PD-1 binding molecule and method of 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 of related applications This application is U.S. Patent Application No. 62 / 198,867 (filed July 30, 2015; pending). (In the 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. (National Patent Application No. 62 / 322,974, filed April 15, 2016; pending) Priority is claimed, and each of these patent applications is referred to in whole as this application. It is used in conjunction with [the text].

[0002] Sequence listing reference This application includes one or more sequence listings under Section 1.821 et seq. of Title 37 of the Federal Code of Regulations. These sequence listings are available in computer-readable media (filename: 1301_0122PCT). _Sequence_Listing_ST25.txt, created July 1, 2016, It is disclosed in (size: 282,789 bytes), and the above file can be referenced. The entirety of this is incorporated herein by reference.

[0003] The present invention relates to a selected anti-P that can bind 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 14 or PD-1 mAb 15 comprising a PD-1 binding domain, The present invention is intended for children. In particular, the present invention is a humanized or chimeric version of such an antibody. or PD-1 binding fragments of such anti-PD-1 antibodies (especially immunoconjugates, diabolic bodies) The present invention relates to a PD-1 binding molecule comprising (BiTE, bispecific antibodies, etc.). In particular, the present invention relates to Epitopes of molecules involved in the regulation of immune checkpoints present on the surface of immune cells The present invention relates to such PD-1 binding molecules that can further bind. The present invention also relates to the detection of PD-1. Or it relates to methods of using such PD-1 binding molecules to stimulate an immune response. The invention also relates to one or more PD-1-conjugated domains of the selected anti-PD-1 antibodies described above. A PD-1 binding molecule containing , one or more effective molecules for stimulating an immune response One or more additional molecules that combine with and / or specifically bind to cancer antigens. This relates to combination therapy, which involves administering additional molecules in combination. [Background technology]

[0004] Explanation of related technologies I. Cell-mediated immune response The immune system of humans and other mammals plays a role in providing protection against infection and disease. Such protection is provided by both humoral and cell-mediated immune responses. Through humoral immune responses, antibodies and other biomolecules that can recognize and neutralize foreign targets (antigens) Production is obtained. In contrast, cell-mediated immune responses involve T cells mediated by macrophages and NATURAL cells. 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 awareness (Non-Patent Document 1).

[0005] The ability of T cells to optimally mediate the immune response against antigens is divided into two distinct signaling phases. Interaction is required (Non-patent documents 2, 3). First, antigen-presenting cells (antigen-present Antigens present on the surface of the cell (APC) are identified by antigen-specific naive CD4 + T It is necessary to present it to the cells. Through such presentation, the presented antigen is specific. T-cell receptors (T-Cell Receptors) instruct T cells to initiate a target immune response. The signal is delivered via the ceptor (TCR). Next, the APC and a separate T cell... A series of co-stimulatory and inhibitory signals mediated by interactions with surface molecules first T It triggers cell activation and proliferation, ultimately leading to T cell inhibition. Therefore, the first signal The first signal confers specificity to the immune response, and the second signal determines the nature, strength, and duration of the above response. It plays a role in determining the duration.

[0006] The immune system is strictly regulated by co-stimulatory and co-inhibitory ligands and receptors. The molecule provides a second signal for T cell activation and also limits the immune response against self. Positive and negative signals to maximize the immune response to infection It provides an equilibrium network (Non-Patent Documents 4, 5). It maintains self-tolerance and the duration of the immune response. The inhibitory pathways that are important for modulating the amplitude of these pathways are collectively called immune checkpoints. The key factors are the B7.1 (CD80) and B7.2 (CD86) ligands of antigen-presenting cells. CD4 + This is the binding between CD28 and CTLA-4 receptors on T lymphocytes (unpatented). References 6, 1, 7). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation. Furthermore, the binding of B7.1 or B7.2 to CTLA-4 inhibits the above activation (non (Patent Documents 1, 7, 8). CD28 is constitutively expressed on the surface of T cells (Non-Patent Document 9). On the other hand, CTLA-4 expression is rapidly upregulated following T cell activation (non-specific). Reference 10). Since CTLA-4 is a high-affinity receptor (Non-Patent Literature 6), binding is first (CD28) initiates T cell proliferation, and subsequently (by the initial expression of CTLA-4) It inhibits T cell proliferation, thereby reducing its effectiveness when proliferation is no longer needed.

