gaaacaccta tttaaat tgg ttcctgcaga agccaggcca gtctccaaag ctcctgatct ac aaagtttc caaccgattt tct ggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtttatttctgc t ctcaaagtac acatgttcca ttcacg ttcg gctcggggac aaagttggaa ataaaa

[0124] I. Anti-human PD-1 antibody PD-1 mAb 9 1. Mouse anti-human PD-1 antibody PD-1 mAb 9 The amino acid sequence of the VH domain of PD-1 mAb 9 (SEQ ID NO: 169) is shown below: (CDR H Residues are underlined): EVMLVESGGG LVKPGGSLKL SCAASGFTFS SYLVS WVRQT PEKRLEWVA T ISGGGGNTYY SDSVKG RFTI SRDNAKNTLY LQISSLRSED TALYYCAR YG FDGAWFAY WG QGTLVTVSS CDRs of PD-1 mAb 9 H 1 (SEQ ID NO: 171): SYLVS CDRs of PD-1 mAb 9 H 2 (SEQ ID NO: 172): TISGGGGNTYYSDSVKG CDRs of PD-1 mAb 9 H 3 (SEQ ID NO: 173): YGFDGAWFAY

[0125] An exemplary polynucleotide encoding the VH Domain of PD-1 mAb 9 is SEQ ID NO: 170 (CDR HThe nucleotides encoding the residues are underlined): gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt agttatcttg tgtct tgggt tcgccagact ccggagaaga ggctggagtg ggtcgcaacc attagtggtg gtggtggtaa cacctactat tcagacagtg tgaagggt CG attcaccatc tccagagaca atgccaagaa caccctgtac ctgcaaatca gcagtctgag gtctgaggac acggccttgt attactgtgc aagg tatggt ttcgacggcg cctggtttgc ttac tggggc caagggactc tggtcactgt ctcttcc is.

[0126] The amino acid sequence of the VL domain of PD-1 mAb 9 (SEQ ID NO: 174) is shown below: (CDR L Residues are underlined): DIQMTQSPAS LSASVGDIVT ITC RASENIY SYLA WYQQKQ EKSPQLLVY N AKTLAA GVPS RFSGSGSGTQ FSLTINSLQP EDFGNYYC QH HYAVPWT FGG GTRLEIT CDRs of PD-1 mAb 9 L 1 (SEQ ID NO: 176): RASENIYSYLA CDRs of PD-1 mAb 9 L 2 (SEQ ID NO: 177): NAKTLAA CDRs of PD-1 mAb 9 L 3 (SEQ ID NO: 178): QHHYAVPWT

[0127] An exemplary polynucleotide encoding the VL Domain of PD-1 mAb 9 is SEQ ID NO: 175 (CDR L The nucleotides encoding the residues are underlined): gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga tattgtcacc atcacatgt c gagcaagtga gaatatttac agtatttag ca tggtatca gcagaaacag gaaaaatctc ctcagctcct ggtctat aat gcaaaaacct tggcagca gg tgtgccatca aggttcagtg gcagtggatc aggcacacag ttttctctga ccatcaacag cctgcagcct gaagattttg ggaattatta ctgt cagcat cattatgctg ttccgtggac g ttcggtgga ggcaccagac tggaaatcac a is.

[0128] 2. Anti-human PD-1 antibody PD-1 mAb to form "hPD-1 mAb 9" Humanization of Ab 9 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 9 was used to identify the antigenic epitope. and further deimmunized to reduce its antigenicity upon administration to a human recipient. We have demonstrated the ability to humanize anti-human PD-1 antibodies to achieve the therapeutic effect of PD-1. Two antibodies designated "PD-1 mAb 9 VH1" and "hPD-1 mAb 9 VH2" were identified. Two humanized VH domains, referred to herein as "hPD-1 mAb 9 VL1" and "hPD Two humanized VL domains, designated "VL-1 mAb 9 VL2" and "VL-1 mAb 9 VL2", were obtained. Any of the VL domains can be paired with any of the humanized VH domains. and one of the humanized VL domains paired with the humanized VH domain. This antibody is commonly referred to as "hPD-1 mAb 9" and contains humanized VH / VL domains. Particular combinations are referred to by reference to the specific VH / VL domains. Humanized hPD-1 mAb 9 VH1 and hPD-1 mAb 9 VL2 The antibody is specifically referred to as "hPD-1 mAb 9(1.2)."

[0129] The amino acid sequence of the VH domain of hPD-1 mAb 9 VH1 (SEQ ID NO: 179) As shown below (CDR H Residues are underlined): EVQLVESGGG LVRPGGSLKL SCAASGFTFS SYLVS WVRQA PGKGLEWVA T ISGGGGNTYY SDSVKG RFTI SRDNAKNSLY LQMNSLRAED TATYYCAR YG FDGAWFAY WG QGTLVTVSS

[0130] An exemplary polynucleotide encoding hPD-1 mAb 9 VH1 has the sequence Number 180 (CDR H The nucleotides encoding the residues are underlined): gaggtgcagc tggtggaaag tgggggcggc ctggtgcgac ccgggggaag tctgaaactg tcctgtgcag catcaggatt tactttttca tcttatctcg tgtct tgggt aagacaagca ccggaaaag gcttggaatg ggtggccact atctccggtg gaggtggcaa cacctactat agcgacagtg tcaaggga ag atttaccatc agtcgcgaca acgctaagaa tagcctgtac ctccagatga actccctgcg cgccgaggac accgccacct attactgtgc acgc tatgga tttgacggcg catggtttgc ctac tgggga cagggcacat tggtaaccgt tagctcc is.

[0131] The amino acid sequence of the VH domain of hPD-1 mAb 9 VH2 (SEQ ID NO: 181) As shown below (CDR H Residues are underlined): EVQLVESGGG LARPGGSLKL SCAASGFTFS SYLVG WVRQA PGKGLEWTA T ISGGGGNTYY SDSVKG RFTI SRDNAKNSLY LQMNSARAED TATYYCAR YG FDGAWFAY WG QGTLVTVSS

[0132] An exemplary polynucleotide encoding hPD-1 mAb 9 VH2 has the sequence Number 182 (CDR H The nucleotides encoding the residues are underlined): gaggtgcagc tggtggaaag tgggggcggc ctggcgcgac ccgggggaag tctgaaactg tcctgtgcag catcaggatt tactttttca tcttatctcg tgggc tgggt aagacaagca ccggaaaag gcttggaatg gacggcc act atctccggtg gaggtggcaa cacctactat agcgacagtg tcaaggga ag atttaccatc agtcgcgaca acgctaagaa tagcctgtac ctccagatga actccgcacg cgccgaggac accgccacct attactgtgc acgc tatgga tttgacggcg catggtttgc ctac tgggga cagggcacat tggtaaccgt tagctcc is.

[0133] CDRs of the VH domain of hPD-1 mAb 9 VH2 H 1. Serine to glycine The amino acid sequence is: SYLV G (SEQ ID NO: 183, substituted glycine (The "A" and "B" are underlined.) Similar substitutions are also found in the PD-1 mAb 9 CDRs described above. H It is believed that it can be incorporated into any of the 1 domains.

[0134] The amino acid sequence of the VL domain of hPD-1 mAb 9 VL1 (SEQ ID NO: 184) As shown below (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASENIY SYLA WYQQKP GKAPKLLIY N AKTLAA GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYAVPWT FGQ GTKLEIK

[0135] An exemplary polynucleotide encoding hPD-1 mAb 9 VL1 has the sequence Number 185 (CDR L The nucleotides encoding the residues are underlined): gacattcaga tgactcagtc tcccagcagt ctgtccgcat ccgtggggga tcgggtcacc atcacctgc c gtgcctcaga aaacatctat tcatacctcg cc tggtatca acagaaacct ggtaaagccc caaaattgct catttac aac gccaagaccc tcgcagct gg cgtgccaagt aggttctcag gcagcggctc agggacagat ttcaccctca ccatatcctc actgcagccc gaggattttg ccacttacta ctgc cagcat cattacgcag tgccctggac c ttcggacaa ggcactaagc tcgagatcaa a is.

[0136] The amino acid sequence of the VL domain of hPD-1 mAb 9 VL2 (SEQ ID NO: 186) As shown below (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASENIY NYLA WYQQKP GKAPKLLIY D AKTLAA GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYAVPWT FGQ GTKLEIK

[0137] An exemplary polynucleotide encoding hPD-1 mAb 9 VL2 has the sequence Number 187 (CDR L The nucleotides encoding the residues are underlined): gacattcaga tgactcagtc tcccagcagt ctgtccgcat ccgtggggga tcgggtcacc atcacctgc c gtgcctcaga aaacatctat aactacctcg cc tggtatca acagaaacct ggtaaagccc caaaattgct catttac gac gccaagaccc tcgcagct gg cgtgccaagt aggttctcag gcagcggctc agggacagat ttcaccctca ccatatcctc actgcagccc gaggattttg ccacttacta ctgc cagcat cattacgcag tgccctggac c ttcggacaa ggcactaagc tcgagatcaa a

[0138] CDRs of the VL domain of hPD-1 mAb 9 VL2 L 1. From Serine to Asparagus The amino acid sequence is: RASENIY N YLA (SEQ ID NO: 188, substituted The asparagine residues are underlined. Similar substitutions are also found in the PD-1 mAb 9 CDR L It is believed that it can be incorporated into any of the 1 domains.

[0139] CDRs of the VL domain of hPD-1 mAb 9 VL2 L 2 is from asparagine to a and the amino acid sequence: D AKTLAA (SEQ ID NO: 189, substitution The aspartic acid residues are underlined. mAb 7 CDR L It is believed that it can be incorporated into either of the two domains.

[0140] J. Mouse anti-human PD-1 antibody PD-1 mAb 10 The amino acid sequence of the VH domain of PD-1 mAb 10 (SEQ ID NO: 190) is shown below: (CDR H Residues are underlined): EVILVESGGG LVKPGGSLKL SCAASGFTFS NYLMSWVRQT PEKRLEWVA S ISGGGSNIYY PDSVKG RFTI SRDNAKNTLY LQMNSLRSED TALYYCAR QE LAFDY WGQGT TLTVSS CDRs of PD-1 mAb 10 H 1 (SEQ ID NO: 192): NYLMS CDRs of PD-1 mAb 10 H 2 (SEQ ID NO: 193): SISGGGSNIYYPDSVKG CDRs of PD-1 mAb 10 H 3 (SEQ ID NO: 194): QELAFDY

[0141] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 10 is , SEQ ID NO: 191 (CDR H Nucleotides encoding the residues are underlined) : gaagtgatac tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt aactatctca tgtct tgggt tcgccagact ccggagaaga ggctggagtg ggtcgca agt attagtggtg gtggtagtaa tatctactat ccagacagtg tgaagggt CG attcaccata tccagggaca atgccaagaa caccctgtac ctgcaaatga acagtctgag gtctgaggac acggccttgt attactgtgc aaga caagaa ctggcttttg actac tgggg ccaaggcacc actctcacag tctcctcc is.

[0142] The amino acid sequence of the VL domain of PD-1 mAb 10 (SEQ ID NO: 195) is shown below: (CDR L Residues are underlined): DIQMTQTTSS LSASLGDRVT ISC RTSQDIS NFLN WYQQKP DGTIKLLIY Y TSRLHS GVPS RFSGSGSGTD YSLTISNLEQ EDIATYFC QQ GSTLPWT FGG GTKLEII CDRs of PD-1 mAb 10 L 1 (SEQ ID NO: 197): RTSQDISNFLN CDRs of PD-1 mAb 10 L 2 (SEQ ID NO: 198): YTSRLHS CDRs of PD-1 mAb 10 L 3 (SEQ ID NO: 199): QQGSTLPWT

[0143] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 10 is , SEQ ID NO: 196 (CDR L Nucleotides encoding the residues are underlined) : gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc atcagttgc a ggacaagtca ggacattagc aattttttaa ac tggtatca gcagaaacca gatggaacta ttaaactcct gatctac tac acatcaagat tacactca gg agtcccatca aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa gaagatattg ccacttactt ttgc caacag ggtagtacgc ttccgtggac g ttcggtgga ggcaccaagc tggaaatcat a is.

[0144] K. Mouse anti-human PD-1 antibody PD-1 mAb 11 The amino acid sequence of the VH domain of PD-1 mAb 11 (SEQ ID NO: 200) is shown below: (CDR H Residues are underlined): EVQLQQSGTV LARPGASVKM SCKTSGYTFT GYWMH WVKQR PGQGLKWMG A IYPGNSDTHY NQKFKG KAKL TAVTSASTAY MELSSLTNED SAIYYCTT GT YSYFDV WGTG TTVTVSS CDRs of PD-1 mAb 11 H 1 (SEQ ID NO: 202): GYWMH CDRs of PD-1 mAb 11 H 2 (SEQ ID NO: 203): AIYPGNSDTHYNQKFKG CDRs of PD-1 mAb 11 H 3 (SEQ ID NO: 204): GTYSYFDV

[0145] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 11 is , SEQ ID NO: 201 (CDR H Nucleotides encoding the residues are underlined) : gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg tcctgcaaga cttctggcta cacatttacc ggctactgga tgcac tgggt aaaacagagg cctggacagagg gtctgaaatg gatgggg gct atttatcctg gaaatagtga tactcactac aaccagaagt tcaagggc aa ggccaaactg actgcagtca catccgccag cactgcctac atggagctca gcagcctgac aaatgaggac tctgcgatct attactgtac tact gggacc tactcgtact tcgatgtc tg gggcacaggg accacggtca ccgtctcctc a is.

[0146] The amino acid sequence of the VL domain of PD-1 mAb 11 (SEQ ID NO: 205) is shown below: (CDR L Residues are underlined): DILLTQSPAI LSVSPGERVS FSC RASQSIG TSIH WYQHRT NGSPRLLIK Y ASESIS GIPS RFSGSGSGTD FTLSINSVES EDIADYYC QQ SNSWLT FGAG TKLELK CDRs of PD-1 mAb 11 L 1 (SEQ ID NO: 207): RASQSIGTSIH CDRs of PD-1 mAb 11 L 2 (SEQ ID NO: 208): YASESIS CDRs of PD-1 mAb 11 L 3 (SEQ ID NO: 209): QQSNSWLT

[0147] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 11 is , SEQ ID NO: 206 (CDR L Nucleotides encoding the residues are underlined) : gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt ttctcctgc a gggccagtca gagcattggc acaagcatac actggtatca gcacagaaca aatggttctc caaggcttct cataaag tat gcttctgagt ctatctct gg gatcccttcc aggtttagtg gcagtggatc agggactgat tttactctta gcatcaacag tgtggagtct gaagatattg cagattatta ctgt caacaa agtaatagct ggctcacg tt cggtgctggg accaagctgg agctgaaa

[0148] L. Mouse anti-human PD-1 antibody PD-1 mAb 12 The amino acid sequence of the VH domain of PD-1 mAb 12 (SEQ ID NO: 210) is shown below: (CDR H Residues are underlined): QGHLQQSGAE LVRPGASVTL SCKASGFTFT DYEMH WVKQT PVHGLEWIG T IDPETGGTAY NQKFKG KAIL TVDKSSTTTY MELRSLTSED SAVFYCSR ER ITTVVEGAYW YFDV WGTGTT VTVSS CDRs of PD-1 mAb 12 H 1 (SEQ ID NO: 212): DYEMH CDRs of PD-1 mAb 12 H 2 (SEQ ID NO: 213): TIDPETGGTAYNQKFKG CDRs of PD-1 mAb 12 H 3 (SEQ ID NO: 214): ERITTVVEGAYWYFDV

[0149] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 12 is , SEQ ID NO: 211 (CDR H Nucleotides encoding the residues are underlined) : cagggtcacc tgcagcagtc tggggctgag ctggtgaggc ctggggcttc agtgacgctg tcctgcaagg cttcgggctt cacatttact gactatgaga tgcac tgggt gaaacagaca cctgtgcatg gcctggaatg gattggg act attgatcctg aaactggtgg tactgcctac aatcagaagt tcaagggc aa ggccatactg acagtagaca aatcttccac tacaacctac atggagctcc gcagcctgac atctgaggac tctgccgtct tttatgttc aaga gagagg attactacgg ttgttgaggg ggcatactgg tacttcgatg tc tggggcac agggaccacg gtcaccgtct cctca is.

[0150] The amino acid sequence of the VL domain of PD-1 mAb 4 (SEQ ID NO:215) is shown below: (CDR L Residues are underlined): DVLMTQTPLS LPVSLGDQAS ISC RSSQNIV HSNGNTYLE W YLQKPGQSPK LLIC KVSTRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHVP YT FGGGTKLE IK CDRs of PD-1 mAb 12 L 1 (SEQ ID NO: 217): RSSQNIVHSNGNTYLE CDRs of PD-1 mAb 12 L 2 (SEQ ID NO: 218): KVSTRFS CDRs of PD-1 mAb 12 L 3 (SEQ ID NO: 219): FQGSHVPYT

[0151] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 12 is , SEQ ID NO: 216 (CDR L Nucleotides encoding the residues are underlined) : gatgttttga tgacccagac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgc a gatctagtca gaacattgta catagtaatg gaaacaccta tttagaa tgg tacctgcaga aaccaggcca gtctccaaag ctcctgatct gc aaagtttc cacccgattt tct ggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tattattgc t ttcaaggttc acatgttccg tacacg ttcg gaggggggac caagctggaa ataaaa is.

[0152] M. Mouse anti-human PD-1 antibody PD-1 mAb 13 The amino acid sequence of the VH domain of PD-1 mAb 13 (SEQ ID NO: 220) is shown below: (CDR H Residues are underlined): EVMLVESGGG LVKPGGSLKL SCAASGFTFS SHTMS WVRQT PEKRLEWVA T ISGGGSNIYY PDSVKG RFTI SRDNAKNTLY LQMSSLRSED TALYYCAR QA YYGNYWYFDV WGTGTTVTVS S CDRs of PD-1 mAb 13 H 1 (SEQ ID NO: 222): SHTMS CDRs of PD-1 mAb 13H 2 (SEQ ID NO: 223): TISGGGSNIYYPDSVKG CDRs of PD-1 mAb 13 H 3 (SEQ ID NO: 224): QAYYGNYWYFDV

[0153] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 13 is , SEQ ID NO: 221 (CDR H Nucleotides encoding the residues are underlined) : gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt agccatacca tgtct tgggt tcgccagact ccggagaaga ggctggagtg ggtcgca acc attagtggtg gtggttctaa tatctactat ccagacagtg tgaagggtcg a ttcaccatc tccagagaca atgccaagaa caccctgtac ctgcaaatga gcagtctgag gtctgaggac acggccttgt attactgtgc aaga caagct tactcggta attack cttcgatgtc tggggcacag ggaccacggt caccgtctcc tcc is.

[0154] The amino acid sequence of the VL domain of PD-1 mAb 13 (SEQ ID NO: 225) is shown below: (CDR L Residues are underlined): DIQMTQSPAT QSASLGESVT ITC LASQTIG TWLA WYQQKP GKSPQLLIY A ATSLADGVPS RFSGSGSGTK FSFKISSLQA EDFVSYYC QQ LDSIPWT FGG GTKLEIK CDRs of PD-1 mAb 13 L 1 (SEQ ID NO: 227): LASQTIGTWLA CDRs of PD-1 mAb 13 L 2 (SEQ ID NO: 228): AATSLAD CDRs of PD-1 mAb 13 L 3 (SEQ ID NO: 229): QQLDSIPWT

[0155] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 13 is , SEQ ID NO: 226 (CDR L Nucleotides encoding the residues are underlined) : gacattcaga tgacccagtc tcctgccacc cagtctgcat ctctgggaga aagtgtcacc atcacgtgc c tggcaagtca gaccattggt acatggttag ca tggtatca gcagaaacca gggaaatctc ctcagctcct gatttat gct gcaaccagct tggcagat gg ggtcccatca aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct gaagattttg taagttatta ctgt cacao cttgacagta ttccgtggac g ttcggtgga ggcaccaagc tggaaatcaa a is.

[0156] N. Mouse anti-human PD-1 antibody PD-1 mAb 14 The amino acid sequence of the VH domain of PD-1 mAb 14 (SEQ ID NO: 230) is shown below: (CDR HResidues are underlined): QVQLQQPGAE LVKPGASVKM SCKASGYNFI SYWIT WVKQR PGQGLQWIG N IYPGTDGTTY NEKFKS KATL TVDTSSSTAY MHLSRLTSED SAVYYCAT GL HWYFDV WGTG TTVTVSS CDRs of PD-1 mAb 14 H 1 (SEQ ID NO: 232): SYWIT CDRs of PD-1 mAb 14 H 2 (SEQ ID NO: 233): NIYPGTDGTTYNEKFKS CDRs of PD-1 mAb 14 H 3 (SEQ ID NO: 234): GLHWYFDV

[0157] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 14 is , SEQ ID NO: 231 (CDR H Nucleotides encoding the residues are underlined) : caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaagatg tcctgcaagg cttctggcta caacttcatc agctactgga taacc tgggt gaaacagagg cctggacaag gccttcagtg gattgga aat atttatcctg gtactgatgg tactacctac aatgagaagt tcaagagc aa ggccacactg actgtagaca catcctccag cacagcctac atgcacctca gtcgcctgac atctgaggac tctgcggtct attactgtgc aact gggcta cactggtact tcgatgtctg gggcacaggg accacggtca ccgtctcctc c is.

[0158] The amino acid sequence of the VL domain of PD-1 mAb 14 (SEQ ID NO: 235) is shown below: (CDR L Residues are underlined): DIVMTQSQKF MSTSVGDRVS VTC KASQSVG TNVA WYQQKP GQSPKALIY S ASSRFS GVPD RFTGSGSGTD FTLTISNVQS EDLAEYFC QQ YNSYPYT FGG GTKLEIK CDRs of PD-1 mAb 14 L 1 (SEQ ID NO: 237): KASQSVGTNVA CDRs of PD-1 mAb 14 L 2 (SEQ ID NO: 238): SASSRFS CDRs of PD-1 mAb 14 L 3 (SEQ ID NO: 239): QQYNSYPYT

[0159] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 14 is , SEQ ID NO: 236 (CDR L Nucleotides encoding the residues are underlined) : gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagt gtcacctgc a aggccagtca gagtgtgggt actaatgtag cc tggtatca acagaagccc ggtcaatctc ctaaagcact gatttac tcg gcatcctccc gattcagt gg cgtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcagtaa tgtgcagtct gaagacttgg cagagtattt ctgt from your fatheratccgtacac g ttcggaggg gggaccaagc tggaaataaa a is.

[0160] O. Anti-human PD-1 antibody PD-1 mAb 15 1. Mouse anti-human PD-1 antibody PD-1 mAb 15 The amino acid sequence of the VH domain of PD-1 mAb 15 (SEQ ID NO: 240) is shown below: (CDR H Residues are underlined): EVMLVESGGG LVKPGGSLKL SCAASGFIFS SYLIS WVRQT PEKRLEWVA A ISGGGADTYY ADSVKG RFTI SRDNAKNTLY LQMSSLRSED TALYYCTR RG TYAMDY WGQG TSVTVSS CDRs of PD-1 mAb 15 H 1 (SEQ ID NO: 242): SYLIS CDRs of PD-1 mAb 15 H 2 (SEQ ID NO: 243): AISGGGADTYYADSVKG CDRs of PD-1 mAb 15 H 3 (SEQ ID NO: 244): RGTYAMDY

[0161] An exemplary polynucleotide encoding the PD-1 mAb 15 VH Domain is SEQ ID NO: 241 (CDR H The nucleotides encoding the residues are underlined): gaagtgatgc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cattttcagt agctatctca tctct tgggt tcgccagact ccggagaaga ggctggagtg ggtcgct gcc attagtggtg gtggtgctga cacctactat gccgacagtg tgaagggt CG attcaccatc tccagagaca atgccaagaa caccctgtat ctgcaaatga gcagtctgag gtctgaggac acggccttat attactgtac aaga cgaggg acctatgcta tggactac tg gggtcaagga acctcagtca ccgtctcctc c is.

[0162] The amino acid sequence of the VL domain of PD-1 mAb 15 (SEQ ID NO: 245) is shown below: (CDR L Residues are underlined): DIQMTQSPAS QSASLGESVT ITC LASQTIG TWLA WYQQKP GKSPQLLIY A ATSLAD GVPS RFSGSGSGTK FSFKISSLQA EDFVNYYC QQ LIGHTINGWT FGG GTKLEIK CDRs of PD-1 mAb 15 L 1 (SEQ ID NO: 247): LASQTIGTWLA CDRs of PD-1 mAb 15 L 2 (SEQ ID NO: 248): AATSLAD CDRs of PD-1 mAb 15 L 3 (SEQ ID NO: 249): QQLYSIPWT

[0163] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 15 is , SEQ ID NO: 246 (CDR LNucleotides encoding the residues are underlined) : gacattcaga tgacccagtc tcccgcctcc cagtctgcat ctctgggaga aagtgtcacc atcacatgc c tggcaagtca gaccattggt acatggttag ca tggtatca gcagaaacca gggaaatctc ctcagctcct gatttat gct gcaaccagct tggcagat gg ggtcccatca aggttcagtg gtagtggatc tggcacaaaa ttttctttca agatcagcag cctacaggct gaagattttg taaattatta ctgt cacao ctttacagta ttccgtggac g ttcggtgga ggcaccaagc tggaaatcaa a is.

[0164] 2. Anti-human PD-1 antibody PD-1 to form "hPD-1 mAb 15" Humanization of mAb 15 The above-mentioned murine anti-human PD-1 antibody PD-1 mAb 15 was used to identify the antigenic epitope. When the antibody is administered to a human recipient, it is humanized and further deimmunized to reduce its antigenicity. We have demonstrated the ability of humanizing anti-human PD-1 antibodies to reduce the incidence of PD-1-associated leukemia. hPD-1 mAb 2 VH1” and one humanized VH domain, referred to herein as “ One humanized VL domain, designated "hPD-1 mAb 1 VL1," was obtained. The antibody comprising the VL domain paired with the humanized VH domain is referred to as "hPD-1 mAb." It is called "b 15".

[0165] Amino acid sequence of the VH domain of hPD-1 mAb 15 VH1 (SEQ ID NO: 250) is shown below (CDR H Residues are underlined): EVQLVESGGG LVRPGGSRLL SCAASGFTFS SYLIS WVRQA PGKGLEWVA A ISGGGADTYY ADSVKG RFTI SRDNAKNSLY LQMNSLRAED TATYYCAR RG TYAMDY WGQG TLVTVSS

[0166] An exemplary polynucleotide encoding hPD-1 mAb 15 VH1 is Column number 251 (CDR H The nucleotides encoding the residues are underlined): gaagtgcaac tggttgaaag tggcggcggg ctggtgcggc caggtggttc actcagactg tcttgtgcag cttcaggctt tacattctcc tcttatctta tctct tgggt gcgccaagcc ccaggtaagg gccttgaatg ggtc gccgcc attagtgggg gtggtgccga tacatattat gccgacagcg tcaaggga CG tttcaccatc agcagggaca acgccaagaa tagcctttac ctgcagatga actcacttag agctgaagac accgctactt attactgtgc ccgg cgcggg acttacgcta tggactat tg gggccagggc accttggtca ctgtctcatc c

[0167] Amino acid sequence of the VL domain of hPD-1 mAb 15 VL1 (SEQ ID NO: 252) is shown below (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC LASQTIG TWLA WYQQKP GKAPKLLIY A ATSLAD GVPS RFSGSGSGTD FTFTISSLQP EDFATYYC QQ LIGHTINGWT FGQ GTKLEIK

[0168] An exemplary polynucleotide encoding hPD-1 mAb 15 VL1 is Column number 253 (CDR H The nucleotides encoding the residues are underlined): gatatccaga tgacccagtc tcccagctct ctcagtgcaa gcgtaggcga ccgtgtgacc atcacctgt c tggccagtca gaccattgga acctggctcg cc tggtatca gcagaaacct ggcaaggccc ctaagctgct gatttacgcc gccacctccc tcgcagat gg agtgccctcc cgatttagcg ggtccgggtc cggcaccgac ttcacattca caatcagcag cctccagccc gaggatttcg ctacatacta ctgt caacag ctctactcca ttccatggac c tttggtcag ggtactaaac tggagatcaa a

[0169] V. Anti-Human PD-1 Antibodies PD-1 mAb 1-15 and PD-1 mAb 1-15 with Engineered Fc Regions Its derivatives In conventional immune function, the interaction between antibody-antigen complexes and cells of the immune system is called antibody-dependent cellular proliferation. From effector functions such as cytotoxicity, mast cell degranulation and phagocytosis to lymphocyte proliferation and antibody secretion These interactions result in a wide range of responses, ranging from immune-regulatory signals such as those that control immune responses. All of these are directed against specialized cell surface receptors on hematopoietic cells, such as the Fc region of an antibody or immune complex. The diversity of cellular responses triggered by antibodies and immune complexes The Fc receptors are FcγRI (CD64), FcγRII (CD32), and Fc This is due to the structural heterogeneity of FcγRIII (CD16). cγRIIA (CD32A) and FcγRIII (CD16) are activating (i.e., enhancing the immune system) ) receptor; FcγRIIB (CD32B) is an inhibitory (i.e., immune system attenuating) receptor Furthermore, interaction with the neonatal Fc receptor (FcRn) promotes delivery of FcRn from endosomes to the cell surface. The IgG1 gene mediates the recycling and release of IgG molecules into the blood. 1), IgG2 (SEQ ID NO: 2), IgG3 (SEQ ID NO: 3) and IgG4 (SEQ ID NO: 4) The amino acid sequence has already been presented.

[0170] Modifications of the Fc region typically result in phenotypic changes, such as altered serum half-life, altered stability, and cell proliferation. This leads to altered sensitivity to enzymes or altered effector functions. Binding molecules may be modified with respect to effector function to enhance the efficacy of such molecules, for example, in the treatment of cancer. It may be desirable to enhance efficacy. For example, the mechanism of action may be to enhance the efficacy of a compound carrying a target antigen. In certain cases, such as antibodies with blocking or antagonizing effects rather than cell killing, Therefore, it is desirable to reduce or eliminate effector function. Tumor and foreign cells express low levels of FcγRIIB, e.g., tumor-specific B cells (e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma), In the above embodiment, effector function activity is generally desirable when targeting cells that do not have effector function activity. The molecules of the invention, given or modified, are desired to have enhanced efficacy of effector function activity. It is useful for the treatment and / or prevention of a disease, disorder or infection.

[0171] In certain embodiments, the PD-1-binding molecules of the invention comprise a wild-type Fc region (e.g., SEQ ID NO: 1) having one or more modifications (e.g., substitutions, deletions, or insertions) to the amino acid sequence of and the modification is in one or more of the Fc regions of the Fc region and thus of the molecule of the invention. In another embodiment, the present invention provides a method for the production of a medicament for the treatment ... The molecule comprises an Fc region having one or more modifications to the amino acids of the wild-type Fc region. The modifications may also enhance the activity of one or more FcγR receptors of the Fc region and, therefore, of the molecules of the invention. In another embodiment, the molecule comprises a variant Fc region, wherein the variant does not include an Fc domain or has a wild-type Fc domain. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity against molecules containing In an alternative embodiment, the molecule confers or mediates increased binding to FcγRIIA. and a variant Fc region, wherein the variant does not contain an Fc region or a wild-type Fc region. and / or In some embodiments, the present invention provides or mediates increased binding to FcγRIIB. The present invention encompasses PD-1 binding molecules comprising a variant Fc region, wherein the variant Fc region is a wild-type It does not exhibit detectable binding to any FcγR compared to comparable molecules containing an Fc region. In another embodiment, the invention encompasses PD-1-binding molecules comprising a variant Fc Region, The variant Fc region may be a single FcγR, preferably FcγRIIA, FcγRIIB, or Such increased affinity and / or The binding activity is preferably greater than that of the parent molecule (without the modified Fc region) in the cell. in cells expressing low levels of FcγR when no binding activity of the FcγR is detectable, or Density: 0,000–20,000 molecules / cell, 20,000–10,000 molecules / cell, 1 Density: 0000-5000 molecules / cell, 5000-1000 molecules / cell, 1000 A density of ~200 molecules / cell, or less than 200 molecules / cell (but at least 10, 5 Cells expressing non-FcγR receptor target antigens at densities of 0, 100, or 150 molecules / cell were cultured. The degree of detectable FcγR binding or FcγR-associated activity is measured in vitro in cells. It is evaluated by determining the

[0172] The PD-1 binding molecules of the present invention have altered affinities for activating and / or inhibitory Fcγ receptors. In one embodiment, the PD-1-binding molecule may comprise a wild-type variant Fc Region. Increased affinity for FcγRIIB relative to equivalent molecules with an Fc region, and a variant Fc region with reduced affinity for FcγRIIIA and / or FcγRIIA; In another embodiment, the PD-1-binding molecules of the present invention comprise an equivalent PD-1-binding molecule comprising a wild-type Fc region. molecules, with reduced affinity for FcγRIIB and FcγRIIIA and / or or a variant Fc region having increased affinity for FcγRIIA. In one embodiment, the PD-1-binding molecules of the invention have a PD-1-binding domain that is substantially identical to that of a comparable molecule with a wild-type Fc Region: Decreased affinity for FcγRIIB and FcγRIIIA and / or FcγRII In yet another embodiment, the present invention provides a variant Fc region that also has reduced affinity for A. PD-1 binding molecules bind to FcγRIIB more effectively than comparable molecules with wild-type Fc regions. The affinity for FcγRIIIA and / or FcγRIIA is unchanged. The antibody comprises a variant Fc region with reduced (or increased) affinity for the antibody.

[0173] In certain embodiments, the PD-1-binding molecules of the invention comprise immunoglobulin-enhanced PD-1-binding molecules. affinity for FcγRIIIA and / or FcγRIIA so as to have effector function Non-limiting examples of effector cell functions include variant Fc regions with altered Fc domains. persistent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, phagocytosis, opsonization, opsonophagocy These include cytotoxicity, cell binding, rosette formation, C1q binding, and complement-determining cell-mediated cytotoxicity. can be.

[0174] In a preferred embodiment, the altered affinity or effector function is a variant of the wild-type Fc region. At least 2-fold, preferably at least 4-fold, at least 5-fold, less than equivalent molecules At least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, At least 50-fold, or at least 100-fold. In other embodiments of the invention, the mutant form The Fc region is at least 65%, preferably at least 10%, of a molecule containing a wild-type Fc region. Also 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, Immunoglobulins that bind to one or more FcRs with 175%, 200%, 225%, or 250% greater affinity Such measurements may be in vivo or in vitro assays. In a preferred embodiment, in vitro assays such as ELISA or surface plasmon resonance assays are used. It is a low assay.

[0175] In various embodiments, the PD-1-binding molecules of the invention comprise a variant Fc Region, The variants stimulate at least one activity of an FcγR receptor or inhibit the activity of an FcγR receptor. In a preferred embodiment, the molecule antagonizes at least one activity of FcγRIIB. one or more activities of, for example, B cell receptor-mediated signaling, B cell activation, B cell proliferation, antibody production, B cell intracellular calcium influx, cell cycle progression, FcεRI signaling FcγRIIB-mediated inhibition of nucleation, phosphorylation of FcγRIIB, SHIP recruitment, S HIP phosphorylation and ligation with Shc, or one or more of the FcγRIIB signaling pathways or the activity of multiple downstream molecules (e.g., MAP kinase, JNK, p38, or Akt). In another embodiment, the PD-1-binding molecules of the present invention comprise antagonizing variants of FcεRI. One or more activities, e.g., mast cell activation, calcium mobilization, degranulation, cytokines Includes variants that stimulate the production or release of serotonin.

[0176] In certain embodiments, the molecule comprises two or more IgG isotypes (e.g., IgG1, The Fc region-containing region of an IgG1 antibody is an Fc region-containing region from an IgG1 antibody (IgG2, IgG3, and IgG4). When used, the Fc region has an amino acid sequence that is different from that of other IgG isotypes. If it has the highest homology with a particular IgG isotype, The various IgG isotypes are described, for example, by Flesch and Nepper t (1999) J. Clin. Lab. Anal. 14: 141-156; Chappel et al. (1993) J. Biol. Chem. 3 3:25124-25131; Chappel et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:9036-904 0; or as described in Bruggemann et al. (1987) J. Exp. Med 166: 1351-1361 , differences in the amino acid sequences of the hinge and / or Fc region can affect serum half-life, complement fixation, Various assays, including FcγR binding affinity and effector function activity (e.g., ADCC, CDC, etc.) This type of variant Fc region can be produced either alone or by combining amino acids. modifications to affect Fc-mediated effector function and / or binding activity. In combination, the amino acid modifications and the IgG hinge / Fc region can be It can exhibit similar functionality (e.g., increased affinity for FcγRIIA) and and act additively, or more preferably synergistically, to enhance the Fc-dependent activity of the molecules of the present invention, as compared to the molecules of the present invention comprising a wild-type Fc region. In another embodiment, amino acid modifications can be used to modify the effector functionality of the molecules of the invention. and IgG Fc regions have opposite functionalities (e.g., affinity for FcγRIIA, respectively). and antibodies of the same isotype that do not contain an Fc region or that do not contain an Fc region. selectively enhances the specific functionality of the molecules of the invention compared to molecules of the invention comprising a wild-type Fc region. It can act to regulate or decrease.

[0177] In a preferred specific embodiment, the PD-1-binding molecules of the invention comprise a variant Fc Region: the variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region; This allows the mutant Fc regions to be synthesized as described in Sondermann et al. (2000) Nature 406:267-73. Based on crystallographic and structural analyses of Fc-FcR interactions such as those shown The parental affinity of the molecule to FcγR is low when it does not have substitutions at positions that directly contact FcγR. An example of a position within the Fc region that makes direct contact with FcγR is amino acid residue 234 ~239), amino acid residues 265-269 (B / C loop), amino acid residues 297-29 9 (C' / E loop) and amino acid residues 327-332 (F / G loop). In some embodiments, the molecules of the invention bind directly to FcγRs based on structural and crystallographic analysis. It involves modification of at least one residue that does not contact, e.g., is not within, the Fc-FcγR binding site. Including variant Fc regions.

[0178] Variant Fc regions are known in the art and include, for example, NK-dependent or macrophage-dependent Fc regions. The Fc region (or parts thereof) as functionally assayed in a peptide-dependent assay In order to confer or modify the effector function exhibited by the molecule of the present invention comprising the Known variant Fc regions may be used in the present invention, e.g., those with altered effector function. The Fc region variants identified as / 088494;WO 07 / 024249;WO 06 / 113665 ;WO 07 / 021841;WO 07 / 106707; and WO 2008 / 140603 and any of the preferred mutations disclosed therein. The body may be used in the molecules of the present invention.

[0179] In certain embodiments, the PD-1-binding molecules of the invention comprise one or more PD-1-binding domains. or a variant Fc region having multiple amino acid modifications, wherein the one or more modifications are Activation FcγR (FcγRIIB, etc.) versus affinity of a heterologous Fc region to inhibitory FcγR The ratio of affinity to a specific FcγR (such as FcγRIIA or FcγRIIIA) was calculated using the wild-type FcγR c) change:

[0180]

number

[0181] A mutant Fc region with an affinity ratio of the mutant Fc region (relative to the wild-type Fc region) greater than 1. Particularly preferred are PD-1 binding molecules of the invention that have a specific targeting region. Such molecules may be used to treat, for example, cancer or Increased efficiency of FcγR-mediated effector cell functions (e.g., ADCC) such as infection Therapeutic or prophylactic treatment of a disease, disorder, or infection for which treatment is desired, or the alleviation of symptoms thereof. In contrast, mutations with affinity ratios less than 1 Atypical Fc regions mediate reduced efficiency of effector cell function. Table 1 shows exemplary single, Double, triple, quadruple, and quintuple mutations were analyzed to determine whether their affinity ratios were greater than or less than 1. are listed by.