[0007] Further investigation into the ligands for the CD28 receptor has revealed a set of related B7 molecules (" Identification and characterization of the "B7 Superfamily" were obtained. (Non-patent documents 11, 6, 8, 12, 13, 3, 14, 15, 16, 17). Currently, the above Several members of the family are publicly known: B7.1 (CD80), B7.2 (CD86) ), inducible costimulatory factor ligand (ICOS-L), programmed death-1 ligand (PD-L) 1;B7-H1), programmed-dead-2 ligand (PD-L2;B7-DC), B7-H3 , B7-H4 and B7-H6 (Non-Patent Documents 12, 18).

[0008] II. Program Death - 1 ("PD-1") Programmed death-1 (also known as "PD-1" or "CD279") broadens the immune response. Approximately 31 of the expanded CD28 / CTLA-4 family of T cell regulatory factors that lower the control of T cells It is a type I membrane protein member of kD (Non-Patent Literature 19, Patent Literature 1-9).

[0009] PD-1 is present on activated T cells, B cells, and monocytes (Non-Patent Documents 20, 21), and Furthermore, it was found at low levels in natural killer (NK) T cells (Non-patent documents 22, 23), It manifests.

[0010] The extracellular domain of PD-1 has 23% identity with the equivalent domain of CTLA-4. It consists of a single immunoglobulin (Ig) V domain (Non-Patent Literature 23). Extracellular I The gV domain is followed by a membrane-permeable region and an intracellular tail. The intracellular tail is an immune receptor. Two motifs located within the tyrosine system inhibitory motif and the immune receptor tyrosine system switch motif It contains a phosphorylation site, which suggests that PD-1 downregulates TCR signaling. (Non-patent documents 19, 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 10-17).

[0012] B7-H1 and B7-DC are found in the heart, placenta, muscles, fetal liver, spleen, lymph nodes, and chest. Glands, as well as human and mouse organisms such as the liver, lungs, kidneys, pancreatic islet cells, and small intestine of mice. It is widely expressed on the surface of fabrics (Non-Patent Literature 23). In humans, B7-H1 protein expression is Human endothelial cells (Non-Patent Literature 25, 26, 27), cardiomyocytes (Non-Patent Literature 28), syncytial nutrient It was observed in cells (Non-Patent Literature 29). The above molecule is also found in the normal tissues of several tissues. Clophages can detect interferon (IFN)-γ or tumor necrosis factor (tumor Macrophages activated by necrosis factor (TNF)-α It is also expressed by (Non-Patent Document 30) and within tumors (Non-Patent Document 31).

[0013] The interaction between B7-H1 and PD-1 is a significant downward co-stimulation of T and B cells. Gunar (Non-Patent Document 23), and its function as a cell death inducer (Non-Patent Documents 19, 32) It is known to provide low concentrations of PD-1 receptors and B7-H. More specifically, it provides low concentrations of PD-1 receptors and B7-H. The interaction between ligand 1 is antigen-specific CD8 + Inhibitory cells that strongly inhibit T cell proliferation It is known to lead to the transmission of Gnál; at high concentrations, interaction with PD-1 is T-cell Although it does not inhibit cell proliferation, it clearly reduces the production of multiple cytokines (non-patent text). Reference 6). Restless CD4 and CD8 T cells and already activated CD4 and CD8 T cells T cell proliferation and cytokine production by both cells, as well as naive T cells derived from umbilical cord blood. It has been found that this is inhibited by soluble B7-H1-Fc fusion protein (non Patent documents 33, 30, 34, 6).