[0182] [Table 1]

[0183] In a specific embodiment, the variant Fc region comprises 235, 240, 241, 24 3, 244, 247, 262, 263, 269, 298, 328, or 330th place Any amino acid modification (e.g., substitution) in any of the following residues: A2 40, I240, L241, L243, H244, N298, I328 or V330 In a different specific embodiment, the variant Fc region has one or more of: 69, 270, 272, 276, 278, 283, 285, 286, 289, 292, 2 93, 301, 303, 305, 307, 309, 331, 333, 334, 335, 3 37, 338, 340, 360, 373, 376, 416, 419, 430, 434, 4 Any amino acid modification at any of positions 35, 437, 438 or 439 (e.g. (e.g., substitution), preferably the following residues: H280, Q280, Y280, G290, S290 , T290, Y290, N294, K295, P296, D298, N298, P298 , V298, I300 or L300.

[0184] In a preferred embodiment, a variant Fc region that binds to an FcγR with altered affinity , 255, 256, 258, 267, 268, 269, 270, 272, 276, 2 78, 280, 283, 285, 286, 289, 290, 292, 293, 294, 2 95, 296, 298, 300, 301, 303, 305, 307, 309, 312, 3 20, 322, 326, 329, 330, 332, 331, 333, 334, 335, 3 37, 338, 339, 340, 359, 360, 373, 376, 416, 419, 4 Any a at any of positions 30, 434, 435, 437, 438 or 439 amino acid modifications (e.g., substitutions). Preferably, the variant Fc region comprises one of the following residues: Contains one of: A256, N268, Q272, D286, Q286, S286, A290, S290, A298, M301, A312, E320, M320, Q320, R320, E322, A326, D326, E326, N326, S326, K330, T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335, Q335, A359, A360 or A430.

[0185] In a different embodiment, it binds to FcγR (via its Fc region) with reduced affinity In the variant Fc region, 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, or any amino acid modification (e.g., substitution) at any of the 439 positions.

[0186] In a different embodiment, a nucleotide sequence that binds to FcγR (via its Fc region) with enhanced affinity is In the mutant Fc region, 280, 283, 285, 286, 290, 294, 295 , 298, 300, 301, 305, 307, 309, 312, 315, 331, 333 , 334, 337, 340, 360, 378, 398, or 430th place In a different embodiment, any amino acid modification (e.g., substitution) in the F In a variant Fc region that binds cγRIIA, any one of the following residues: A 255, A256, A258, A267, A268, N268, A272, Q272, A 276, A280, A283, A285, A286, D286, Q286, S286, A 290, S290, M301, E320, M320, Q320, R320, E322, A 326, D326, E326, S326, K330, A331, Q335, A337 or A430.

[0187] Preferred variants are 228, 230, 231, 232, 233, 234, 235, 23 9, 240, 241, 243, 244, 245, 247, 262, 263, 264, 26 5, 266, 271, 273, 275, 281, 284, 291, 296, 297, 29 8, 299, 302, 304, 305, 313, 323, 325, 326, 328, 33 Contains one or more modifications at either position 0 or 332.

[0188] Particularly preferred variants include one or more modifications selected from groups A-AI:

[0189] [Table 2]

[0190] Further particularly preferred variants include one or more modifications selected from groups 1 to 105:

[0191] [Table 3] JPEG2024178244000006.jpg83163

[0192] In one embodiment, the multi-PD-1 binding molecules of the invention comprise at least one PD-1-binding domain in the Fc region. In certain embodiments, the variant Fc domain comprises a variant Fc region having a modification: Consists of L235V, F243L, R292P, Y300L, V305I, and P396L It contains at least one substitution selected from the group:

[0193] In a specific embodiment, the variant Fc region comprises: (A) A group consisting of F243L, R292P, Y300L, V305I, and P396L at least one substitution selected from; (B)(1)F243L and P396L; (2) F243L and R292P; and (3) R292P and V305I; at least two substitutions selected from the group consisting of: (C)(1) F243L, R292P, and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P, and P396L; and (4) R292P, V305I and P396L; at least three substitutions selected from the group consisting of: (D)(1) F243L, R292P, Y300L, and P396L; and (2) F243L, R292P, V305I and P396L; at least four substitutions selected from the group consisting of: (E)(1) F243L, R292P, Y300L, V305I, and P396L; and to (2) L235V, F243L, R292P, Y300L and P396L At least five substitutions selected from the group consisting of:

[0194] In another specific embodiment, the variant Fc region comprises the following substitutions: (A) F243L, R292P, and Y300L; (B) L235V, F243L, R292P, Y300L, and P396L; or (C) F243L, R292P, Y300L, V305I, and P396L.

[0195] In one embodiment, the PD-1-binding molecules of the invention comprise a PD-1-binding molecule comprising a wild-type IgG1 Fc region (SEQ ID NO: 1). No. 1) exhibits binding similar to that exhibited by FcγRIA (CD64), FcγRIIA (CD32A) ), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRII It has a mutant Fc region that has reduced (or almost no) binding to IB (CD16b). In one embodiment, the PD-1-binding molecules of the present invention bind to FcγR (e.g., FcγRIII). A) and ADCC effector function was reduced. In certain embodiments, the variant Fc region has a variant Fc region that does not have the above function. The Fc region consists of L234A, L235A, D265A, N297Q, and N297G. In a specific embodiment, the variant comprises at least one substitution selected from the group consisting of: The Fc region is: L234A; L235A; L234A and L235A; D265A; N29 Contains the 7Q or N297G substitution.

[0196] Preferred IgG1 Sequences for the CH2 and CH3 Domains of the PD-1-Binding Molecules of the Invention has a L234A / L235A substitution (SEQ ID NO: 5): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X where X is lysine (K) or absent.

[0197] In a different embodiment, the PD-1-binding molecules of the invention comprise a wild-type IgG1 Fc region (sequence Reduced binding to FcγRIIIA (CD16a) (relative to binding shown in column 1) It has an Fc region that does not bind (or barely binds) and / or has reduced effector function. In a specific embodiment, the PD-1-binding molecules of the present invention comprise an IgG2 Fc region (SEQ ID NO:2 ) or IgG4 Fc region (SEQ ID NO: 4). When an IgG4 Fc region is used, The present invention provides an IgG4 hinge region S228P substitution (e.g., For example, SEQ ID NO: 13: ESKYGPPCP P CP(Lu et al., (2008) “The Effect Of A Point Mutati on On The Stability Of Igg4 As Monitored By Analytical Ultracentrifugation,” J. This also includes the introduction of stabilizing mutations such as those described in Pharmaceutical Sciences 97:960-969. Other stabilizing mutations known in the art can be introduced into the IgG4 Fc region to (Peters, P et al., (2012) “Engineering an Improved IgG4 Molecule with Re duced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability,” J. Biol. Chem., 287:24525-24533; WO 2008 / 145142).

[0198] Other references include:Jefferis, BJ et al. (2002) Interaction Sites On Human IgG-Fc For FcgammaR: Current Models," Immunol. Lett. 82:57-65; Presta, LG . . . . et al. (2002) "Engineering Therapeutic Antibodies For Improved Function " Bioc hem. Soc. Trans. 30:487-90; Idusogie, EE et al. (2001) "Engineered Antibodies With Increased Activity To Recruit Complement," J. Immunol. 166:2571-75; Shields , RL et al. (2001) "High Resolution Mapping of the Binding Site on Human IgGl." For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And Design Of IgGl Varian ts With Improved Binding To The Fc Gamma R " J. Biol. Chem. 276:6591-6604; gie, EE et al. (2000) "Mapping Of The Clq Binding Site On Rituxan, A Chimeric." Antibody With A Human IgG Fc," J. Immunol. 164:4178-84; Reddy, MP et al ) "Elimination Of Fc Receptor-Dependent Effector Functions Of A Modified IgG4 Mo." noclonal Antibody To Human CD4 " J. Immunol. 164: 1925-1933; Xu, D. et al. (2000 ) "In Vitro Characterization of Five Humanized OKT3 Effector Function Variant An tibodies " Cell. Immunol. 200: 16-26; Armour, K.L. et al. (1999) "Recombinant hu man IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Act ivities " Eur. J. Immunol. 29:2613-24; Jefferis, R. et al. (1996) "Modulation Of Fc(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interac tions " Immunol. Lett. 54: 101-04; Lund, J. et al. (1996) "Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Ch ains " J. Immunol. 157:4963-4969; Hutchins et al. (1995) "Improved Biodistributi on, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant O f Campath-IH," Proc. Natl. Acad. Sci. (U.S.A.) 92: 11980-84; Jefferis, R. et al. (1995) "Recognition Sites On Human IgG For Fc Gamma Receptors: The Role Of Glyc osylation," Immunol. Lett. 44: 111-17; Lund, J. et al. (1995) "Oligosaccharide-P rotein Interactions In IgG Can Modulate Recognition By Fc Gamma Receptors " FASE B J. 9: 115-19; Alegre, M . et al. (1994) "A Non-Activating "Humanized" Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo," Transplantat ion 57: 1537-1543; Lund et al. (1992) "Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma RII," Mol. Immunol. 29:53-59; Lund et al. (1991) "Huma n Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On H uman IgG," J. Immunol. 147:2657-2662; Duncan, AR et al. (1988) "Localization O f The Binding Site For The Human High-Affinity Fc Receptor On IgG," Nature 332:5 63-564; U.S. Patent No. 5,624,821; U.S. Patent No. 5,885,573; U.S. Patent No. 6,194,551; U.S. Patent No. 7,276,586; and U.S. Patent No. 7,317 ,091; and WO 00 / 42072 and WO 99 / 58572 The use of any known variant Fc region, such as those disclosed, is encompassed.

[0199] In some embodiments, the molecules of the invention further comprise one or more glycosylation sites. whereby one or more carbohydrate moieties are covalently attached to the molecule. Preferably, the Fc region contains one or more glycosylation sites and / or one or more The molecules of the invention, which have one or more modifications, exhibit enhanced antibody-mediated activity compared to unmodified antibodies. confers or has an effector function, such as enhanced ADCC activity. In some embodiments, the present invention further provides a method for the preparation of a polypeptide that interacts directly or indirectly with the carbohydrate moiety of an Fc region. It is known that, 241, 243, 244, 245, 245, 249, 256, 258 , 260, 262, 264, 265, 296, 299 and 301 amino acids. The present invention also includes molecules containing one or more modifications of amino acids, but not limited to the following: Amino acids that interact directly or indirectly with the compound moiety are known in the art. See, e.g., Jefferis et al. (1995) Immunology Letters, 44:111-7 (which is incorporated by reference). (which is incorporated herein by reference in its entirety).

[0200] In another embodiment, the present invention provides a method for determining the functionality of a molecule, preferably a target molecule or an Fc One or more glycosylation sites can be added to the molecule without altering its binding activity to γR. Glycosyl groups are also included in the molecule, which have been modified by introducing at one or more sites The amino acid sequence may be introduced into the variable and / or constant regions of the molecules of the invention. When used herein, "glycosylation site" refers to an Oligosaccharides (i.e., carbohydrates containing two or more monosaccharides linked together) are specifically covalently bonded to Thus, the oligosaccharide side chains may be attached to any specific amino acid sequence in the antibody. N-linked glycosylation is linked to the antibody backbone via an N- or O-linkage. refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. It refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, such as serine or threonine. The molecule may contain one or more glycosylation sites, including N-linked and O-linked glycosylation sites. Any suitable glycosylation site for N-linked or O-linked glycosylation known in the art may be provided. Any glycosylation site may be used in accordance with the present invention. An exemplary N-linked glycosylation site that can be used is the amino acid sequence: Asn-X-Thr / Ser. where X can be any amino acid and Thr / Ser is threonine or serine. Such one or more moieties are known in the art to which the present invention pertains. Methods may be used to introduce the molecules of the invention (e.g., in vitro mutagenesis, recombinant T DNA: A SHORT COURSE, JD Watson, et al. WH Freeman and Company, New York, 1983, chapter 8, pp. 106-116, the entire contents of which are incorporated herein by reference. An exemplary method for introducing a glycosylation site into a molecule of the invention is to: The amino acid sequence is modified or mutated to give the desired Asn-X-Thr / Ser sequence. may include the step of obtaining

[0201] In some embodiments, the present invention provides a method for the preparation of glycosylated proteins by adding or deleting glycosylation sites. The present invention also encompasses methods for modifying the carbohydrate content of the molecules of the invention. Methods for modifying the carbohydrate content of molecules (e.g., molecules containing an Fc region) are known in the art. See, e.g., U.S. Pat. No. 6,218,149; European Patent No. 0 359096B1; U.S. Patent Application No. 2002 / 0028486; International Publication No. 03 / 0 35835; U.S. Patent Application No. 2003 / 0115614; U.S. Patent No. 6,218,1 No. 49; U.S. Pat. No. 6,472,511 (each of which is incorporated herein by reference in its entirety). In other embodiments, the present invention provides a method for detecting one or more endogenous genes of a molecule of interest. Methods for modifying the carbohydrate content of the molecules of the invention by deleting carbohydrate moieties are also included. In a specific embodiment, the present invention provides a method for modifying the position adjacent to position 297. Therefore, the step of shifting the glycosylation site of the Fc region of the antibody is included. In one embodiment, the present invention provides a modification at position 296, thereby providing a nucleotide sequence at position 296 rather than position 297. The method includes the step of glycosylating the

[0202] Effector function can also be enhanced by introducing one or more cysteine ​​residues into the Fc region. This allows for interchain disulfide bonding in this region, resulting in improved absorption capacity. Homodimeric antibodies that may improve cell proliferation and / or enhance complement-mediated cell killing and ADCC (Caron, PC et al. (1992) "Engineered Humanize d Dimeric Forms Of IgG Are More Effective Antibodies J. Exp. Med. 176: 1191-1195 ; Shopes, B. (1992) "A Genetically Engineered Human IgG Mutant With Enhanced Cyt" "Biolytic Activity," J. Immunol. 148(9):2918-2922). Homodimers with enhanced antitumor activity. Monoclonal antibodies are also described in Wolff, EA et al. (1993) "Monoclonal Antibody Homodimers: Enhanced d Antitumor Activity in Nude Mice," Cancer Research 53:2560-2565 Alternatively, dual Fc domains can be prepared using heterobifunctional cross-linkers such as Antibodies can be engineered that have enhanced complement lysis and ADCC capabilities (Stev enson, GT et al. (1989) "A Chimeric Antibody With Dual Fc Regions (bisFabFc) P repared By Manipulations At The IgG Hinge "Anti-Cancer Drug Design 3:219-230) .

[0203] The serum half-life of the molecules of the invention comprising an Fc region is determined by the binding affinity of the Fc region for FcRn. As used herein, the term "antioxidant" can be used to refer to an agent that is capable of acting as an antioxidant. The term "half-life" is a measure of the average survival time of a molecule after administration. The half-life refers to the pharmacokinetic properties of a molecule. The half-life is the time it takes for a molecule to be released into the blood (i.e., circulating half-life) or into other tissues. When measured at 50 percent (50%) of the known amount of the molecule is present in the subject's body (e.g. the time required for the substance to be expelled from the body of a human patient or other mammal or a specific body cavity thereof. Generally, an increase in half-life increases the mean residence time of an administered molecule in the circulation. This leads to an increase in travel time (MRT).

[0204] In some embodiments, the PD-1-binding molecules of the invention comprise a variant Fc Region, and The variant Fc region contains at least one amino acid modification relative to the wild-type Fc region and therefore As a result, the molecule has an increased half-life (relative to the wild-type Fc region).

[0205] In some embodiments, the PD-1-binding molecules of the invention comprise a variant Fc Region, The mutant Fc regions are 238, 250, 252, 254, 256, 257, 256, and 265. , 272, 286, 288, 303, 305, 307, 308, 309, 311, 312 , 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413 , 424, 428, 433, 434, 435 and 436 Contains half-life extending amino acid substitutions at multiple positions. Increases the half-life of Fc region-containing molecules. Many specific mutations that can be made are known in the art, for example, M25 2Y, S254T, T256E, and combinations thereof. For example: U.S. Pat. No. 6,277,375; U.S. Patent No. 7,083,784; U.S. Patent No. 7,217, 797; U.S. Patent No. 8,088,376; U.S. Patent Publication No. 2002 / 0147311 No.; U.S. Patent Publication No. 2007 / 0148164; and International Publication No. 98 / 23289 WO 2009 / 058492; and WO 2010 / 033279 ( See the mutations described in the above, which are incorporated herein by reference in their entireties. Fc region-containing molecules with enhanced shear life include those containing Fc region residues 250, 252, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 27 56, 257, 288, 307, 308, 309, 311, 378, 428, 433, 4 substitutions in two or more of 34, 435 and 436, in particular T250Q, M252Y; S254T, T256E, K288D, T307Q, V308P, A378V, M428 Those having two or more substitutions selected from L, N434A, H435K and Y436I Other examples include:

[0206] In a specific embodiment, the variant Fc region comprises the following substitutions: (A) M252Y, S254T and T256E; (B) M252Y and S254T; (C) M252Y and T256E; (D) T250Q and M428L; (E) T307Q and N434A; (F) A378V and N434A; (G) N434A and Y436I; (H) V308P and N434A; or (I) K288D and H435K.

[0207] The present invention further comprises: (A) one or more mutations that alter effector function and / or FcγR; and to (B) one or more mutations that increase serum half-life The present invention also encompasses variant Fc regions comprising:

[0208] VI. Bispecific Anti-Human PD-1 Binding Molecules One embodiment of the present invention is capable of binding to a "first epitope" and a "second epitope." bispecific binding molecules, wherein the first epitope is an epitope of human PD-1. wherein the second epitope is the same or a different epitope of PD-1; or present on the surface of immune cells (e.g., T lymphocytes) and involved in the regulation of immune checkpoints In one embodiment, the second epitope is an epitope of another molecule involved. -H3, B7-H4, BTLA, CD3, CD8, CD16, CD27, CD32, CD 40, CD40L, CD47, CD64, CD70, CD80, CD86, CD94, C D137, CD137L, CD226, CTLA‐4, galectin‐9, GITR, GI TRL, HHLA2, ICOS, ICOSL, KIR, LAG-3, LIGHT, MHC Class I or II, NKG2a, NKG2d, OX40, OX40L, PD1H, P D-1, PD-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM- In one embodiment, the second epitope is an epitope of PD-3 or VISTA. In a specific embodiment, the second epitope is not an epitope of CD13 7, CTLA-4, LAG-3, OX40, TIGIT, or TIM-3. In embodiments, the bispecific molecule comprises three or more epitope binding sites. A bispecific molecule is one that binds two or more different epitopes of LAG-3 and one or more non-LAG-3 molecules. The antibody may bind to at least one epitope of the antibody.

[0209] The present invention relates to a method for detecting PD-1 and the second epitope (e.g., B7-H3, B7-H4, BT7-H8, BT7-H9, BT7-H10, BT7-H11, BT7-H12, BT7-H13, BT7-H14, BT7-H15, BT7-H16, BT7-H17, BT7-H18, BT7- LA, CD40, CD80, CD86, CD137, CTLA‐4, ICOS, KIR, LAG-3, MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc.) In some embodiments, the bispecific antibodies include those capable of simultaneously binding to PD-1 and The bispecific antibody capable of simultaneously binding to the second epitope is described in WO 1998 / 023444. 002463, WO 2005 / 070966, WO 2006 / 1077 86, International Publication No. 2007 / 024715, International Publication No. 2007 / 075270, International Publication No. 2006 / 107617, International Publication No. 2007 / 046893, International Publication International Publication No. 2007 / 146968, International Publication No. 2008 / 003103, International Publication No. 200 8 / 003116, WO 2008 / 027236, WO 2008 / 02 4188, WO 2009 / 132876, WO 2009 / 018386 No., International Publication No. 2010 / 028797, International Publication No. 2010028796, International Publication International Publication No. 2010 / 028795, International Publication No. 2010 / 108127, International Publication No. 201 0 / 136172, International Publication No. 2011 / 086091, International Publication No. 2011 / 13 3886, WO 2012 / 009544, WO 2013 / 003652 No. 2013 / 070565, No. 2012 / 162583, No. 2012 / 162583, Publication No. 2012 / 156430, International Publication No. 2013 / 174873, and International Publication and any of the methods described in the publications of US Pat. No. 2014 / 022540, which are incorporated herein by reference. Each is incorporated by reference in its entirety into this application.

[0210] A. Bispecific diabodies without an Fc region One embodiment of the present invention comprises a first polypeptide chain and a second polypeptide chain, Preferably, the double specific polypeptide chain is composed of the first polypeptide chain and the second polypeptide chain. For isomeric diabodies, the sequences are such that the polypeptide chains are covalently linked to each other. , a first epitope and a second epitope (these epitopes are not identical to each other) This allows the formation of covalent diabodies that can simultaneously bind to the The bispecific diabody has a "VL1" / "VH1" domain capable of binding to the first epitope. " domain and a "VL2" / "VH2" domain capable of binding to the second epitope. The notation "VL1" and "VH1" respectively refer to such bispecific diabodies. The term "variable light chain domain" refers to the variable light chain domain and variable heavy chain domain that bind to the "first" epitope of the antibody. Similarly, the notations "VL2" and "VH2," respectively, refer to such bispecific diabodies. The term "variable light chain domain" refers to a variable light chain domain and a variable heavy chain domain that bind to a "second" epitope of a Whether a particular epitope is designated as a primary or secondary epitope The designation is irrelevant and such designation is used to designate domains of the polypeptide chains of the binding molecules of the invention. In one embodiment, one of the epitopes is Another such epitope is the epitope of PD-1. Not a target (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, C D86, CD137, CTLA‐4, ICOS, KIR, LAG‐3, MHC class I or are epitopes such as II, OX40, PD-L1, TCR, and TIM-3).

[0211] The VL domain of the first polypeptide chain is linked to the VH domain of the second polypeptide chain. interacts with the first antigen (i.e., PD-1 or an antigen containing the second epitope) ) to form a first functional antigen-binding site specific for the second polypeptide chain. The VL domain interacts with the VH domain of the first polypeptide chain to bind to a second antigen. a second functional antigen specific for (i.e., the antigen containing the second epitope or PD-1) Thus, the VL and VH domains of the first and second polypeptide chains form a binding site. The main choice is that the two polypeptide chains of the diabody together bind to the PD-1 It has VL and VH domains that can bind to both the epitope and the second epitope. (i.e., these are VL PD‐1 / VH PD‐1 and VL2 / VH2 (where PD-1 is the "first (first) epitope), or VL1 / VH1 and VL PD‐1 / VH PD‐1 ( Here, PD-1 comprises a "second epitope").

[0212] The first polypeptide chain of one embodiment of such bispecific diabodies comprises an N-terminal In the direction from the C-terminus: the N-terminus; a monoclonal antibody capable of binding to the first or second epitope The VL1 domain of a clonal antibody (i.e., VL PD‐1 or VL Epitope 2 ); First intervening spacer peptide (linker 1); (the first polypeptide chain is VL PD‐1 (If it contains the second epitope, or (wherein the first polypeptide chain is a VL Epitope 2 If it contains a) a VH2 domain of a monoclonal antibody capable of binding to a first epitope; optionally, a system a second intervening spacer peptide (linker 2) containing a methyl group residue; and the C-terminus (Figure 1).

[0213] The second polypeptide chain of this embodiment of the bispecific diabody is arranged from the N-terminus to the C-terminus Towards: N-terminus; monoclonal antibody capable of binding to PD-1 or the second epitope described above The VL2 domain (i.e., VL PD‐1 or VL Epitope 2 and the VL domain is the the first intervening spacer is selected not to be included in the first polypeptide chain of the aorta body; a subpeptide (linker 1); (the second polypeptide chain is VL PD‐1 If it contains ) the second epitope, or (the second polypeptide chain is a VL Epitope 2 Contains the VH1 domain of a monoclonal antibody capable of binding to PD-1 (if present); optionally, a cysteine a second intervening spacer peptide (Linker 2) containing a methylamino acid residue; and the C-terminus (Figure 1).

[0214] Most preferably, an intervening linker peptide separating the VL and VH domains as described above. The length of the linker (e.g., linker 1) is such that the VL and VH domains of the polypeptide chain are spaced apart from each other. Therefore, the first polypeptide is selected to substantially or completely prevent binding to the The VL and VH domains of the peptide chains are substantially or completely unable to bind to each other. Similarly, the VL and VH domains of the second polypeptide chain bind to each other. A preferred intervening spacer peptide (Linker 1) is It has the sequence (SEQ ID NO: 14): GGGSGGGG.

[0215] The length and composition of the second intervening linker peptide (Linker 2) determines the heterodimer-promoting activity. Typically, the second intervening linker peptide ( Linker 2) contains 3 to 20 amino acid residues. In particular, the heterodimer-promoting domain If the linker peptide does not contain a cysteine ​​residue, a cysteine-containing second intervening linker peptide (linker A second intervening cysteine-containing spacer peptide (linker 2) is used. Preferred cysteine-containing spacers include one, two, three, or four or more cysteines. The peptide (linker 2) has the sequence SEQ ID NO: 15: GGCGGG. Alternatively, the linker 2 does not contain cysteine ​​(e.g., GGG, GGGS (SEQ ID NO: 29), LGGGSG (SEQ ID NO: 26) 1), GGGSGGGSGGG (SEQ ID NO: 262), ASTKG (SEQ ID NO: 30), LEPKSS (SEQ ID NO: 33 ), APSSS (SEQ ID NO: 34), etc.), cysteine-containing heterodimer-promoting domains described below. Optionally, a cysteine-containing linker 2 and a cysteine-containing heterodimer are used. Both the body facilitating domains are used.

[0216] The Heterodimer-Promoting Domain comprises GVEPKSC (SEQ ID NO: 16) or VEPKSC (SEQ ID NO: 17) or AEPKS C (SEQ ID NO: 18), and on the other polypeptide chain GFNRGEC (SEQ ID NO: 19) or FNRGEC (SEQ ID NO: 20) (U.S. Pat. No. 2007 / 003999). No. 004909).

[0217] More preferably, however, the Heterodimer-Promoting Domain of such diabodies , at least six, at least seven, or at least eight amino acid residues, It is formed from one, two, three or four tandemly repeated coil domains, The heterodimer-promoting domain has a net charge (Apostolovic, B. et al. (2008) “pH ‐Sensitivity of the E3 / K3 Heterodimeric Coiled Coil,” Biomacromolecules 9:3173 -3180; Arndt, KM et al. (2001) “Helix‐stabilized Fv (hsFv) Antibody Fragment s: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coile d‐coil Domain,” J. Molec. Biol. 312:221‐228; Arndt, KM et al. (2002) “Comp arison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils,” Structure 10:1235‐1248; Boucher, C. et al. (2010) “Protein Detection By Weste rn Blot Via Coiled-Coil Interactions,” Analytical Biochemistry 399:138‐140; Ca chia, P.J. et al. (2004) “Synthetic Peptide Vaccine Development: Measurement Of Polyclonal Antibody Affinity And Cross‐Reactivity Using A New Peptide Capture And Release System For Surface Plasmon Resonance Spectroscopy,” J. Mol. Recogni t. 17:540‐557; De Crescenzo, G.D. et al. (2003) “Real‐Time Monitoring of the Interactions of Two‐Stranded de novo Designed Coiled‐Coils: Effect of Chain Le ngth on the Kinetic and Thermodynamic Constants of Binding,” Biochemistry 42:17 54‐1763; Fernandez‐Rodriquez, J. et al. (2012) “Induced Heterodimerization An d Purification Of Two Target Proteins By A Synthetic Coiled‐Coil Tag,” Protein Science 21:511‐519; Ghosh, T.S. et al. (2009) “End‐To‐End And End‐To‐Midd le Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein Structures,” Acta Crystallographica D65:1032‐1041; Grigoryan, G. et al. (2008) “Structural Specificity In Coiled‐Coil Interactions,” Curr. Opin. Struc. Bio l. 18:477‐483; Litowski, J.R. et al. (2002) “Designing Heterodimeric Two‐Stra nded α‐Helical Coiled‐Coils: The Effects Of Hydrophobicity And α‐Helical Pr opensity On Protein Folding, Stability, And Specificity,” J. Biol. Chem. 277:37 272‐37279; Steinkruger, J.D. et al. (2012) “The d′‐‐d‐‐d′ Vertical Triad is Less Discriminating Than the a′‐‐a‐‐a′ Vertical Triad in the Antiparal lel Coiled‐coil Dimer Motif,” J. Amer. Chem. Soc. 134(5): 2626-2633; Straussma n, R. et al. (2007) “Kinking the Coiled Coil - Negatively Charged Residues at t he Coiled‐coil Interface,” J. Molec. Biol. 366:1232‐1242; Tripet, B. et al. ( 2002) “Kinetic Analysis of the Interactions between Troponin C and the C‐termi nal Troponin I Regulatory Region and Validation of a New Peptide Delivery / Captur e System used for Surface Plasmon Resonance,” J. Molec. Biol. 323:345-362; Wool fson, DN (2005) “The Design Of Coiled‐Coil Structures And Assemblies,” Adv. Prot. Chem. 70:79‐112; Zeng, Y. et al. (2008) “A Ligand‐Pseudoreceptor System m Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors Wi th Altered Tropism,” J. Gene Med. 10:355-367).

[0218] Such repeating coil domains may be perfect repeats or may have substitutions. For example, the coil domain of the Heterodimer-Promoting Domain of the first polypeptide chain may be Eight amino acid residues selected to confer a negative charge to the heterodimer-promoting domain sequence, and the coiled heterodimer-promoting domain of the second polypeptide chain The main domains are eight amino acids selected to confer a positive charge to the heterodimer-promoting domain. It may contain a sequence of amino acid residues of opposite charge (or vice versa). When using a silyl group for the other polypeptide chain, which silyl group is used for the first or second polypeptide chain? It is not important which coil is used. Positively charged amino acids are lysine, arginine, and histidine. and / or the negatively charged amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine, and / or the negatively charged amino acid is Preferably, it is glutamic acid. (Such domains inhibit homodimerization and are involved in this.) Therefore, only a single heterodimer-promoting domain can be employed to promote heterodimerization. However, both the first and second polypeptide chains of the diabodies of the invention are heterodimer-promoting. It is preferred that the domain is contained.

[0219] In a preferred embodiment, one of the Heterodimer-Promoting Domains comprises four tandem " E-coil" helical domain (SEQ ID NO: 21: E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K) The glutamic acid residues form a negative charge at pH 7, while the hetero The other dimer-promoting domain contains four tandem "K-coil" domains (SEQ ID NO: No. 22: K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E), and the lysine residue is The presence of such a charged domain allows the first polypeptide to be linked to the second polypeptide. The linkage between the second polypeptide is promoted, thus promoting heterodimer formation. Of the four tandem "E-coil" helical domains of SEQ ID NO: 21, One is a cysteine ​​residue: E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E Contains K (SEQ ID NO: 23) Also particularly preferred are Heterodimer-Promoting Domains that have been modified to is a sequence in which one of the four tandem "K-coil" helical domains of SEQ ID NO: 22 is in cis Tein residue: K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 24) It is a heterodimer-promoting domain decorated with

[0220] As disclosed in WO 2012 / 018687, the in vivo administration of diabodies To improve the pharmacokinetic properties of the diabody, the diabody may be modified by adding a hydroxylase to one or more of the termini of the diabody. The polypeptide may be modified to contain multiple polypeptide portions of serum-binding proteins. Preferably, the polypeptide portion of such serum-binding protein is located at the C-terminus of the diabody. Albumin is the most abundant protein in plasma and is found in humans. Albumin has a half-life of 19 days in the endothelial cell. Albumin can then bind non-covalently to other proteins to extend serum half-life. The albumin-binding domain 3 (ABD3) of protein G in G148 is a stable triple helix. It consists of 46 amino acid residues that form a bundle and has broad albumin binding specificity (Jo hansson, MU et al. (2002) “Structure, Specificity, And Mode Of Interaction Fo r Bacterial Albumin-Binding Modules,” J. Biol. Chem. 277(10):8114‐8120). and the use of serum-binding proteins, particularly diabodies, to improve the in vivo pharmacokinetic properties of the diabodies. A preferred polypeptide moiety is the albumin binding domain from streptococcal protein G (A BD), and more preferably, Streptococcus dysgalactiae strain G148. Albumin binding domain 3 (ABD3) of protein G (SEQ ID NO: 25): LAEAKVLANR ELD KYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

[0221] The method disclosed in WO 2012 / 162068 (which is incorporated herein by reference) As mentioned above, the "deimmunized" variant of SEQ ID NO: 25 binds to the MHC The ability to attenuate or eliminate class II binding. Based on this, the following combinations of substitutions are preferred for forming such deimmunized ABDs: The preferred substitutions are: 66D / 70S+71A; 66S / 70S+71A; 6S / 70S+79A;64A / 65A / 71A;64A / 65A / 71A+66S;6 4A / 65A / 71A+66D;64A / 65A / 71A+66E;64A / 65A / 7 9A+66S;64A / 65A / 79A+66D;64A / 65A / 79A+66E. Repair with modifications L64A, I65A and D79A, or N66S, T70S and D79A Mutant ABD. Amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI D 66 NAK S 70 A 71 EGVKALIDE ILAALP (SEQ ID NO: 26) or the amino acid sequence: LAEAKVLANR ELDKYGVSDY YKN A 64 A 65 NNAKT VEGVKALI A 79 E ILAALP (SEQ ID NO: 27) or the amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI S 66 NAK S 70 VEGVKALI A 79 E. ILAALP (SEQ ID NO: 28) Mutant deimmunized ABDs are particularly preferred, since such deimmunized ABDs are This is because they exhibit near wild-type binding while providing attenuation of HC class II binding. The first polypeptide chain of such a diabody having an ABD preferably comprises Located C-terminal to the E-coil (or K-coil) domain of a polypeptide chain such as By this, the E coil (or K coil) domain and the ABD (which is preferably is a deimmunized ABD). A preferred sequence for the peptide linker is SEQ ID NO: 29: GGGS.

[0222] B. Fc Region-Containing Bispecific Diabodies One embodiment of the present invention is directed to a PD-1 antibody that binds to a second epitope (e.g., B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, B7-H8, B7-H9, B7-H10, B7-H11, B7-H12, B7-H13, B7-H14, B7-H15, B7-H16, B7-H17 4, BTLA, CD40, CD80, CD86, CD137, CTLA‐4, ICOS, KIR, LAG‐3, MHC class I or II, OX40, PD‐1, PD‐L1, TC Regarding bispecific diabodies, the Fc region can simultaneously bind to multiple antigens (e.g., TIM-3, TIM-4, etc.) One or both of the diabody polypeptide chains are IgG CH2-CH3 domains are added to allow the complexation of the diabody chains to form an Fc region. This increases the biological half-life and / or alters the valency of the diabody. Incorporating IgG CH2-CH3 domains into both of the diabody polypeptides This allows the formation of bispecific Fc region-containing diabodies (Figure 2).

[0223] Alternatively, one of the diabody polypeptides may contain an IgG CH2-CH3 domain. Incorporation of this amino acid allows the formation of more complex four-chain bispecific Fc-region-containing diabodies. Figure 3C shows the structure of the constant light chain (CL) domain and the constant heavy chain CH1 domain. A representative four-chain diabody having a polyclonal domain is shown, but fragments of such domains and other polyclonal domains are also shown. Alternatively, peptides may be employed (see, e.g., Figures 3A and 3B, U.S. Patent Publication No. 2013-003334). 0295121; U.S. Patent Publication No. 2010-0174053 and U.S. Patent Publication No. 20 09-0060910; European Patent Publication No. 2714079; European Patent Publication No. 26012 16; European Patent Publication No. 2376109; European Patent Publication No. 2158221, and International Publication No. 2012 / 162068; International Publication No. 2012 / 018687; International Publication No. Thus, for example, instead of the CH1 domain, a human Ig The amino acid sequence GVEPKSC (SEQ ID NO: 16), VEPKSC (SEQ ID NO: 1) derived from the hinge domain of G 7) or a peptide having AEP KSC (SEQ ID NO: 18) may be employed, and a peptide having a CL domain Alternatively, the C-terminal six amino acids of the human kappa light chain, GFNRGEC (SEQ ID NO: 19) or FNRGEC (SEQ ID NO: 20) A representative peptide containing a four-chain diabody is shown in Figure 3A. Alternatively, or in addition, tandem coil domains of opposite charge, e.g., "E coils" Helical domain (SEQ ID NO: 21: E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K or SEQ ID NO: 23: E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K); and the "K coil" domain (SEQ ID NO: 22: K V AAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E or SEQ ID NO: 24: K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VA AL K E) may be employed. The main part is shown in Figure 3B.

[0224] The Fc Region-containing diabody molecules of the present invention generally include an intervening linker peptide (linker). Typically, this additional linker comprises 3 to 20 amino acid residues. Additional or alternative linkers that can be employed in Fc Region-containing diabody molecules include: : GGGS (SEQ ID NO: 29), LGGGSG (SEQ ID NO: 261), GGGSGGSGGGG (SEQ ID NO: 262) , ASTKG (SEQ ID NO: 30), DKTHTCPPCP (SEQ ID NO: 31), EPKSCDKTHTCPPCP (SEQ ID NO: 3 2), LEPKSS (SEQ ID NO: 33), APSSS (SEQ ID NO: 34), and APSSSPME (SEQ ID NO: 35) ), LEPKSADKTHTCPPC (SEQ ID NO: 36), GGC, and GGG. For ease of use, SEQ ID NO: 33 may be used in place of GGG or GGC. GG or SEQ ID NO: 33 immediately followed by SEQ ID NO: 31 to form the alternative linker: GGGD KTHTCPPCP (SEQ ID NO: 263); and LEPKSSDKTHTCPPCP (SEQ ID NO: 37). The Fc Region-containing diabody molecules of the present invention may further comprise, in addition to or instead of a linker, An IgG hinge region may be incorporated. Exemplary hinge regions include: EPKS from IgG1 CDKTHTCPPCP (SEQ ID NO: 32); ERKCCVECPPCP (SEQ ID NO: 11) from IgG2; IgG ESKYGPPCPSCP (SEQ ID NO: 12) from 4; and containing stabilizing substitutions to reduce strand exchange ESKYGPPCPPCP (SEQ ID NO: 13) from an IgG4 hinge mutant.

[0225] As presented in Figures 3A-3C, the diabodies of the present invention comprise four different chains. The first and third polypeptide chains of such diabodies may comprise four domains: (i) VL1-containing domain; (ii) VH2-containing domain; (iii) heterodimer-promoting domain; and (iv) a domain containing a CH2-CH3 sequence. The four polypeptide chains comprise: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a Heterodimer-Promoting Domain, wherein the Heterodimer-Promoting Domain is a first Promotes dimerization of the third polypeptide chain with the second / fourth polypeptide chain. and the VL and / or VH domains of the first and second polypeptide chains, The VL and / or VH domains of the peptide chains may be the same or different, This allows for monospecific, bispecific or tetraspecific tetravalent binding. Notation "VL3" and "VH3," which bind to the "third" epitope of such a diabody, respectively. The variable light and heavy domains are referred to as "VL4" and "VH4" respectively. " respectively represent the variable light chain domains that bind the "fourth" epitope of such diabodies. The variable heavy chain domains are the PI domain and the variable heavy chain domain. The general structure of the polypeptide chain is presented in Table 2.