[0014] The roles of B7-H1 and PD-1 in T cell activation and proliferation are due to these biomolecules This suggests that it may function as a therapeutic target for the treatment of inflammation and cancer. The use of anti-PD-1 antibodies for the treatment of staining and tumors, as well as for the upmodulation of adaptive immune responses, has been proposed. (See Patent Documents 18, 19, 20, 2, 21, 22, 23, 24, 25, 26) Antibodies that can specifically bind to PD-1 have been reported in 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 This more strongly instructs the body's immune system to attack infected cells, especially at relatively low therapeutic concentrations. There continues to be a demand for improved compositions that can do so, because the adaptive immune system Although it may be a powerful protective mechanism against cancer and disease, the expression of PD-1 and other tumor micro-effects This is because it may be hindered by immunosuppressive mechanisms in the environment. Furthermore, in the tumor environment Co-inhibitory molecules expressed by tumor cells, immune cells, and stromal cells affect T cell response against cancer cells. This can predominantly weaken the answer. Therefore, there is a need for strong PD-1 binding molecules. It continues to exist. In particular, it has the desired binding kinetic profile, and also PD-1 / PD-L 1. Antagonizing the PD-1 / PD-L1 axis by blocking the interaction (this is cancer (or can provide improved therapeutic value to patients separated from other diseases and conditions), There continues to be a demand for powerful PD-1 binding molecules. This invention addresses these and other This targets the following objectives. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] U.S. Patent Application Publication No. 2007 / 0202100 [Patent Document 2] U.S. Patent Application Publication No. 2008 / 0311117 [Patent Document 3] U.S. Patent Application Publication No. 2009 / 0110667 [Patent Document 4] U.S. Patent No. 6,808,710 [Patent Document 5] U.S. Patent No. 7,101,550 [Patent Document 6] U.S. Patent No. 7,488,802 [Patent Document 7] U.S. Patent No. 7,635,757 [Patent Document 8] U.S. Patent No. 7,722,868 [Patent Document 9] WO 01 / 14557 [Patent Document 10] U.S. Patent No. 6,803,192 [Patent Document 11] U.S. Patent No. 7,794,710 [Patent Document 12] U.S. Patent Application Publication No. 2005 / 0059051 [Patent Document 13] U.S. Patent Application Publication No. 2009 / 0055944 [Patent Document 14] U.S. Patent Application Publication No. 2009 / 0274666 [Patent Document 15] U.S. Patent Application Publication No. 2009 / 0313687 [Patent Document 16] WO 01 / 39722 [Patent Document 17] WO 02 / 086083 [Patent Document 18] U.S. Patent Application Publication No. 2010 / 0040614 [Patent Document 19] U.S. Patent Application Publication No. 2010 / 0028330 [Patent Document 20] U.S. Patent Application Publication No. 2004 / 0241745 [Patent Document 21] U.S. Patent Application Publication No. 2009 / 0217401 [Patent Document 22] U.S. Patent No. 7,521,051 [Patent Document 23] U.S. Patent No. 7,563,869 [Patent Document 24] U.S. Patent No. 7,595,048 [Patent Document 25] WO 2004 / 056875 [Patent Document 26] WO 2008 / 083174 [Patent Document 27] U.S. Patent No. 8,008,449

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Patent Document 44

Non-Patent Document

[0017]

Non-Patent Document 1

[0018] The present invention relates to a selected anti-P that can bind 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 14 or PD-1 mAb 15 comprising a PD-1 binding domain, The present invention is intended for children. In particular, the present invention is a humanized or chimeric version of such an antibody. or PD-1 binding fragments of such anti-PD-1 antibodies (especially immunoconjugates, diabolic bodies) The present invention relates to a PD-1 binding molecule comprising (BiTE, bispecific antibodies, etc.). In particular, the present invention relates to To an epitope of a molecule involved in the control of immune checkpoints present on the surface of immune cells Relates to such PD-1 binding molecules that can further bind. The present invention also relates to methods of using such PD-1 binding molecules for the detection or stimulation of an immune response of PD-1. The present invention also relates to a combination therapy of administering a PD-1 binding molecule comprising one or more PD-1 binding domains of a selected anti-PD-1 antibody as described above, in combination with one or more additional molecules effective to stimulate an immune response, and / or in combination with one or more additional molecules that specifically bind to a cancer antigen.

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

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

[0021] The present invention further includes the heavy chain variable domain as specified by SEQ ID NOs. 79, SEQ ID NOs. 93, and SEQ ID NOs. 14. 7. Amino acid combination of SEQ ID NO: 149, SEQ ID NO: 179, SEQ ID NO: 181, or SEQ ID NO: 250 This relates to embodiments of such anti-human PD-1 binding molecules having columns.

[0022] The present invention further includes the above light chain variable domain as specified by SEQ ID NOs. 81, 95, and 15. 1, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 184, SEQ ID NO: 186, or SEQ ID NO: 2 This 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 relates that the anti-human PD-1 binding molecule is linked to human PD-1 and a second epitope. Regarding embodiments of bispecific binding molecules that can bind simultaneously, in particular, the second epitope described above P is a molecule involved in the control of immune checkpoints present on the surface of immune cells. This is a type of epitope (in particular, the second epitope mentioned above 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 For more information, see II, NKG2a, NKG2d, OX40, OX40L, PD1H, PD-1, P D-L1, PD-L2, PVR, SIRPa, TCR, TIGIT, TIM-3 or VI It is an epitope of STA, and more specifically, the second epitope mentioned above is CD137, CT (Implementation of LA-4, LAG-3, OX40, TIGIT, or TIM-3 epitopes) Regarding form.