[0226] [Table 4]

[0227] In a specific embodiment, a diabody of the invention comprises a total of four polypeptide chains. bispecific, tetravalent (i.e., having four epitope-binding sites), Fc-containing diabody The bispecific, tetravalent, Fc-containing diabodies of the present invention are shown in Figures 3A-3C. Two epitope-binding sites immunospecific for PD-1 (these are the same epitope or can bind to a different epitope on PD-1), and a second epitope (e.g., B7-H 3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4 , ICOS, KIR, LAG‐3, MHC class I or II, OX40, PD‐L1, T It has two epitope binding sites specific for CR, TIM-3, etc.

[0228] In a further embodiment, the bispecific Fc Region-containing diabody comprises three polypeptides. The first polypeptide of such a diabody may comprise three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a CH2-CH The second polypeptide of such a diabody contains a domain containing three sequences. (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) the above dye. a domain that promotes heterodimerization and covalent binding with the first polypeptide chain of the abodi The third polypeptide of such a diabody comprises a CH2-CH3 sequence. Thus, the first and second polypeptide chains of such diabodies are linked together. and a VL1 / VH1 binding site capable of binding to the first epitope and a VL1 / VH1 binding site capable of binding to the second epitope. The first and second polypeptides form a VL2 / VH2 binding site capable of binding to the polypeptide. are connected to each other by disulfide bonds involving cysteine ​​residues in their respective third domains. In particular, the first and third polypeptide chains are complexed with each other to form disulfide bonds. The Fc region is stabilized by peptide bonds. Figures 4A and 4B show the structure of such a diabody. Suitable Fc Region-containing bispecific diabodies have either of two orientations (Table 3). good.

[0229] [Table 5]

[0230] In a specific embodiment, a diabody of the invention comprises a total of three polypeptide chains. bispecific, bivalent (i.e., having two epitope-binding sites), Fc-containing diabody The bispecific, bivalent Fc-containing diabodies of the present invention are PD- One epitope binding site immunospecific for 1, a second epitope (e.g., B7-H3, B 7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, IC OS, KIR, LAG‐3 MHC class I or II, OX40, PD‐L1, TCR, It has one epitope binding site specific for TIM-3, etc.

[0231] In a further embodiment, the bispecific Fc Region-containing diabody comprises a total of five polypeptides. In certain embodiments, two of the five polypeptide chains may be The first polypeptide chain of such a diabody has the same amino acid sequence. (i) a VH1-containing domain; (ii) a CH1-containing domain; and (iii) a CH2-C The first polypeptide chain contains a domain containing a VH1 and a heavy chain H3 sequence. The second and fifth chains of such diabodies may be heavy chains of antibodies containing constant regions. The polypeptide chain contains: (i) a VL1-containing domain; and (ii) a CL-containing domain. The second and / or fifth polypeptide chains of such diabodies may be the first and / or fifth polypeptide chains. It may be a light chain of an antibody containing a VL1 that is complementary to the VH1 of the third polypeptide chain. The first, second and / or fifth polypeptide chains may be isolated from a naturally occurring antibody. Alternatively, they can be constructed recombinantly. The polypeptide chain of: (i) a VH1-containing domain; (ii) a CH1-containing domain; i) a domain containing a CH2-CH3 sequence; (iv) a VL2-containing domain; (v) a VH (vi) a Heterodimer-Promoting Domain; and The nucleotide domain promotes dimerization of the third chain with the fourth chain. The fourth polypeptide of the body comprises: (i) a VL3-containing domain; (ii) a VH2-containing domain; and (iii) heterodimerization with a third polypeptide chain of the diabody and Contains a domain that promotes covalent binding.

[0232] Thus, the first and second polypeptide chains and the third and fourth polypeptide chains of such diabodies The fifth polypeptide chain is a chain of two VLs that can be linked to each other and bind to the first epitope. The third and fourth polypeptides of such diabodies form a VH1 / VH1 binding site. The VL2 / VH2 binding site can bind to a second epitope, and the VL2 / VH2 binding site can bind to a second epitope. and a third epitope. The polypeptides are held together by disulfide bonds involving cysteine ​​residues in the respective constant regions. In particular, the first and third polypeptide chains are complexed with each other. The Fc region is formed by the diabody. Such diabodies have enhanced potency. The structures of diabodies such as VL1 / VH1, VL2 / VH2 and VL3 / VH are shown. The three domains can be identical or different, allowing for monospecific, bispecific, It will be understood that monospecific or trispecific binding is possible. As provided herein, these domains preferably bind PD-1 to a second epitope. Topics (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, C D137, CTLA‐4, ICOS, KIR, LAG‐3, MHC class I or II, O X40, PD-L1, TCR, TIM-3, etc.

[0233] The VL and VH domains of the polypeptide chains are VL / VH domains specific for a desired epitope. The V formed by the linkage of the polypeptide chains is selected to form an H-binding site. The L / VH binding sites may be identical or different, allowing for monospecific, bispecific, or Tetravalent binding, which may be bispecific, trispecific or tetraspecific, is possible. In particular, the VL and V The H domain allows the bispecific diabody to have two binding sites for the first epitope and and two binding sites for the second epitope, or three binding sites for the first epitope. One binding site for the first epitope and one binding site for the second epitope, or (as shown in Figure 5) two binding sites for a first epitope, one binding site for a second epitope and one binding site for a third epitope The five chains of the present invention may be selected to have one binding site for the epitope. The general structure of the polypeptide chains of Fc-region-containing diabodies is shown in Table 4.

[0234] [Table 6]

[0235] In a specific embodiment, the diabodies of the invention comprise two diabodies directed to a first epitope. A total of five polypeptides each having a binding site for a second epitope and two binding sites for a second epitope. bispecific, tetravalent (i.e., has four epitope-binding sites), Fc-containing antibody consisting of a 2-chain In one embodiment, the bispecific, tetravalent, Fc-containing diabodies of the present invention The antibody contains two epitope-binding sites immunospecific for PD-1 (these are the same epitope on PD-1). may bind to a different epitope on PD-1 or a different epitope on PD-1), and a second epitope (e.g. For example, B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA‐4, ICOS, KIR, LAG‐3, MHC class I or II, OX40, P It has two epitope binding sites specific for other antigens (D-L1, TCR, TIM-3, etc.). In embodiments, the bispecific, tetravalent, Fc-containing diabodies of the invention are immunospecific for PD-1. Three distinct epitope-binding sites (which may be the same epitope on PD-1 or may bind to a different epitope), and a second epitope (e.g., B7-H3, B7- H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS , KIR, LAG‐3 MHC class I or II, OX40, PD‐L1, TCR, T In another embodiment, the present invention provides a method for the preparation of a human IgG1-associated ... This bispecific, tetravalent, Fc-containing diabody targets one epitope immunospecific for PD-1. The B7-binding site and a second epitope (e.g., B7-H3, B7-H4, BTLA, CD4 0, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 3 specific for MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc. It has two epitope binding sites.

[0236] C. Bispecific Trivalent Binding Molecules Containing an Fc Region A further embodiment of the present invention is a polypeptide comprising an Fc region and a first epitope, a second epitope and and a third epitope simultaneously, wherein the above At least one of the epitopes is not identical to another. The specificity of the diabody is determined by the VL1 / VH1 domains that can bind to the first epitope. a "VL2" / "VH2" domain capable of binding to a second epitope, and a "VL3" / "VH4" domain capable of binding to a third epitope. In one embodiment, the epitope comprises a "VL3" / "VH3" domain capable of binding to the epitope. One or two of the epitopes are epitopes of PD-1, and another of the above epitopes (or others) are not epitopes of PD-1 (e.g., B7-H3, B7-H4 , BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, K IR, LAG-3, MHC class I or II, OX40, PD-1, PD-L1, TCR Such bispecific trivalent binding molecules contain three epitopes. Two of the dimers provide binding site A and binding site B. a diabody-type binding domain, and a non-diabody-type binding domain that provides binding site C. (See, e.g., Figures 6A-6F, and PCT Application No. PCT / US15 / 33081 No. and PCT Application No. PCT / US15 / 33076).

[0237] Typically, a trivalent binding molecule of the invention comprises four distinct polypeptide chains (Figures 6A-6C). 6B), but such molecules may be, for example, those polypeptide chains joined together (e.g., by peptide bonds). These polypeptides can be synthesized by fusing them together (by "dividing" them) or by "splitting" them. by "mixing" to form additional polypeptide chains, or by forming fewer or additional By linking additional polypeptide chains by disulfide bonds, fewer or more 6B to 6F show three polypeptide chains. This aspect of the invention is illustrated by a schematic representation of a molecule having: As presented in Figure F, the trivalent binding molecules of the invention comprise the diabody-type binding domains described above. are N-terminal (Figures 6A, 6C, and 6D) or C-terminal (Figures 6B, 6E, and 6F) to the Fc region. ) and the like.

[0238] In certain embodiments, the first polypeptide chain of such a trivalent binding molecule of the invention comprises: (i) a VL1-containing domain; (ii) a VH2-containing domain; (iii) a heterodimer-promoting domain and (iv) a domain containing a CH2-CH3 sequence. The VL2 domain is composed of the CH2-CH3 domains as shown in Table 5 (FIGS. 6A and 6B). The second polypeptide of such an embodiment is located N-terminal or C-terminal to the containing domain. The peptide chain comprises: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) The third polypeptide chain of such embodiments contains a Heterodimer-Promoting Domain. (i) a VH3-containing domain; (ii) a CH1-containing domain; and (iii) a CH2-CH The third polypeptide chain contains a domain containing VH3 and heavy chain constants. The fourth polypeptide of such an embodiment may be a heavy chain of an antibody containing a constant region. (i) a VL3-containing domain; and (ii) a CL-containing domain. The polypeptide chain contains a VL3 complementary to the VH3 of the third polypeptide chain. The third or fourth polypeptide chain may be a light chain of an antibody. They can be isolated from the host, or they can be recombinantly, synthetically, or by other means. This can be configured as follows.

[0239] The variable light domains of the first and second polypeptide chains are separated by an intervening spacer linker. Thus, the variable heavy domains of such polypeptide chains are separated by the intervening spacer region. The anchor links these VL1 / VH2 (or these VL2 / VH1) domains together. The first and second epitopes can be bound to each other to form an epitope-binding site. The length of the intervening spacer peptide ( Linker 1) has the sequence (SEQ ID NO: 14): GGGSGGGG. The main chain is separated by one or more intervening spacer peptides, optionally containing cysteine ​​residues. Exemplary linkers useful for generating trivalent binding molecules are provided herein. and PCT Application No. PCT / US15 / 33081; and PCT Application No. PC Therefore, the first such trivalent binding molecule is also proposed in T / US15 / 33076. and the second polypeptide chain is linked together to form a VL1 / VL2 polypeptide chain capable of binding to the first epitope. Forms a VH1 binding site and a VL2 / VH2 binding site capable of binding to a second epitope The third and fourth polypeptide chains of such a trivalent binding molecule are linked together to form a third Forms a VL3 / VH3 binding site that can bind to epitopes. The VH2 and VL3 / VH3 domains may be the same or different, thereby allowing for a single It will be understood that binding that is monospecific, bispecific or trispecific is possible.

[0240] As mentioned above, a trivalent binding molecule of the present invention may comprise three polypeptides. The trivalent binding molecule containing the polypeptide chains binds the N-terminal domain of the fourth polypeptide to the N-terminal domain of the third polypeptide. Alternatively, the VH3-containing domain of the peptide may be linked to the VH3-containing domain of the peptide. The bottom three domains: (i) the VL3-containing domain; (ii) the VH3-containing domain; and iii) a third domain of the trivalent binding molecule of the present invention containing a domain containing a CH2-CH3 sequence; The polypeptide chains of VL3 and VH3 are used, where VL3 and VH3 are linked by these domains. The length of the fragment is sufficient (at least 9 amino acids) to allow the formation of an epitope binding site. The amino acids are separated from each other by an intervening spacer peptide having a 2-amino acid residue.

[0241] The VL1 / VH1, VL2 / VH2 and VL3 / VH3 domains may be identical or different. This allows for binding that is monospecific, bispecific, or trispecific. However, as presented herein, this These domains preferably bind to PD-1 and a second epitope (or a second and a third epitope). (Preferably, the epitope is selected to bind B7-H3, B7 -H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICO S, KIR, LAG‐3, MHC class I or II, OX40, PD‐L1, TCR, T epitopes such as IM-3).

[0242] In particular, the VL and VH domains form a trivalent binding molecule that binds two different molecules to a first epitope. a binding site and one binding site for a second epitope, or one binding site for a first epitope a binding site for one epitope and two binding sites for a second epitope, or one binding site for a second epitope, one binding site for a third epitope, The polypeptides of exemplary trivalent binding molecules of the present invention may be selected to contain one binding site corresponding to the The general structure of the peptide chains is presented in Figures 6A-6F and Table 5.

[0243] [Table 7]

[0244] One embodiment of the present invention is a method for detecting two epitope binding sites for PD-1 and one epitope binding site for PD-1 or other antigens. Other molecules (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA‐4, ICOS, KIR, LAG‐3, MHC class I or II, Related to a second epitope present on OX40, PD-L1, TCR, TIM-3, etc. Bispecific trivalent binding molecules with one epitope-binding site. The two epitope-binding sites may bind to the same epitope or different epitopes. Another embodiment of the present invention is directed to a single epitope binding site for PD-1 and a PD-1 Other molecules (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86 , CD137, CTLA‐4, ICOS, KIR, LAG‐3, MHC class I or II , OX40, PD-L1, TCR, TIM-3, etc.) The present invention relates to a bispecific trivalent binding molecule comprising an epitope-binding site of the second antigen. The two epitope-binding sites may bind to the same epitope or different epitopes ( For example, the antigen-binding domains may bind to the same or different epitopes of LAG-3. Such bispecific trivalent binding molecules may comprise three or four polypeptide chains.

[0245] VII. Constant Domains and Fc Region Presented herein are PD-1 binding molecules (e.g., antibodies, diabodies, 3 and antibody constant domains useful for generating valent binding molecules, etc.

[0246] A preferred CL domain is a human IgG CLκ domain. The amino acid sequence of the Lκ domain is (SEQ ID NO:8): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC is.

[0247] Alternatively, an exemplary CL domain is a human IgG CLλ domain. The amino acid sequence of the human IgG CLλ domain is (SEQ ID NO: 9): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS is.

[0248] As provided herein, the PD-1-binding molecules of the present invention comprise an Fc region. The Fc region of such molecules of the present invention may be of any isotype (e.g., IgG1 The PD-1-binding molecules of the present invention may be of the following classes: IgG1, IgG2, IgG3, or IgG4. It may further comprise a CH1 domain and / or a hinge region. If a hinge region is present, the CH1 domain and / or hinge region may be any of the amino acid sequences. It may be of any isotype (e.g., IgG1, IgG2, IgG3, or IgG4). , preferably of the same isotype as the desired Fc region.

[0249] An exemplary CH1 domain is a human IgG1 CH1 domain. The amino acid sequence of the G1 CH1 domain is (SEQ ID NO: 10): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV is.

[0250] An exemplary CH1 domain is a human IgG2 CH1 domain. The amino acid sequence of the G2 CH1 Domain is (SEQ ID NO:257): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV is.

[0251] An exemplary CH1 domain is a human IgG4 CH1 domain. The amino acid sequence of the G4 CH1 domain is (SEQ ID NO:254): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV is.

[0252] One exemplary hinge region is a human IgG1 hinge region. The amino acid sequence of the range region is (SEQ ID NO: 32): EPKSCDKTHTCPPCP.

[0253] Another exemplary hinge region is a human IgG2 hinge region. The amino acid sequence of the range region is (SEQ ID NO: 11): ERKCCVECPPCP.

[0254] Another exemplary hinge region is a human IgG4 hinge region. The amino acid sequence of the range region is (SEQ ID NO: 12): ESKYGPPCPSCP. As previously mentioned, the IgG4 hinge region may contain stabilizing mutations such as the S228P substitution. An exemplary stabilized IgG4 hinge region amino acid sequence is (SEQ ID NO: 13): ES KYGPPCP P It is CP.

[0255] The Fc Region of the Fc Region-containing molecules (e.g., antibodies, diabodies, and trivalent molecules) of the present invention may comprise: It may be a complete Fc region (e.g., a complete IgG Fc region) or just a fragment of the Fc region. Optionally, the Fc region of the Fc region-containing molecules of the invention may contain a C-terminal lysine amino acid In particular, the Fc region of the Fc region-containing molecule of the present invention does not contain engineered variant Fc regions. The Fc Region of the bispecific Fc Region-containing molecules of the present invention may be one or more F The antibody may be capable of binding to a FcγR or FcγRs, but more preferably is capable of binding to a FcγR or FcγRs. Alternatively, the variant Fc region exhibits FcγRIA binding (relative to that exhibited by a wild-type Fc region). (CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), Fcγ Altered binding to RIIIA (CD16a) or FcγRIIIB (CD16b) or have a reduced ability to bind to one or more inhibitory receptors or Therefore, the Fc region of the bispecific Fc region-containing molecule of the present invention does not have the above-mentioned ability. Some or all of the CH2 domain and / or the CH3 domain of the Fc region or may comprise some or all of the CH2 or CH3 of a complete Fc region. one or more insertions and / or one or more deletions in the CH3 domain The nucleotide sequence may comprise a variant CH2 and / or a variant CH3 sequence (which may contain a deletion). A suitable Fc region may comprise non-Fc polypeptide portions or may be a non-naturally occurring complete Fc region. may comprise portions of the CH2 and / or CH3 domains, or may comprise non-naturally occurring sequences of the CH2 and / or CH3 domains. It may have two CH2 domains or two CH3 regions, or an N-terminal From the C-terminus, the CH3 domain and the CH2 domain to which it is connected, etc. ).

[0256] Fc domain modifications that manifest as altered effector function are known in the art. and modifications that enhance binding to activating receptors (e.g., FcγRIIA (CD1 6A)), and modifications that reduce binding to inhibitory receptors (e.g., FcγRIIB(C D32B) (see, for example, Stavenhagen, JB et al. (2007) "Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro An d Controls Tumor Expansion In Vivo Via Low‐Affinity Activating Fcgamma Receptor s,” Cancer Res. 57(18):8882-8890). An exemplary variant of the human IgG1 Fc region with increased binding to CD16A is F2 These contain 43L, R292P, Y300L, V305I or P296L substitutions. The amino acid substitutions may be made in any combination or subcombination within the human IgG1 Fc region. In one embodiment, the human IgG1 Fc region variant may be present in In another embodiment, the human IgG1 Fc The regional variants include F243L, R292P, Y300L, V305I, and P296L substitutions. Contains.

[0257] In particular, with respect to the Fc region of the polypeptide chain of the Fc region-containing molecule of the present invention, (wild-type Ig FcγRIA (CD64) binding, Fc γRIIA(CD32A), FcγRIIB(CD32B), FcγRIIIA(CD1 6a) or FcγRIIIB (CD16b) binding is reduced (or only slightly binds to these) It is preferred that the variant Fc region and its ability to mediate such altered binding be Mutational forms are described above. In a specific embodiment, the Fc domains of the present invention The domain-containing molecule comprises an IgG Fc region with reduced ADCC effector function. In a preferred embodiment, the first and / or third polypeptide chains of such an Fc region-containing molecule The CH2-CH3 domain of the present invention contains the following substitutions: L234A, L235A, N297Q, and In another embodiment, the human I The gG Fc region variants include N297Q substitution, N297G substitution, L234A and L235A The IL-16A and IL-16B mutations were not detected in the IL-16A and IL-16B subunits, but contained a D265A or D265A substitution, because these mutations abolished FcR binding. Alternatively, the Fc region of wild-type IgG1 (SEQ ID NO: 1) may be used to inhibit the binding of the Fc region. and) inherently low (or almost no) binding to FcγRIIIA (CD16a), and / or utilize the CH2-CH3 domain of the Fc region, which has inherently low effector function. In a specific embodiment, the Fc region-containing molecule of the invention comprises an IgG2 Fc region (SEQ ID NO:2 ) or IgG4 Fc region (SEQ ID NO: 4). When an IgG4 Fc region is used, The present invention also provides a stabilizing polypeptide, such as the hinge region S228P substitution described above (see, for example, SEQ ID NO: 13). This also includes the introduction of natural mutations such as N297G, N297Q, L234A, L235A, and D26 The 5A substitution abolishes effector function, so in situations where effector function is desired, this It is preferred to avoid these substitutions.

[0258] In particular, with respect to the Fc region of the polypeptide chain of the Fc region-containing molecule of the present invention, Preferably, the serum half-life is increased relative to the half-life exhibited by the native Fc region. Extended phase variant Fc regions and mutant forms are described above. In some embodiments, the first and / or third polypeptide chains of such Fc region-containing molecules The CH2-CH3 domain contains the following substitutions: M252Y, S254T, and T256E. The present invention further includes any one, two or three of: (A) One or more mutations that alter effector function and / or FcγR; and (B) one or more mutations that increase serum half-life The present invention also encompasses Fc region-containing molecules comprising a variant Fc region comprising:

[0259] Preferred IgG1 Sequences for the CH2 and CH3 Domains of the Fc Region-Containing Molecules of the Invention is the substitution L234A / L235A / M252Y / S254T / T256E (SEQ ID NO: 25 8): APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X wherein X is lysine (K) or absent.

[0260] Preferred IgG4 Sequences for the CH2 and CH3 Domains of the Fc Region-Containing Molecules of the Invention M252Y / S254T / T256E substitutions (SEQ ID NO: 259): APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X wherein X is lysine (K) or absent.

[0261] Regarding diabodies and trivalent binding molecules in which the first and third polypeptide chains are not identical between the CH2-CH3 domains of two first polypeptide chains, or between the CH2-CH3 domains of two third polypeptide chains. It is possible to reduce or prevent homodimerization between the CH2-CH3 domains of peptide chains. Preferably, the CH2 and / or CH3 domains of such polypeptide chains have the sequence They need not be identical, but are advantageously of a similar nature to facilitate complex formation between the two polypeptide chains. For example, amino acid substitutions (preferably "knobs") in the CH2 or CH3 domains are made. nob) with amino acids containing bulky side groups, e.g., tryptophan The introduction of the mutated domain prevents interaction with the similarly mutated domain by steric hindrance. The modified domain is then subjected to complementary or adaptive mutations (e.g., substitution with glycine). A series of such domains can be paired with a "hole." The mutations may occur in any of the polypeptides containing the CH2-CH3 domains that form the Fc region. The protein can be used to suppress homodimerization and promote heterodimerization. Methods of protein processing are known in the art, particularly for processing immunoglobulin-like molecules. , which are encompassed herein (see, e.g., Ridgway et al. (1996) "'Knobs-Into-Holes' Engine" eering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,” Protein Eng r. 9:617-621; Atwell et al. (1997) “Stable Heterodimers From Remodeling The Dom ain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35; and Xie et al. (2005) “A New Format of Bispecific Antibody: Highly Efficient ient Heterodimerization, Expression And Tumor Cell Lysis,” J. Immunol. Methods 296:95-101, each of which is incorporated herein by reference in its entirety. Preferably, the "knob" is engineered into the CH2-CH3 domain of the first polypeptide chain and The "hole" is the CH of the third polypeptide chain of the diabody containing these polypeptide chains. The "knob" is therefore the first polypeptide chain to process its CH This may play a role in preventing homodimerization via the CH2 and / or CH3 domains. The third polypeptide chain preferably contains a "hole" substituent, so that the first polypeptide chain It heterodimerizes with the peptide chain and homodimerizes with itself. This strategy is For example, this may be used for diabodies and trivalent binding molecules containing three, four, or five chains. Here, the "knob" is processed into the CH2-CH3 domain of the first polypeptide chain, The "hole" is processed into the CH2-CH3 domain of the third polypeptide chain.

[0262] A preferred knob is prepared by modifying the IgG Fc region to contain the modification group T366W. A preferred hole is generated by modifying the IgG Fc region to include the modified groups T366S, L The hole-bearing third polypeptide was prepared by incorporating 368A and Y407V. Assists in purifying peptide chain homodimers from bispecific heterodimeric Fc region-containing molecules. To achieve this, it is preferred to provide a hole-bearing CH2 and CH3 domain of the third polypeptide chain. The protein A binding site was mutated by an amino acid substitution at position 435 (H435R). In this way, the hole-bearing third polypeptide chain homodimer is attached to protein A. Bispecific monovalent Fc diabodies do not bind to the tandem structure of the first polypeptide chain. The antibody remains capable of binding to protein A via the protein A binding site. In this embodiment, the hole-bearing third polypeptide chain comprises amino acid substitutions at positions 434 and 435. It can be used (N434A / N435K).

[0263] Regarding the CH2 and CH3 domains of the first polypeptide chain of the Fc region-containing molecule of the present invention A preferred IgG1 amino acid sequence is the "knob-bearing" sequence (SEQ ID NO: 6): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W C L VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHN H YTQKS LSLSPG X where X is lysine (K) or absent.

[0264] Fc domains with two polypeptide chains (or three, four, or five polypeptide chains) The second polypeptide chain of an Fc Region-containing molecule of the present invention has a nucleotide sequence similar to that of a nucleotide sequence ... The preferred IgG1 amino acid sequences for the CH2 and CH3 domains of the peptide chain are "Carrying" sequence (SEQ ID NO: 7): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS LSLSPG X where X is lysine (K) or absent.

[0265] As mentioned above, the CH2-CH3 domains of SEQ ID NO:6 and SEQ ID NO:7 are substituted with alanine. and a substitution at position 235 with alanine, thus (wild-type Fc region FcγRIA (CD64), FcγRIIA (CD 32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or Fcγ Fc region with reduced (or substantially no) binding to RIIIB (CD16b) The present invention also provides a method for modifying the effector function and / or FγR binding activity of the Fc region. Also encompassed are such CH2-CH3 domains containing alternative and / or additional substitutions. The present invention also provides such Cs that further comprise one or more half-life extending amino acid substitutions. The H2-CH3 domain is also encompassed. In particular, the present invention relates to the M252Y / S254T / T256E and further including such hole-bearing and knob-bearing CH2-CH3 domains.

[0266] The first polypeptide chain has a "knob-bearing" CH2-CH3 sequence, such as that of SEQ ID NO:6. However, as will be appreciated, it is preferred that the first polypeptide chain not contain a " A "knob-bearing" CH2-CH3 domain (e.g., SEQ ID NO: 7) can be employed, in which case a "knob-bearing" CH2-CH3 domain can be employed. The CH2-CH3 domain (e.g., SEQ ID NO: 6) is a protein having two polypeptide chains. In the second polypeptide chain (or three, four, or five polypeptides) of the Fc Region-containing molecule of the invention In the third polypeptide chain of an Fc region-containing molecule having a peptide chain.

[0267] As mentioned above, the present invention provides a method for the production of a medicament having wild-type CH2 and CH3 domains or substituted as described above. and Fc region-containing molecules (e.g., antibodies) having CH2 and CH3 domains comprising a combination of and Fc region-containing diabodies). An exemplary amino acid sequence of the H2-CH3 domain is (SEQ ID NO:260): APEX1X2GGPSV FLFPPKPKDT LX3IX4RX5PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLX6CX7VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLX8SKL TVDKSRWQQG NVFSCSVMHE ALHX9X 10 YTQKS LSLSPGX 11 where: (a) X1 and X2 are both L (wild type) or both A (low FcγR binding). below); (b) X3, X4, and X5 are M, S, and T (wild type), respectively, or Y, T, and E (extended half-life); (c) X6, X7, and X8 are T, L, and Y (wild type), respectively, or W, L, and Y (knob), or S, A and V (hole); (d) X9 and X 10 are N and H (wild type) or N and R (protein protein A binding), or A and K (no protein A binding); (e)X 11 is K or absent.

[0268] In other embodiments, the present invention provides a method for treating a disease caused by a disease, such as a stroke, stroke, or stroke injury, as described in WO 2007 / 110205; WO 2007 / 110206; 11 / 143545; International Publication No. 2012 / 058768; International Publication No. 2013 / 0 No. 6867, all of which are incorporated herein by reference in their entireties. Mutations known in the art, such as those described herein, can be used to favor heterodimerization over homodimerization. PD-1 binding proteins with engineered CH2 and / or CH3 domains to direct merization. This includes synthetic molecules.

[0269] VIII.PD-1×LAG-3 bispecific binding molecule The present invention particularly relates to anti-PD-1 antibodies, preferably the novel anti-human PD-1 antibodies provided herein. an epitope-binding fragment of one of the PD-1 antibodies and an anti-human LAG-3 antibody, preferably Epitope binding of one of the novel anti-human LAG-3 antibodies provided herein and PD-1 x LAG-3 bispecific binding molecules (e.g., bispecific antibodies, bispecific antibodies, and PD-1 x LAG-3 fragments). Preferred PD-1 x LAG-3 bispecific antibodies of the present invention are PD-1 x LAG-3 diabodies. The combined molecule binds this PD-1×LAG-3 bispecific binding molecule to two distinct epitopes: By cooperatively binding to the PD-1 epitope and the LAG-3 epitope, It has an epitope-binding fragment of the antibody that can attenuate the inhibitory activity of the molecule. As used herein, such attenuation is defined as a detectable At least 20% attenuation of PD-1 and / or LAG-3 inhibitory activity, at least 50 % attenuation, at least 80% attenuation, or at least 90% attenuation, or detectable P This refers to the complete elimination of D-1 and / or LAG-3 inhibitory activity. The selection of epitope-binding fragments (e.g., VL and VH domains) of anti-LAG-3 antibodies is based on this The polypeptide chains that make up such a PD-1×LAG-3 bispecific binding molecule assemble to form at least one functional antibody specific for a first antigen (i.e., PD-1 or LAG-3); The primary binding site and the second antigen (i.e., PD-1 or LA, depending on the identity of the first antigen) G-3) and form at least one functional antigen-binding site specific for the antigen. It will be adjusted.

[0270] In certain embodiments, the PD-1 x LAG-3 bispecific binding molecules of the invention isomeric diabodies, which preferably comprise 2, 3, or 4 hydroxybenzoates, as described herein. In another specific embodiment, the PD-1×L polypeptide of the present invention comprises 4 or 5 polypeptide chains. The AG-3 bispecific binding molecule is a bispecific antibody, which preferably has a structure similar to that described herein. As described in WO 2005 / 023994, the polypeptides may contain two, three, or four polypeptide chains. 7 / 024715; WO 2007 / 110205; WO 2009 / 08 0251; WO 2009 / 080254; WO 2009 / 089004 No.;WO 2011 / 069104;WO 2011 / 117329;International Publication No. 2011 / 131746; International Publication No. 2011 / 133886; International Publication No. 011 / 143545; International Publication No. 2012 / 023053; International Publication No. 2013 / See also US Pat. No. 6,060,867, all of which are incorporated herein by reference in their entireties. (see).

[0271] A. Anti-human LAG-3 antibody Exemplary antibodies that are immunospecific for human LAG-3 are listed below. The antibodies were secreted by antibody-secreting hybrids elicited with LAG-3 or its peptide fragments. by isolating the antibody or by detecting its binding to LAG-3 or a peptide fragment thereof. It can be produced by screening a recombinant antibody library using the 28 amino acid sequence. It contains a 497 amino acid residue signal sequence (shown underlined) and a 497 amino acid residue mature protein. Human LAG-3 has the amino acid sequence (SEQ ID NO: 38): MWEAQFLGLL FLQPLWVAPV KPLQPGAEVP VVWAQEGAPA QLPCSPTIPL QDLSLLRRAG VTWQHQPDSG PPAAAPGHPL APGPHPAAPS SWGPRPRRYT VLSVGPGGLR SGRLPLQPRV QLDERGRQRG DFSLWLRPAR RADAGEYRAA VHLRDRALSC RLRLRLGQAS MTASPPGSLR ASDWVILNCS FSRPDRPASV HWFRNRGQGR VPVRESPHHH LAESFLFLPQ VSPMDSGPWG CILTYRDGFN VSIMYNLTVL GLEPPTPLTV YAGAGSRVGL PCRLPAGVGT RSFLTAKWTP PGGGPDLLVT GDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGS PGSLGKLLCE VTPVSGQERF VWSSLDTPSQ RSFSGPWLEA QEAQLLSQPW QCQLYQGERL LGAAVYFTEL SSPGAQRSGR APGALPAGHL LLFLILGVLS LLLLVTGAFG FHLWRRQWRP RRFSALEQGI HPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL It has.

[0272] 1. LAG‐3 mAb A The anti-human LAG-3 antibody BMS-9, referred to herein as "LAG-3 mAb A" 86016 (25F7; Medarex / BMS) and its variants have already been described. (See, e.g., WO 2014 / 008218). LAG-3 mAb The amino acid sequence of the heavy chain variable domain of A is the amino acid sequence (SEQ ID NO: 39) (CDRs are shown below) (shown with a line): QVQLQQWGAG LLKPSETLSL TCAVYGGSFS DYYWN WIRQP PGKGLEWIG E INHNGNTNSN PSLKS RVTLS LDTSKNQFSL KLRSVTAADT AVYYCAF GYS DYEYNWFDP W GQGTLVTVSS It has.

[0273] The amino acid sequence of the light chain variable domain of LAG-3 mAb A is shown in Table 1 (SEQ ID NO: 1). No. 40) (CDRs are underlined): EIVLTQSPAT LSLSPGERAT LSC RASQSIS SYLA WYQQKP GQAPRLLIY D ASNRAT GIPA RFSGSGSGTD FTLTISSLEP EDFAVYYC QQ RSNWPLT FGQ GTNLEIK It has.

[0274] Additional mouse anti-human LAG-3 antibodies with unique binding properties have recently been identified (US (See Patent Application No. 62 / 172,277). Preferred PD-1 x LAG-3 antibodies of the present invention The bispecific binding molecule may be the anti-human LAG-3 antibody LAG-3 mAb 1 or LAG-3 m It contains an epitope-binding fragment of Ab 6, which binds to a novel epitope and binds to LAG-3. Particularly preferred is the LAG-3 mAb, which does not compete with BMS-986016 for binding. The present invention provides a method for the preparation of a humanized antibody having a humanized VH and / or VL domain of LAG-3 mAb 6 or LAG-3 mAb 1. It is a PD-1×LAG-3 bispecific binding molecule.

[0275] 2. LAG-1 mAb 1 The amino acid sequence of the VH domain of LAG-3 mAb 1 (SEQ ID NO: 41) is shown below: (CDR H Residues are underlined): QIQLVQSGPE LKKPGETVKI SCKASGYTFR NYGMN WVKQA PGKVLKWMG W INTYTGESTY ADDFEG RFAF SLGTSASTAY LQINILKNED TATYFCAR ES LYDYYSMDY W GQGTSVTVSS CDRs of LAG-3 mAb 1 H 1 (SEQ ID NO: 42): RNYGMN CDRs of LAG-3 mAb 1 H 2 (SEQ ID NO: 43): WINTYTGESTYADDFEG CDRs of LAG-3 mAb 1 H 3 (SEQ ID NO: 44): ESLYDYYSMDY

[0276] The amino acid sequence of the VL domain of LAG-3 mAb 1 (SEQ ID NO: 45) is shown below: (CDR L Residues are underlined): DVVVTQTPLT LSVTIGQPAS ISC KSSQSLL HSDGKTYLN W LLQRPGQSPE RLIY LVSELD S GVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYC WQGTHFP YT FGGGTKLE IK CDRs of LAG-3 mAb 1 L 1 (SEQ ID NO: 46): KSSQSLLHSDGKTYLN CDRs of LAG-3 mAb 1 L 2 (SEQ ID NO: 47): LVSELDS CDRs of LAG-3 mAb 1 L 3 (SEQ ID NO: 48): WQGTHFPYT

[0277] As used herein, "hLAG-3 mAb 1 VH1" and "hLAG-3 mAb 1 Two exemplary humanized VH domains of LAG-3 mAb 1, designated "VH1" and "VH2"; and four exemplary humanized VL domains of LAG-3 mAb 1, "hLAG-3 m Ab 1 VL1”, “hLAG-3 mAb 1 VL2”, “hLAG-3 mAb The human mAb 1 VL3 and human mAb 1 VL4 are listed below. Any of the humanized VL domains can be paired with any of the humanized VH domains to detect LAG-3 binding. Thus, the humanized VL domain paired with the humanized VH domain can be generated. Any antibody containing one of these amino acids is commonly referred to as "hLAG-3 mAb 1." , a particular combination of humanized VH / VL domains can be identified by a specific VH / VL domain. For example, hLAG-3 mAb 1 VH1 and hLAG-3 The humanized antibody comprising mAb 1 VL2 is specifically referred to as "hLAG-3 mAb 1(1. 2)"

[0278] The amino acid sequence of the VH domain of hLAG-3 mAb 1 VH1 (SEQ ID NO: 49) As shown below (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W INTYTGESTY ADDFEG RFVF SMDTSASTAY LQISSLKAED TAVYYCAR ES LYDYYSMDY W GQGTTVTVSS

[0279] The amino acid sequence of the VH domain of hLAG-3 mAb 1 VH2 (SEQ ID NO: 50) As shown below (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W INTYTGESTY ADDFEG RFVF SMDTSASTAY LQISSLKAED TAVYFCAR ES LYDYYSMDY W GQGTTVTVSS

[0280] The amino acid sequence of the VL domain of hLAG-3 mAb 1 VL1 (SEQ ID NO: 51) As shown below (CDR L Residues are underlined): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQKPGQSPE RLIY LVSELD S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCW QGTHFP YT FGGGTKVE IK

[0281] The amino acid sequence of the VL domain of hLAG-3 mAb 1 VL2 (SEQ ID NO: 52) As shown below (CDR L Residues are underlined): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQRPGQSPE RLIY LVSELD S GVPDRFSGSGSGTDFTLKI SRVEAEDVGV YYC WQGTHFP YT FGGGTKVE IK

[0282] The amino acid sequence of the VL domain of hLAG-3 mAb 1 VL3 (SEQ ID NO: 53) As shown below (CDR L Residues are underlined): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQKPGQPPE RLIY LVSELD S GVPDRFSGSGSGTDFTLKI SRVEAEDVGV YYC WQGTHFP YT FGGGTKVE IK

[0283] The amino acid sequence of the VL domain of hLAG-3 mAb 1 VL4 (SEQ ID NO: 54) As shown below (CDR L Residues are underlined): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDAKTYLN W LLQKPGQPPE RLIY LVSELD S GVPDRFSGSGSGTDFTLKI SRVEAEDVGV YYC WQGTHFP YT FGGGTKVE IK

[0284] CDRs of the VL domain of hLAG-3 mAb 1 VL4 L 1 is glycine to ara The amino acid sequence is: KSSQSLLHSD A KTYLN (SEQ ID NO: 55, substituted The same substitutions were made in the LAG-3 mAb described above. 1 CDR L It is believed that it can be incorporated into any of the 1 domains.