[0024] The present invention further comprises the above anti-human PD-1 binding molecule with a LAG-3 epitope binding site. It is a bispecific molecule, and in particular the above LAG-3 epitope binding site comprises the following, Regarding form: (A)(1) Amino acid sequences: Sequence IDs 42, 43 and 44 respectively The variable heavy chain CDR of LAG-3 mAb 1 possesses H 1 domain, CDR H 2 domains and and CDR H 3 domains; and (2) Amino acid sequences: Sequence IDs 46, 47, and 48 respectively The variable light chain CDR of LAG-3 mAb 1 possesses L 1 domain, CDR L 2 domains and and CDR L 3 domains; or (B)(1) Amino acid sequences: Sequence IDs 42, 43 and 44 respectively The variable heavy chain CDR of hLAG-3 mAb 1 VH1 possesses H 1 domain, CDRH 2 Domains and CDRs H 3 domains; and (2) Amino acid sequences: Sequence IDs 55, 47, and 48 respectively The variable light chain CDR of hLAG-3 mAb 1 VL4 possesses L 1 domain, CDR L 2 Domains and CDRs L 3 domains; or (C)(1) Amino acid sequences: Sequence IDs 57, 58 and 59 respectively The variable heavy chain CDR of LAG-3 mAb 6 possesses H 1 domain, CDR H 2 domains and and CDR H 3 domains; and (2) Amino acid sequences: Sequence IDs 61, 62 and 63 respectively The CDR possesses a 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 sequence numbers 57, 58, and 59 respectively Variable heavy chain CDR of G-3 mAb 6 VH1 H 1 domain, CDR H 2 domains and C DR H 3 domains; and (2) Amino acid sequences: Sequence IDs 298, 62 and 63 respectively This has a variable light chain CDR of LAG-3 mAb 6. L 1 domain, CDR L 2 domains and CDR L 3 domains.

[0025] The present invention further relates that the above molecule is a diamond body, and in particular, that there are two of the above diamond bodies. Or such an antibody is a covalent complex containing three, four, or five polypeptide chains. This invention relates to embodiments of human PD-1 binding molecules. The present invention further relates to the above molecule being a trivalent binding molecule. Furthermore, the above trivalent bond molecule contains three, four, five, or six or more polypeptide chains. This invention relates to an embodiment of such an anti-human PD-1 binding molecule, which is a covalent complex. Akira further describes embodiments of such anti-human PD-1 binding molecules in which the above molecule comprises an Fc region. The present invention further relates to the above molecule having an albumin-binding domain, particularly deimmunized albumin This relates to embodiments of such anti-human PD-1 binding molecules that include a binding domain.

[0026] The present invention further comprises the above molecule having an Fc region, and the above Fc region is a mutant Fc region. The above mutant Fc region reduces the affinity of the above mutant Fc region to FcγR, and It includes one or more amino acid modifications that increase the serum half-life, and more specifically, 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; This numbering includes at least one amino acid substitution selected from the group consisting of Ka This is the numbering of the EU index, as in bat, for all such anti-human PDs. -1. This relates to embodiments of the bonded molecule.

[0027] The present invention further uses any of the above-mentioned PD-1 binding molecules to enable T cell-mediated immunotherapy. The present invention relates to embodiments that stimulate the response. The present invention further relates to any of the above-mentioned PD-1 binding molecules This is used in the treatment of diseases or conditions associated with a suppressed immune system, particularly cancer or infections. Regarding the form of implementation.