[0285] 3. LAG-3 mAb 6 The amino acid sequence of the VH domain of LAG-3 mAb 6 (SEQ ID NO: 56) is shown below: (CDR H Residues are underlined): EVLLQQSGPE LVKPGASVKI PCKASGYTFT DYNMD WVKQS HGESLEWIG D INPDNGVTIY NQKFEGKATL TVDKSSSTAY MELRSLTSED TAVYYCAR EA DYFYFDY WGQ GTTLTVSS CDRs of LAG-3 mAb 6 H 1 (SEQ ID NO: 57): DYNMD CDRs of LAG-3 mAb 6 H 2 (SEQ ID NO: 58): DINPDNGVTIYNQKFEG CDRs of LAG-3 mAb 6 H 3 (SEQ ID NO: 59): EADYFYFDY

[0286] The amino acid sequence of the VL domain of LAG-3 mAb 6 (SEQ ID NO: 60) is shown below: (CDR residues are underlined): DIVMTQSHRF MSTSVGDRVS ITC KASQDVS SVVA WYQQKP GQSPKLLIF S ASYRYT GVPD RFTGSGSGTD FTFTISSVQA ADLAVYYCQQ HYSTPWT FGG GTKLEIK CDRs of LAG-3 mAb 6 L 1 (SEQ ID NO: 61): KASQDVSSVVA CDRs of LAG-3 mAb 6 L 2 (SEQ ID NO: 62): SASYRYT CDRs of LAG-3 mAb 6 L 3 (SEQ ID NO: 63): HYSTPWT

[0287] As used herein, "hLAG-3 mAb 6 VH1" and "hLAG-3 mAb 6 Two exemplary humanized VH domains of LAG-3 mAb 6, designated "VH2" and "VH3" respectively, and two exemplary humanized VL domains of LAG-3 mAb 6, "hLAG-3 m Ab 6 VL1" and "hLAG-3 mAb 6 VL2" are listed below. Any of the humanized VL domains can be paired with any of the humanized VH domains to produce LAG-3 Thus, the humanized VL domain paired with the humanized VH domain can be used to generate a binding domain. Any antibody comprising one of the domains is generally referred to as "hLAG-3 mAb 6." A particular combination of humanized VH / VL domains is referred to as a specific VH / VL domain. For example, hLAG-3 mAb 6 VH1 and hLAG- The humanized antibody comprising the VL2 of 3 mAb 6 is specifically referred to as "hLAG-3 mAb 6( 1.2).

[0288] Amino acid sequence of the VH domain of hLAG-3 mAb 6 VH1 (SEQ ID NO: 294) is shown below (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT DYNMD WVRQA PGQGLEWMG D INPDNGVTIY NQKFEG RVTM TTDTSTSTAY MELRSLRSDD TAVYYCAR EA DYFYFDY WGQ GTTLTVSS

[0289] Amino acid sequence of the VH domain of hLAG-3 mAb 6 VH2 (SEQ ID NO: 295) is shown below (CDR H Residues are underlined): EVQLVESGGG LVKPGGSLRL SCAASGFTFS DYNMD WVRQA PGKGLEWVS D INPDNGVTIY NQKFEG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR EA DYFYFDY WGQ GTTLTVSS

[0290] Amino acid sequence of the VL domain of hLAG-3 mAb 6 VL1 (SEQ ID NO: 296) is shown below (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASQDVS SVVA WYQQKP GKAPKLLIY S ASYRYT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWT FGG GTKLEIK

[0291] Amino acid sequence of the VL domain of hLAG-3 mAb 6 VL2 (SEQ ID NO: 297) is shown below (CDR L Residues are underlined): DIVMTQSPSS LSASVGDRVT ITC RASQDVS SVVA WYQQKP GKAPKLLIY S ASYRYT GVPD RFSGSGSGTD FTFTISSLQP EDIAVYYCQQ HYSTPWT FGG GTKLEIK

[0292] CDRs of the VL domains of hLAG-3 mAb 6 VL1 and VL2 L 1 is lysine to arginine, and the amino acid sequence: R ASQDVSSVVA (SEQ ID NO: 298 The substituted arginine is shown underlined). 3 mAb 6 CDR L It is believed that it can be incorporated into any of the 1 domains.

[0293] Exemplary Four-Chain Fc Region-Containing Diabodies with BE / K Coils (referred to as "DART A," "DART B," "DART C," and "DART I" PD-1×LAG-3 dual specificity with an E / K-coil heterodimer-promoting domain (known as We generated isomeric four-chain Fc region-containing diabodies. These exemplary PD-1 x LAG-3 diabodies are described in detail below. The scope is intended to be illustrative and not limiting.

[0294] 1. DART A DART A has two binding sites specific for PD-1 and one specific for LAG-3. two complementary binding sites, a variant IgG4 Fc region engineered for extended half-life, and A bispecific four-chain Fc domain with a cysteine-containing E / K-coil heterodimer-promoting domain The first and third polypeptide chains of DART A are N-terminally linked to the ATP-binding domain. From the N-terminus to the C-terminus: VL domain of a monoclonal antibody capable of binding to LAG-3 N (VL LAG‐3 hLAG-3 mAb 1 VL4) (SEQ ID NO: 54); intervening linker Peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); Monoclonal antibody capable of binding to PD-1 VH domain of the cloned antibody (VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 1 47); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)) ; Cysteine-containing heterodimer-promoting (E-coil) domain (EVAACEK-EVAALEK-EVAALEK - EVAALEK (SEQ ID NO: 23); stabilized IgG4 hinge region (SEQ ID NO: 13); substitution M2 Mutant IgG4 CH2 containing 52Y / S254T / T256E and no C-terminal residues - the CH3 domain (SEQ ID NO: 259); and the C-terminus.

[0295] The amino acid sequences of the first and third polypeptide chains of DART A are shown in SEQ ID NO: 267. Variant: DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDX1KTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSG GCGGGEVAAC EKEVAALEKE VAALEKEVAA LEKESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLX2IX3R X4PEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G wherein X1, X2, X3, and X4 are independently selected, X1 is A or G; and X2 is X3 is T or S; X4 is E or T.

[0296] The amino acid sequence of the first and third polypeptide chains of DART A is SEQ ID NO: 267. where X1 is A; X2 is Y; X3 is T; and X4 is E.

[0297] The second and fourth polypeptide chains of DART A are arranged in the N-terminal to C-terminal direction as follows: The VL domain (VL) of a monoclonal antibody capable of binding to PD-1 PD‐1 hPD‐1 mAb 7 VL2) (SEQ ID NO: 153); intervening linker peptide (Linker 1: GGGS GGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 ( VH LAG‐3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49); cysteine-containing intervening Linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); cysteine-containing heterocyclic Dimer-promoting (K-coil) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 24) ); and the C-terminus.

[0298] The amino acid sequence of the second and fourth polypeptide chains of DART A is (SEQ ID NO:268): : EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTSASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSG GCGGGKVAAC KEKVAALKEK VAALKEKVAA LKE is.

[0299] 2.DART B DART B is identical to DART A, but the first and third polypeptides of DART B are the tide chain comprises the VL domain of hLAG-3 mAb 1 VL3 (SEQ ID NO: 53); This is a CDR L The only difference is that it contains an amino acid substitution in 1. The first and third polypeptide chains are arranged in the N-terminal to C-terminal direction as follows: N-terminus; LAG-3 The VL domain (VL) of a monoclonal antibody that can bind LAG‐3 hLAG‐3 mAb 1 VL3) (SEQ ID NO: 53); intervening linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); VH domain of a monoclonal antibody capable of binding to PD-1 (VH PD‐1 HP D-1 mAb 7 VH1) (SEQ ID NO: 147); intervening linker peptide (linker 2 GGCGGG (SEQ ID NO: 15); cysteine-containing heterodimer-promoting (E coil) domain ( EVAACEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 23); stabilized IgG4 hinge region ( SEQ ID NO: 13); containing substitutions M252Y / S254T / T256E and no C-terminal residues , a variant of the IgG4 CH2-CH3 domain (SEQ ID NO: 259); and the C-terminus.

[0300] The amino acid sequence of the first and third polypeptide chains of DART B is SEQ ID NO:267 , where X1 is G; X2 is Y; X3 is T; and X4 is E.

[0301] The amino acid sequence of the second and fourth polypeptide chains of DART B is SEQ ID NO: 268. do.

[0302] 3. DART C DART C is identical to DART B, but the first and third polypeptides of DART C are The tide chains consist of wild-type IgG4 CH2-CH3 domains (SEQ ID NO: 4) without the C-terminal residues. ) The first and third polypeptide chains of DART C therefore differ from each other only in that they comprise: In the N-terminal to C-terminal direction: N-terminus; V of a monoclonal antibody capable of binding to LAG-3 L domain (VL LAG‐3 hLAG‐3 mAb 1 VL3) (SEQ ID NO: 53); Linker peptide (Linker 1: GGGSGGGG (SEQ ID NO: 14)); a molecule capable of binding to PD-1 The VH domain of the monoclonal antibody (VH PD‐1 hPD-1 mAb 7 VH1) Sequence number 147); intervening linker peptide (linker 2: GGCGGG (SEQ ID NO: 15)); cis The tein-containing heterodimer-promoting (E-coil) domain (EVAACEK-EVAALEK-EVAALEK-EVAAL) EK (SEQ ID NO: 23); stabilized IgG4 hinge region (SEQ ID NO: 13); including C-terminal residues the IgG4 CH2-CH3 domain (SEQ ID NO: 4); and the C-terminus.

[0303] The amino acid sequence of the first and third polypeptide chains of DART C is SEQ ID NO: 267 , where X1 is G; X2 is M; X3 is S; and X4 is T.

[0304] The amino acid sequence of the second and fourth polypeptide chains of DART C is SEQ ID NO: 268. do.

[0305] 4. DART I DART I has two binding sites specific for PD-1 and one specific for LAG-3. two complementary binding sites, a variant IgG4 Fc region engineered for extended half-life, and A bispecific four-chain Fc domain with a cysteine-containing E / K-coil heterodimer-promoting domain The first and third polypeptide chains of DART I are N-terminally linked to the ATP-binding domain. From the N-terminus to the C-terminus: VL domain of a monoclonal antibody capable of binding to LAG-3 N (VL LAG‐3 hLAG‐3 mAb 6 VL1) (SEQ ID NO: 296); intervening linker -peptide (linker 1: GGGSGGGG (SEQ ID NO: 14)); monoclonal antibody capable of binding to PD-1 The VH domain of a monoclonal antibody (VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); a cysteine-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15) ); Cysteine-containing heterodimer-promoting (E-coil) domain (EVAACEK-EVAALEK-EVAALE) K-EVAALEK (SEQ ID NO: 23); stabilized IgG4 hinge region (SEQ ID NO: 13); substitution M2 Mutant IgG4 CH2 containing 52Y / S254T / T256E and no C-terminal residues - the CH3 domain (SEQ ID NO: 259); and the C-terminus.

[0306] The amino acid sequence of the first and third polypeptide chains of DART I is (SEQ ID NO:290): : DIQMTQSPSS LSASVGDRVT ITCRASQDVS SVVAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWTFGG GTKLEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYSFTSYWMN WVRQAPGQGL EWIGVIHPSD SETWLDQKFK DRVTITVDKS TSTAYMELSS LRSEDTAVYY CAREHYGTSP FAYWGQGTLV TVSSGGCGGG EVAACEKEVA ALEKEVAALE KEVAALEKES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG is.

[0307] The second and fourth polypeptide chains of DART I are arranged in the N-terminal to C-terminal direction as follows: VL domain of a monoclonal antibody capable of binding to PD-1 (VL PD‐1 hPD‐ 1 mAb 7 VL2) (SEQ ID NO: 153); intervening linker peptide (Linker 1: GG GSGGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 (VH LAG‐3 hLAG-3 mAb 6 VH1) (SEQ ID NO: 294); cysteine-containing Intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); cysteine-containing heterocycle Dimer-promoting (K-coil) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 2) 4); and the C-terminus.

[0308] The amino acid sequence of the second and fourth polypeptide chains of DART I is (SEQ ID NO:291): :EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTD YNMDWVRQAP GQGLEWMGDI NPDNGVTIYN QKFEGRVTMT TDTSTSTAYM ELRSLRSDDT AVYYCAREAD YFYFDYWGQG TTLTVSSGGC GGGKVAACKE KVAALKEKVA ALKEKVAALK E is.

[0309] C. Exemplary Four-Chain Fc Region-Containing Diabodies with CL / CH1 Domains Referred to as "DART D", "DART E", "DART J" and "DART 1" containing a PD-1 x LAG-3 bispecific four-chain Fc region with CL / CH1 domains Diabodies containing the Fc region were generated and the structures of these Fc region-containing diabodies are detailed below. These exemplary PD-1 x LAG-3 diabodies are intended to illustrate the scope of the present invention. is intended to be illustrative and not limiting.

[0310] 1. DART D DART D has two binding sites specific for PD-1 and one specific for LAG-3. Two specific binding sites, a CL / CH1 domain, and mutations engineered to extend half-life It is a bispecific four-chain Fc region-containing diabody with an IgG4 Fc region. The first and third polypeptide chains of RTD are arranged in the N-terminal to C-terminal direction as follows: N-terminus; P The VL domain (VL) of a monoclonal antibody capable of binding to D-1 PD‐1 hPD‐1 mA b 7 VL2) (SEQ ID NO: 153); intervening linker peptide (Linker 1: GGGSGGGG ( SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 (VH LA G‐3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49); intervening linker peptide ( Linker 2: LGGGSG (SEQ ID NO: 261); IgG4 CH1 domain (SEQ ID NO: 254 ); stabilized IgG4 hinge region (SEQ ID NO: 13); substitutions M252Y / S254T / T25 A mutant form of the IgG4 CH2-CH3 domain (SEQ ID NO: 6E) containing 6E but excluding the C-terminal residue No. 259); and the C-terminus.

[0311] The amino acid sequence of the first and third polypeptide chains of DART D is (SEQ ID NO: 269): : EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTSASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSL GGGSGASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLYITR EPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G is.

[0312] The second and fourth polypeptide chains of DART D are arranged in the N-terminal to C-terminal direction as follows: The VL domain (VL) of a monoclonal antibody capable of binding to LAG-3 LAG‐3 hLAG -3 mAb 1 VL4) (SEQ ID NO: 54); intervening linker peptide (Linker 1: GG GSGGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to PD-1 ( VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); intervening linker peptide tide (linker 2: LGGGSG (SEQ ID NO: 261)); κCL domain (SEQ ID NO: 8); and Includes the C-terminus.

[0313] The amino acid sequence of the second and fourth polypeptide chains of DART D is (SEQ ID NO: 270) : DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSL GGGSGRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC is.

[0314] 2. DART E DART E has two specific binding sites for PD-1 and one specific binding site for LAG-3. Two specific binding sites, a CL / CH1 domain, and mutations engineered to extend half-life It is another bispecific four-chain Fc region-containing diabody with an IgG4-type Fc region. The positions of the PD-1 and LAG-3 binding sites in DART E are inverted compared to DART D It has been done.

[0315] The first and third polypeptide chains of DART E are, in the N-terminal to C-terminal direction: N-terminus The VL domain (VL) of a monoclonal antibody capable of binding to LAG-3 LAG‐3 hLAG -3 mAb 1 VL4) (SEQ ID NO: 54); intervening linker peptide (Linker 1: GG GSGGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to PD-1 ( VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); intervening linker peptide IgG4 CH1 domain (Linker 2: LGGGSG (SEQ ID NO: 261)); IgG4 CH1 domain (SEQ ID NO: 254); stabilized IgG4 hinge region (SEQ ID NO: 13); substitutions M252Y / S254T / A mutant form of the IgG4 CH2-CH3 domain containing T256E and excluding the C-terminal residue ( SEQ ID NO: 259); and the C-terminus.

[0316] The first and third polypeptide chains of DART E are (SEQ ID NO:271): DIVMTQTPLS LSVTPGQPAS ISCKSSQSLL HSDAKTYLNW LLQKPGQPPE RLIYLVSELD SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCWQGTHFP YTFGGGTKVE IKGGGSGGGG QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSL GGGSGASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTKTYTCNV DHKPSNTKVD KRVESKYGPP CPPCPAPEFL GGPSVFLFPP KPKDTLYITR EPEVTCVVVD VSQEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KGLPSSIEKT ISKAKGQPRE PQVYTLPSQ EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL G is.

[0317] The second and fourth polypeptide chains of DART E are arranged in the N-terminal to C-terminal direction as follows: N-terminus The VL domain (VL) of a monoclonal antibody capable of binding to PD-1 PD‐1 hPD‐1 mAb 7 VL2) (SEQ ID NO: 153); intervening linker peptide (Linker 1: GGGS GGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 ( VH LAG‐3 hLAG-3 mAb 1 VH1 (SEQ ID NO: 49); intervening linker peptide tide (linker 2: LGGGSG (SEQ ID NO: 261)); κCL domain (SEQ ID NO: 8); and Includes the C-terminus.

[0318] The amino acid sequence of the second and fourth polypeptide chains of DART E is (SEQ ID NO: 272): : EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTSASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSL GGGSGRTVAA PSVFIFPPSD EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC is.

[0319] 3.DART J DART J has two specific binding sites for PD-1 and one specific binding site for LAG-3. Two specific binding sites, a CL / CH1 domain, and mutations engineered to extend half-life It is a bispecific four-chain Fc region-containing diabody with an IgG4 Fc region. The first and third polypeptide chains of RT J are arranged in the N-terminal to C-terminal direction as follows: N-terminus; L The VL domain (VL) of a monoclonal antibody capable of binding to AG-3 LAG‐3 hLAG‐3 mAb 6 VL1) (SEQ ID NO: 296); intervening linker peptide (Linker 1: GGGSGG GG (SEQ ID NO: 14); the VL domain of a monoclonal antibody capable of binding to PD-1 (VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); intervening linker peptide (Linker 2: LGGGSG (SEQ ID NO: 261)); IgG4 CH1 domain (SEQ ID NO: 2 54); stabilized IgG4 hinge region (SEQ ID NO: 13); substitutions M252Y / S254T / T A mutant form of the IgG4 CH2-CH3 domain containing 256E and excluding the C-terminal residue (sequence column number 259); and the C-terminus.

[0320] The amino acid sequence of the first and third polypeptide chains of DART J is (SEQ ID NO: 292): : DIQMTQSPSS LSASVGDRVT ITCRASQDVS SVVAWYQQKP GKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYSTPWTFGG GTKLEIKGGG SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYSFTSYWMN WVRQAPGQGL EWIGVIHPSD SETWLDQKFK DRVTITVDKS TSTAYMELSS LRSEDTAVYY CAREHYGTSP FAYWGQGTLV TVSSLGGGSG ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG is.

[0321] The second and fourth polypeptide chains of DART J are arranged in the N-terminal to C-terminal direction as follows: The VL domain (VL) of a monoclonal antibody capable of binding to PD-1 PD‐1 hPD‐1 mAb 7 VL2) (SEQ ID NO: 153); intervening linker peptide (Linker 1: GGGS GGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 ( VH LAG‐3 hLAG-3 mAb 6 VH1) (SEQ ID NO: 294); peptide (linker 2: LGGGSG (SEQ ID NO: 261)); κCL domain (SEQ ID NO: 8); and and the C-terminus.

[0322] The amino acid sequence of the second and fourth polypeptide chains of DART J is (SEQ ID NO: 293): : EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTD YNMDWVRQAP GQGLEWMGDI NPDNGVTIYN QKFEGRVTMT TDTSTSTAYM ELRSLRSDDT AVYYCAREAD YFYFDYWGQG TTLTVSSLGG GSGRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC is.

[0323] 4. DART 1 DART 1 has two specific binding sites for PD-1 and one specific binding site for LAG-3. It contains two specific binding sites, the CL / CH1 domain and a domain engineered for reduced FcγR binding. A bispecific four-chain Fc region-containing diabody with a mutant IgG1 Fc region. The first and third polypeptide chains of DART 1 are arranged in the N-terminal to C-terminal direction: The VL domain (VL) of a monoclonal antibody capable of binding to PD-1 PD‐1 PD-1 mAb A VL) (SEQ ID NO: 65); intervening linker peptide (Linker 1: GGGSGGGG ( SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 (VH LA G‐3 LAG-3 mAb A VH1) (SEQ ID NO: 39); intervening linker peptide ( anchor 2: LGGGSG (SEQ ID NO: 261); IgG1 CH1 domain (SEQ ID NO: 10); IgG1 hinge region (SEQ ID NO: 32); containing substitutions L234A / L235A and C-terminal residues a variant of the IgG1 CH2-CH3 domain (SEQ ID NO: 5) that does not contain the C-terminus; nothing.