[0028] The present invention particularly relates to the above-mentioned uses in the treatment, diagnosis, or prognosis of cancer, wherein the cancer is Adrenal cancer; AIDS-related cancer; alveolar soft part sarcoma; astrocytic neoplasm; 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; colorectal cancer; colorectal cancer; benign fibrous histiocytoma; fibrinogenic small round cell tumor; Ependymoma; Ewing's tumor; Extraskeletal myxoid chondrosarcoma; Osteid fibrodysplasia; Fibrous dysplasia Diseases; gallbladder or bile duct cancer; gastrointestinal cancer; gestational trophoblastic disease; germ cell tumors; head and neck cancer; hepatocyte Cancer; pancreatic islet cell tumor; Kaposi's sarcoma; kidney cancer, leukemia, lipoma / benign liposomal tumor, liposarcoma Malignant liposomal tumors; liver cancer; lymphoma; lung cancer; medulloblastoma; melanoma; meningioma; multiple endocrine tumors Urinary tumors; multiple myeloma; myelodysplastic syndrome; neuroblastoma; neuroendocrine tumors; ovarian cancer; pancreatic Thoracic cancer; papillary thyroid carcinoma; parathyroid tumor; childhood cancer; peripheral nerve sheath tumor; pheochromocytoma; pituitary tumor Surgery; prostate cancer; posterior uveal melanoma; rare hematological disorders; renal metastasis; rhabdoid tumors Rhabdomyosarcoma; sarcoma; skin cancer; soft tissue sarcoma; squamous cell carcinoma; gastric cancer; synovial sarcoma; testicular cancer; Cancer cells selected from the group consisting of thymic carcinoma; thymoma; metastatic thyroid cancer; and uterine cancer cells. Its existence is its defining characteristic.

[0029] The present invention particularly relates to the above-mentioned uses in the treatment, diagnosis, or prognosis of cancer, wherein the cancer is Colorectal cancer; hepatocellular carcinoma; glioma; kidney cancer; breast cancer; multiple myeloma; bladder cancer; neuroblastoma Tumors; sarcomas; 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-cell leukemia Lymphoblastic leukemia (B-ALL); Chronic lymphocytic leukemia (CLL); Pilocytic cell leukemia ( HCL; blastic plasmacytoid dendritic cell neoplasm (BPDCN); mantle cell leukemia (MC) Non-Hodgkin lymphoma (non-Hodgkin's L), including small lymphocytic lymphoma (SLL). Lymphomas (NHL); Hodgkin lymphoma; systemic mastocytosis; or Burkitt lymphoma be.

[0030] The present invention further involves detecting and labeling any of the above-mentioned PD-1 binding molecules, and PD-1 This relates to embodiments used for detection. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1 is a schematic diagram of a typical covalent diabody having two epitope-binding sites, each consisting of two polypeptide chains, one having an E-coil or the other a K-coil heterodimer-promoting domain. As shown in Figure 3B, cysteine ​​residues may be present in the linker and / or the heterodimer-promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. [Figure 2] Figure 2 is a schematic diagram of a typical covalent diabody molecule having two epitope-binding sites, each consisting of two polypeptide chains, one with a CH2 domain and the other with a CH3 domain, such that the linked chain forms all or part of the Fc region. The VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. [Figure 3A-3C]Figures 3A-3C are schematic diagrams of a typical tetravalent diabody having four epitope-binding sites, consisting of two pairs of polypeptide chains (i.e., a total of four polypeptide chains). One polypeptide in each pair has CH2 and CH3 domains such that the linked chains form all or part of the 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 may 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 divalent for each epitope it binds to. In embodiments where the VL and VH domains recognize the same epitope (e.g., using the same VL domain CDR and the same VH domain CDR 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 may be different. In embodiments where the VL and VH domains of each polypeptide pair recognize different epitopes (as shown in Figure 3C), the resulting molecule has four epitope-binding sites, is quadruple-specific, and is monovalent for each epitope it binds to. Figure 3A shows an Fc diabody containing a peptide heterodimer-promoting domain with a cysteine ​​residue. Figure 3B shows an Fc region-containing diabody, which contains E-coil and K-coil heterodimer-promoting domains with a cysteine ​​residue and a linker (having any cysteine ​​residue). Figure 3C shows an Fc region-containing diabody containing antibody CH1 and CL domains. [Figure 4A-4B] Figures 4A and 4B are schematic diagrams of typical covalent diabody molecules having two epitope-binding sites consisting of three polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains such that the linked chains form all or part of the Fc region. The polypeptide chain having VL and VH domains further comprises a heterodimer-promoting domain. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. [Figure 5]Figure 5 is a schematic diagram of a typical covalent diabody having four epitope-binding sites consisting of five polypeptide chains. Two of the polypeptide chains have CH2 and CH3 domains such that the linked chains form an Fc region containing all or part of the Fc region. Polypeptide chains having VL and VH domains further have heterodimer-promoting domains. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. [Figures 6A-6F] Figures 6A-6F are schematic diagrams of typical Fc-domain-containing trivalent binding molecules having three epitope-binding sites. Figures 6A and 6B show the domains of trivalent binding molecules comprising two diabody-type binding domains and a Fab-type binding domain, each having a different domain orientation where the diabody-type binding domain is N-terminus or C-terminus to the Fc domain. The molecules in Figures 6A and 6B have four chains. Figures 6C and 6D show the domains of trivalent binding molecules comprising two diabody-type binding domains that are N-terminus to the Fc domain, and a Fab-type binding domain or an scFv-type binding domain in which the light and heavy chains are linked via polypeptide spacers, respectively. The trivalent binding molecules in Figures 6E and 6F schematically show the domains of trivalent binding molecules comprising two diabody-type binding domains that are C-terminus to the Fc domain, and a linked Fab-type binding domain or an scFv-type binding domain in which the diabody-type binding domains are located. The trivalent binding molecules in Figures 6C-6F have three chains. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. [Figures 7A-7D]Figures 7A to 7D show that the anti-PD-1 antibodies PD-1 mAb 1 to 15 bind to human PD-1. Binding curves for 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). The binding curves for 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 that the anti-PD-1 antibodies PD-1 mAb 1-15 bind to cynomolgus monkey PD-1. Binding curves for 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 mAb 1-15 to block the binding of human PD-L1 to human PD-1. The inhibition curves are shown in Figures 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). [Figure 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. Figures 10A, panels i-vi, show the results of tissue incubated with labeled PD-1 mAb 7 (0.313 μg / mL). Figures 10A, panels vii-xii, show the results of tissue incubated with labeled isotype control mAb (0.314 μg / mL). Figure 10B shows histological staining of skin (panels i and iv), tonsils (panels ii and v), and PD-1-expressing NSO cells (panels iii and vi). Figure 10B, panels i-iii, show the results of tissue 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 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, which