[0324] The amino acid sequence of the first and third polypeptide chains of DART 1 is (SEQ ID NO: 284): : EIVLTQSPAT LSLSPGERAT LSCRASQSIS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGQ GTNLEIKGGG SGGGGQVQLV ESGGGVVQPG RSLRLDCKAS GITFSNSGMH WVRQAPGKGL EWVAVIWYDG SKRYYADSVK GRFTISRDNS KNTLFLQMNS LRAEDTAVYY CATNDYWGQ GTLVTVSSLG GGSGASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLYI TREPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPG is.

[0325] The second and fourth polypeptide chains of DART 1 are arranged in the N-terminal to C-terminal direction: The VL domain (VL) of a monoclonal antibody capable of binding to LAG-3 LAG‐3 LAG‐ 3 mAb A VL) (SEQ ID NO: 40); intervening linker peptide (Linker 1: GGGSGG GG (SEQ ID NO: 14); the VH domain of a monoclonal antibody capable of binding to PD-1 (VH PD‐1 PD-1 mAb A VH) (SEQ ID NO: 64); intervening linker peptide ( Car2: LGGGSG (SEQ ID NO: 261); κCL domain (SEQ ID NO: 8); and C-terminus nothing.

[0326] The amino acid sequence of the second and fourth polypeptide chains of DART 1 is (SEQ ID NO: 285): : EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKGGG SGGGGQVQLQ QWGAGLLKPS ETLSLTCAVY GGSFSDYYWN WIRQPPGKGL EWIGEINHNG NTNSNPSLKS RVTLSLDTSK NQFSLKLRSV TAADTAVYYC AFGYSDYEYN WFDPWGQGTL VTVSSLGGGS GRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC is.

[0327] D. Exemplary Five-Chain Fc Region-Containing Diabodies The CL / CH1 domain and E / K domain, referred to as "DART F" and "DART G," Two exemplary PD-1 x LAG-3 bispecifics containing a coiled-coil heterodimer-promoting domain We generated five-chain Fc region-containing diabodies. The structures of these Fc region-containing diabodies are shown in Fig. These exemplary PD-1 x LAG-3 diabodies are within the scope of the present invention. are intended to be illustrative and not limiting.

[0328] 1.DART F DART F has three binding sites specific for PD-1 and one specific for LAG-3. The antibody has one binding site, the CL / CH1 domain, which reduces FcγR binding and extends half-life. The mutant knob / hole-bearing IgG1 Fc region and E / K coil heterotransferases were engineered for It is a bispecific five-chain Fc Region-containing diabody with a Dimer-Promoting Domain. The first polypeptide chain of RTF is arranged in the N-terminal to C-terminal direction as follows: N-terminus; The VH domain of a monoclonal antibody that can bind to PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); IgG1 CH1 domain (SEQ ID NO: 10); IgG1 human Range region (SEQ ID NO: 32); substitutions L234A / L235A / M252Y / S254T / T Hole-bearing IgG1 containing 256E / N434A / H435K and no C-terminal residues CH2-CH3 domain (SEQ ID NO: 260, where X1 is A, X2 is A, and X3 is Y, X4 is T, X5 is E, X6 is S, X7 is A, and X8 is V , and X9 is A, and X 10 is K and X 11 is absent); and the C-terminus.

[0329] The amino acid sequence of the first polypeptide chain of DART F is (SEQ ID NO:273): QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLVS KLTVDKSRWQ QGNVFSCSVM HEALHAKYTQ KSLSLSPG is.

[0330] The second and fifth polypeptide chains of DART F are, in the N-terminal to C-terminal direction: N-terminus The VL domain (VL) of a monoclonal antibody capable of binding to PD-1 PD‐1 hPD‐1 mAb 7 VL2) (SEQ ID NO: 153), κC domain (SEQ ID NO: 8); and C-terminus Including the edges.

[0331] The amino acid sequence of the second and fifth polypeptide chains of DART F is (SEQ ID NO: 274): : EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC is.

[0332] The third polypeptide chain of DART F is composed of the following polypeptides in the N- to C-terminal direction: N-terminus; PD The VH domain (VH) of a monoclonal antibody capable of binding to IgG-1 PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); IgG1 CH1 domain (SEQ ID NO: 10); Ig G1 hinge region (SEQ ID NO: 32); substitutions L234A / L235A / M252Y / S254 Knob-bearing IgG1 CH2-CH3 domain containing T / T256E and excluding the C-terminal residue X1 is A, X2 is A, X3 is Y, and X4 is T X5 is E, X6 is W, X7 is L, X8 is Y, and X9 is N , X 10 is H and X 11 is absent); the intervening linker peptide (GGGSGGGSGGG (SEQ ID NO: No. 262)); VL domain (VL) of a monoclonal antibody capable of binding to LAG-3 LAG‐3 hLAG-3 mAb 1 VL4) (SEQ ID NO: 54); an intervening linker peptide ( Car1: GGGSGGGG (SEQ ID NO: 14); VH of a monoclonal antibody capable of binding to PD-1 Domain (VH PD‐1hPD-1 mAb 7 VH1) (SEQ ID NO: 147); Yne-containing intervening linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); heterodimer Facilitatory (E-coil) domain (EVAALEK-EVAALEK-EVAALEK-EVAALEK (SEQ ID NO: 21)); and the C-terminus.

[0333] The amino acid sequence of the third polypeptide chain of DART F is (SEQ ID NO:275): QVQLVQSGAE VKKPGASVKV SCKASGYSFT SYWMNWVRQA PGQGLEWIGV IHPSDSETWL DQKFKDRVTI TVDKSTSTAY MELSSLRSED TAVYYCAREH YGTSPFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGGG GSGGGSGGGD IVMTQTPLSL SVTPGQPASI SCKSSQSLLH SDAKTYLNWL LQKPGQPPER LIYLVSELDS GVPDRFSGSG SGTDFTLKIS RVEAEDVGVY YCWQGTHFPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYSFTS YWMNWVRQAP GQGLEWIGVI HPSDSETWLD QKFKDRVTIT VDKSTSTAYM ELSSLRSEDT AVYYCAREHY GTSPFAYWGQ GTLVTVSSGG CGGGEVAALE KEVAALEKEV AALEKEVAAL EK is.

[0334] The fourth polypeptide chain of DART F is arranged in the N-terminal to C-terminal direction as follows: N-terminus; PD The VL domain (VL) of a monoclonal antibody capable of binding to β-1 PD‐1 hPD-1 mAb 7 VL2) (SEQ ID NO: 153); intervening linker peptide (Linker 1: GGGSGG GG (SEQ ID NO: 14); VH domain of a monoclonal antibody capable of binding to LAG-3 ( VH LAG‐3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49); cysteine-containing intervening Linker peptide (Linker 2: GGCGGG (SEQ ID NO: 15)); heterodimer promotion (K yl) domain (KVAALKE-KVAALKE-KVAALKE-KVAALKE (SEQ ID NO: 22)); and C-terminus Includes:

[0335] The amino acid sequence of the fourth polypeptide chain of DART F is (SEQ ID NO:276): EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGMSFMNWF QQKPGQPPKL LIHAASNQGS GVPSRFSGSG SGTDFTLTIS SLEPEDFAVY FCQQSKEVPY TFGGGTKVEI KGGGSGGGGQ VQLVQSGAEV KKPGASVKVS CKASGYTFTN YGMNWVRQAP GQGLEWMGWI NTYTGESTYA DDFEGRFVFS MDTSASTAYL QISSLKAEDT AVYYCARESL YDYYSMDYWG QGTTVTVSSG GCGGGKVAAL KEKVAALKEK VAALKEKVAA LKE is.

[0336] 2.DART G DART G has two binding sites specific for PD-1 and one specific for LAG-3. Two specific binding sites, the CL / CH1 domain, and the CL / CH2 domain, are used for reduced FcγR binding and extended half-life. Mutant knob / hole-bearing IgG1 Fc regions and E / K coil heterodimers engineered for A bispecific five-chain Fc Region-containing diabody with a Dimer-Promoting Domain. The first polypeptide chain of TG is arranged in the following order from N-terminus to C-terminus: N-terminus; LAG-3 The VH domain of a monoclonal antibody that can bind to LAG‐3 hLAG‐3 mAb 1 VH1) (SEQ ID NO: 49); IgG1 CH1 domain (SEQ ID NO: 10); IgG1 human Range region (SEQ ID NO: 32); substitutions L234A / L235A / M252Y / S254T / T Hole-bearing IgG1 containing 256E / N434A / H435K and no C-terminal residues CH2-CH3 domain (SEQ ...

Claims

1. 1. An anti-human PD-1 binding monospecific monoclonal antibody comprising a variable heavy chain domain and a variable light chain domain, The variable heavy chain domain comprises CDR H 1 domain, CDR H 2 Domains and CDRs H The variable light chain domain comprises three domains, the variable light chain domain comprising: L 1 domain, CDR L 2 Domains and CDRs L It has three domains: (A) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 1 and comprise the amino acid sequences: SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 1 and comprise the amino acid sequences: SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; or (B) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 2 and comprise the amino acid sequences: SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 2 and comprise the amino acid sequences: SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92, respectively; or (C) (1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 3 and comprise the amino acid sequences: SEQ ID NO:99, SEQ ID NO:100, and SEQ ID NO:101, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 3 and comprise the amino acid sequences: SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106; or (D) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 4 and comprise the amino acid sequences: SEQ ID NO:109, SEQ ID NO:110, and SEQ ID NO:111, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 4 and comprise, respectively, the amino acid sequences: SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116; or (E) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 5 and comprise the amino acid sequences: SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 5 and comprise the amino acid sequences: SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, respectively; or (F) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 6 and comprise the amino acid sequences: SEQ ID NO:129, SEQ ID NO:130, and SEQ ID NO:131, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 6 and comprise the amino acid sequences: SEQ ID NO:134, SEQ ID NO:135, and SEQ ID NO:136, respectively; or (G) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 7 and comprise the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 7 and comprise the amino acid sequences: SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146, respectively; or (H)(1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 8 and comprise the amino acid sequences: SEQ ID NO:161, SEQ ID NO:162, and SEQ ID NO:163; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 8 and comprise the amino acid sequences: SEQ ID NO:166, SEQ ID NO:167, and SEQ ID NO:168, respectively; or (I) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 9 and comprise the amino acid sequences: SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 9 and comprise the amino acid sequences: SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178, respectively; or (J) (1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 10 and comprise the amino acid sequences: SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 10 and comprise the amino acid sequences: SEQ ID NO:197, SEQ ID NO:198, and SEQ ID NO:199, respectively; or (K) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 11 and comprise the amino acid sequences: SEQ ID NO:202, SEQ ID NO:203, and SEQ ID NO:204; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 11 and comprise the amino acid sequences: SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:209; or (L) (1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 12 and comprise the amino acid sequences: SEQ ID NO:212, SEQ ID NO:213, and SEQ ID NO:214; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 12 and comprise the amino acid sequences: SEQ ID NO:217, SEQ ID NO:218, and SEQ ID NO:219; or (M) (1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 13 and comprise the amino acid sequences: SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 13 and comprise the amino acid sequences: SEQ ID NO:227, SEQ ID NO:228, and SEQ ID NO:229, respectively; or (N) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 14 and comprise the amino acid sequences: SEQ ID NO:232, SEQ ID NO:233, and SEQ ID NO:234; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 14 and comprise the amino acid sequences: SEQ ID NO:237, SEQ ID NO:238, and SEQ ID NO:239, respectively; or (O) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 15 and comprise the amino acid sequences: SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 15 and comprise the amino acid sequences: SEQ ID NO:247, SEQ ID NO:248, and SEQ ID NO:249, respectively; or (P) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the heavy chain CDRs of hPD1 mAb 7 (1.3) and comprise the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of hPD1 mAb 7 (1.3) and comprise the amino acid sequences: SEQ ID NO:157, SEQ ID NO:158, and SEQ ID NO:145, respectively; or (Q) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of hPD1 mAb 9 (2.2) and comprise the amino acid sequences: SEQ ID NO:183, SEQ ID NO:172, and SEQ ID NO:173, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the light chain CDRs of hPD1 mAb 9 (2.2) and comprise the amino acid sequences: SEQ ID NO:188, SEQ ID NO:189, and SEQ ID NO:178, respectively; Anti-human PD-1 binding monospecific monoclonal antibody.

2. (A) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 1 and comprise the amino acid sequences: SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 1 and comprise the amino acid sequences: SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; or (B) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 2 and comprise the amino acid sequences: SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 2 and comprise the amino acid sequences: SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92, respectively; or (C) (1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 5 and comprise the amino acid sequences: SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 5 and comprise the amino acid sequences: SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, respectively; or (D) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 7 and comprise the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 7 and comprise the amino acid sequences: SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146, respectively; or (E) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the heavy chain CDRs of hPD1 mAb 7 (1.3) and comprise the amino acid sequences: SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of hPD1 mAb 7 (1.3) and comprise the amino acid sequences: SEQ ID NO:157, SEQ ID NO:158, and SEQ ID NO:145, respectively; or (F) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 9 and comprise the amino acid sequences: SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 9 and comprise the amino acid sequences: SEQ ID NO:176, SEQ ID NO:177, and SEQ ID NO:178, respectively; or (G) (1) the CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of hPD1 mAb 9 (2.2) and comprise the amino acid sequences: SEQ ID NO:183, SEQ ID NO:172, and SEQ ID NO:173, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of hPD1 mAb 9 (2.2) and comprise the amino acid sequences: SEQ ID NO:188, SEQ ID NO:189, and SEQ ID NO:178, respectively; or (H)(1) Said CDR H 1 domain, said CDR H 2 domain and said CDRs H The 3 Domains are the Heavy Chain CDRs of PD1 mAb 15 and comprise the amino acid sequences: SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244, respectively; (2) the CDR L 1 domain, said CDR L 2 domain and said CDRs L The 3 Domains are the Light Chain CDRs of PD1 mAb 15 and comprise the amino acid sequences: SEQ ID NO:247, SEQ ID NO:248, and SEQ ID NO:249, respectively.

2. The anti-human PD-1 binding monospecific monoclonal antibody of claim 1.

3. 3. The anti-human PD-1 binding monospecific monoclonal antibody of claim 1 or 2, wherein the antibody is a chimeric antibody or a humanized antibody.

4. 4. The anti-human PD-1 -binding monospecific monoclonal antibody of any one of claims 1-3, wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:79, SEQ ID NO:93, SEQ ID NO:149, SEQ ID NO:179, SEQ ID NO:181, or SEQ ID NO:

250.

5. 5. The anti-human PD-1 -binding monospecific monoclonal antibody of any one of claims 1-4, wherein the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO:81, SEQ ID NO:95, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:184, SEQ ID NO:186, or SEQ ID NO:

251.

6. 6. The anti-human PD-1 binding monospecific monoclonal antibody of any one of claims 1-5, wherein the antibody comprises an Fc Region.

7. 7. The anti-human PD-1 -binding monospecific monoclonal antibody of claim 6, wherein the Fc Region is of the IgG1, IgG2, IgG3, or IgG4 isotype.

8. 8. The anti-human PD-1 binding monospecific monoclonal antibody of claim 7, wherein the antibody further comprises a hinge domain.

9. the Fc region and the hinge domain are of the IgG4 isotype; 9. The anti-human PD-1 -binding monospecific monoclonal antibody of claim 8, wherein said hinge domain comprises a stabilizing mutation.

10. The Fc region is a variant Fc region, The variant Fc Region comprises: (a) one or more amino acid modifications that reduce the affinity of the variant Fc Region for an FcγR; and / or (b) one or more amino acid modifications that increase the serum half-life of the variant Fc region.

10. The anti-human PD-1 binding monospecific monoclonal antibody of any one of claims 6 to 9, comprising:

11. 11. The anti-human PD-1 -binding monospecific monoclonal antibody of claim 10, wherein said modification that reduces the affinity of said variant Fc Region for an FcγR comprises the substitutions L234A; L235A; or L234A and L235A, wherein the numbering of said substitutions is the EU index numbering as in Kabat.

12. 12. The anti-human PD-1 -binding monospecific monoclonal antibody of claim 10 or 11, wherein said modifications that increase the serum half-life of the variant Fc Region comprise the following substitutions: M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T, and T256E; or K288D and H435K, wherein the numbering of said substitutions is the EU index numbering as in Kabat.

13. 13. The anti-human PD-1 binding monospecific monoclonal antibody of any one of claims 1 to 12, wherein the antibody is detectably labeled and used to detect PD-1.

14. 13. A composition comprising the anti-human PD-1 binding monospecific monoclonal antibody of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

15. 15. The anti-human PD-1 binding monospecific monoclonal antibody of any one of claims 1 to 12, or the composition of claim 14, for use in the treatment of cancer.

16. The cancers include: adrenal cancer; AIDS-related cancer; alveolar soft part sarcoma; astrocytic tumor; bladder cancer; Bone cancer; brain and spinal cord cancer; metastatic brain tumors; breast cancer; carotid artery tumors; cervical cancer; chondrosarcoma; chordoma; chromogenic renal cell carcinoma; clear cell carcinoma; colon cancer; colorectal cancer; cutaneous benign fibrous histiocytoma; desmoplastic small round cell tumor; ependymoma; Ewing's tumor; extraskeletal myxoid chondrosarcoma; fibrous imperfecta osseous; fibrous dysplasia; gallbladder or bile duct cancer; gastrointestinal cancer; gestational trophoblastic disease; germ cell tumors; head and neck cancer; hepatocellular carcinoma; islet cell tumors; Kaposi's sarcoma; kidney cancer, leukemia, lipoma / benign lipomatous tumor, liposarcoma / malignant lipomatous tumor, liver cancer; lymphoma; Lung cancer; Medulloblastoma; Melanoma; Meningioma; Multiple endocrine neoplasms; Multiple myeloma; Myelodysplastic syndromes; Neuroblastoma; Neuroendocrine tumors; Ovarian cancer; 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, characterized by the presence of a cancer cell selected from the group consisting of: pancreatic cancer; papillary thyroid carcinoma; parathyroid tumor; pediatric cancer; peripheral nerve sheath tumor; pheochromocytoma; pituitary tumor; prostate cancer; posterior uveal melanoma; rare hematological disorder; renal metastatic cancer; rhabdoid tumor; rhabdomyosarcoma; sarcoma; skin cancer; soft tissue sarcoma; squamous cell carcinoma; gastric cancer; synovial sarcoma; testicular cancer; thymic carcinoma; thymoma; thyroid metastatic cancer; and uterine cancer cells.

17. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is: colorectal cancer; hepatocellular carcinoma; glioma; renal cancer; breast cancer; multiple myeloma; bladder cancer; neuroblastoma; sarcoma; non-Hodgkin's lymphoma; non-small cell lung cancer; ovarian cancer; pancreatic cancer; rectal cancer; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); acute B-lymphoblastic leukemia (B-ALL); chronic lymphocytic leukemia (CLL); hairy cell leukemia (HCL); blastic plasmacytoid dendritic cell neoplasm (BPDCN); non-Hodgkin's lymphoma (NHL), including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL); Hodgkin's lymphoma; systemic mastocytosis; or Burkitt's lymphoma.

18. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is uterine cancer.

19. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is squamous cell carcinoma.

20. 16. The anti-human PD-1 binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is glioma.

21. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is cervical cancer.

22. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is renal cancer.

23. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is lung cancer.

24. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is non-small cell lung cancer.

25. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is head and neck cancer.

26. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is renal metastatic cancer.

27. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is chromogenic renal cell carcinoma.

28. 16. The anti-human PD-1 -binding monospecific monoclonal antibody or composition of claim 15, wherein the cancer is a skin cancer.

29. 13. A nucleic acid expression vector encoding the anti-human PD-1 binding monospecific monoclonal antibody of any one of claims 1 to 12.

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