contain 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, which contain IgG1(AA) or IgG4(P), to antagonize the PD-1 / PD-L1 axis by blocking PD-1 / PD-L1 interactions and preventing downregulation of the T cell response in the 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 equivalent to or higher than those of 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 Staphylococcus 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, which contain 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 anti-PD-1 antibodies are also shown. [Figures 16A-16B] Figures 16A-16B show that the PD-1×LAG-3 bispecific diabody components DART A, DART D, DART E, DART F, DART G, and DART H can stimulate cytokine production to levels equivalent to, or even higher than, those observed with the administration of the anti-PD-1 mAb + anti-LAG-3 mAb combination (PD-1 mAb A + LAG-3 mAb A), and that the PD-1×LAG-3 bispecific diabody components DART A, DART D, DART E, DART F, and DART G provided the greatest enhancement of cytokine release. The IFNγ secretion profiles of PBMCs stimulated with low concentrations of SEB (0.2 ng / mL) and treated with the PD-1×LAG-3 bispecific diabody, or single and combined anti-PD-1 and anti-LAG-3 antibodies, are plotted. Results using PBMCs from two representative donors are shown in Figures 16A and 16B. [Figures 17A-17B] Figures 17A and 17B show that the PD-1×LAG-3 bispecificity diabody components DART A, DART B, and DART C can stimulate cytokine production to levels equivalent to, or even higher than, those observed with the administration of an anti-PD-1 mAb + anti-LAG-3 mAb combination (PD-1 mAb A + LAG-3 mAb A). The IFNγ secretion profiles of PBMCs from two representative donors stimulated with high concentrations of SEB (85 ng / mL) and treated with the PD-1×LAG-3 bispecificity diabody, or single and combined anti-PD-1 and anti-LAG-3 antibodies, are plotted. Results using PBMCs from the two representative donors are shown in Figures 17A and 17B. [Figures 18A-18B] Figures 18A and 18B demonstrate that the PD-1×LAG-3 bispecificity diabody components DART A, DART B, and DART C can stimulate cytokine production to levels equivalent to, or even higher than, those observed with the administration of an anti-PD-1 mAb + anti-LAG-3 mAb combination (PD-1 mAb A + LAG-3 mAb A). The IFNγ secretion profiles of PBMCs from two representative donors stimulated with moderate concentrations of SEB (0.5 ng / mL) and treated with the PD-1×LAG-3 bispecificity diabody, or single and combined anti-PD-1 and anti-LAG-3 antibodies, are plotted. Results using PBMCs from the two representative donors are shown in Figures 18A and 18B. [Figure 19] Figure 19 shows that the PD-1×LAG-3 bispecificity diabody components DART D and DART H can stimulate cytokine production to levels equivalent to, or even higher than, those observed with the administration of an anti-PD-1 mAb + anti-LAG-3 mAb combination (PD-1 mAb A + LAG-3 mAb A), and that DART D provides the greatest enhancement of cytokine release. The IL-2 secretion profiles of PBMCs from representative donors stimulated with high concentrations of SEB (85 ng / mL) and treated with the PD-1×LAG-3 bispecificity diabody, or single and combined anti-PD-1 and anti-LAG-3 antibodies, are plotted. [Figure 20]Figure 20 shows that the PD-1×LAG-3 bispecificity diabody components DART B and DART I can stimulate cytokine production to levels equivalent to, or even higher than, those observed with 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)). The IFNγ secretion profiles of PBMCs from representative donors stimulated with moderate concentrations of SEB (0.5 ng / mL) and treated with the PD-1×LAG-3 bispecificity diabody, or single and combined anti-PD-1 and anti-LAG-3 antibodies, are plotted. [Figure 21A-21D] Figures 21A–21D show that the PD-1×LAG-3 bispecific diabody DART I can stimulate cytokine production to levels equivalent to, or even higher than, those observed with the administration of an anti-PD-1 mAb + anti-LAG-3 mAb combination (PD-1 mAb A + LAG-3 mAb A). The IFNγ (Figures 21A and 21C) and IL-2 (Figures 21B and 21D) secretion profiles of CD4 memory cells from two representative donors stimulated with tetanus toxoid (5 μg / mL) and treated with PD-1×LAG-3 bispecific diabody DART-I, combined anti-PD-1 and anti-LAG-3 antibodies, or isotype controls are plotted. The 7-day 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×LAG-3 bispecific molecule, DART I, are equivalent to those of the cynomolgus monkey anti-PD-1 antibody, PD-1 mAb A IgG4(P). This 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 against DART I (triangle) and PD-1 mAb A (circle). [Figures 23A-23C]Figures 23A-23C show the serum antibody concentrations and percentages of bound PD-1 on the surface of CD4+ or CD8+ T cells over time in animals treated 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, where the symbols represent the percentage of bound PD-1 on T cells for each individual animal, and the dashed line represents the mean. Serum mAb concentrations are plotted on the left Y-axis, where the symbols represent the serum levels for each individual animal, and the solid line represents the non-linear fit of the data. Each panel represents data for animals (n=1 / sex / group) that received 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) via IV infusion on day 1. [Modes for carrying out the invention]

[0032] The present invention relates to a selected anti-P that can bind 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 14 or PD-1 mAb 15 comprising a PD-1 binding domain, The present invention is intended for children. In particular, the present invention is a humanized or chimeric version of such an antibody. or PD-1 binding fragments of such anti-PD-1 antibodies (especially immunoconjugates, diabolic bodies) The present invention relates to a PD-1 binding molecule comprising (BiTE, bispecific antibodies, etc.). In particular, the present invention relates to Epitopes of molecules involved in the regulation of immune checkpoints present on the surface of immune cells The present invention relates to such PD-1 binding molecules that can further bind. The present invention also relates to the detection of PD-1. Or it relates to methods of using such PD-1 binding molecules to stimulate an immune response. The invention also relates to one or more PD-1-conjugated domains of the selected anti-PD-1 antibodies described above. A PD-1 binding molecule containing , one or more effective molecules for stimulating an immune response One or more additional molecules that combine with and / or specifically bind to cancer antigens. This relates to combination therapy, which involves administering additional molecules in combination.

[0033] I. Antibodies and their binding domains The antibody of the present invention has at least one located in the variable domain of the immunoglobulin molecule Depending on the antigen recognition site, it can target carbohydrates, polynucleotides, lipids, polypeptides, and other materials. An immunoglobulin molecule that can bind heterologously. In this specification, the term "Antibodies" include monoclonal antibodies and multiplex antibodies. Specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, lact D-type antibody, single-chain Fv(scFv), single-chain antibody, Fab fragment, F(ab') fragment, disulf Idiode-binding bispecific Fv (sdFv), intracellular antibodies, and any of the above epithelial cells It contains linkage fragments. In particular, antibodies contain immunoglobulin molecules and immunoglobulin molecules. It contains epidemiologically active fragments, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules are Any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subc It can be considered a Lass product. Antibodies, in addition to their known uses in diagnostics, can also be used for therapeutic purposes. Antibodies have been shown to be useful as therapeutic agents. Antibodies are polypeptides or proteins or non- It can bind to protein molecules in an immune-specific manner. This is because certain domestic molecules can bind to such molecules. This is because an element, part, or form ("epitope") exists. Pitope-containing molecules can possess immunological activity, thereby enabling antibody formation in animals. It triggers a production response. Such molecules are called "antigens." For the past few decades... Interest in the therapeutic potential of antibodies is rising again, and antibodies are a biotechnology-derived drug. It is one of the leading classes (Chan, CE et al. (2009) “The Use Of Anti bodies In The Treatment Of Infectious Diseases,” Singapore Med. J. 50(7):663-66 6) More than 200 antibody-based drugs are approved for use or are under development.

[0034] The term "monoclonal antibody" refers to a homogeneous antibody. It refers to a group of bodies, and the monoclonal antibodies mentioned above are involved in the selective binding of antigens (which occur naturally). Monoclonal antibodies are composed of amino acids (which do not occur naturally or are naturally occurring). It has targeting for a single epitope (or antigen site). Term: "monoclonal antibody" "Body" refers not only to complete monoclonal antibodies and full-length monoclonal antibodies, but also to these fragments. Fractional cells (Fab, Fab', F(ab')2, Fv, etc.), single-chain cells (scFv), and their mutants. Fusion proteins containing an antibody moiety, humanized monoclonal antibodies, chimeric monoclonal antibodies An immunoglobulin comprising a body and an antigen recognition site having the necessary specificity and ability to bind to the antigen. It includes any other modified configuration of the molecule. A source of antigen or a method for producing an antigen (for example, Regarding hybridomas, phage selection, recombinant expression, genetically modified animals, etc., This term is not intended to define immunoglobulins as a whole, and in the definition of "antibodies". This includes the aforementioned fragments, etc. Methods for producing monoclonal antibodies are publicly known in the relevant art. One method that can be adopted is Kohler, G. et al. (1975) “Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity,” Nature 256:495-497 This is a method or a modified version thereof. Typically, monoclonal antibodies are used in mice, rats, or rabbits. It is expressed in the animal. The above antibody is expressed in animals in a finely tuned immunogenicity amount containing the desired epitope. Immunogens are produced by immunization with cells, cell extracts, or protein preparations. This can include cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissues. These are some, but are not limited to these. Cells that may be used for immunization can be those that can be used as immunogens. Cells may be cultured for a certain period of time (for example, at least 24 hours) before use. It may be used as an immunogen in Germany, or in combination with undenatured adjuvants such as Ribi. (Jennings, VM (1995) “Review of Selected Adjuvants Used in Antibody Producti on,” (see ILAR J. 37(3):119-125). Generally, when cells are used as immunogens, they are completely They must be kept in a state that is viable and preferably able to survive. Complete cells must not rupture. To enable immune animals to detect antigens more effectively than cells that have been treated. This can be done. The use of denaturation or strong adjuvants, such as Freund's adjuvant, can cause cells to denature. It may cause rupture and is therefore not recommended. Immunogens should be administered once every two weeks or once a week. It may be administered multiple times at periodic intervals, such as once, or produced in animals (e.g., during tissue recombination). It may be administered in a manner that maintains survival capacity, or to provide immunity against the desired pathogenic epitope. Existing monoclonal antibodies and other equivalent antibodies with disease specificity are known in the art. Recombinant sequences and production can be achieved by any of the following means. In one embodiment, such an antibody The body is arranged, and then the polynucleotide sequence is cloned into a vector for expression or reproduction. The sequence encoding the antibody of interest is retained in the vector within the host cell, and continues... The above host cells can then be expanded and frozen for future use. Such antibody poly The nucleotide sequence is single-specific or multiple-specific (e.g., bi-specific, triply specific) according to the present invention. (Sex and quadruspecificity) molecules, as well as affinity-optimized, chimeric antibodies, humanized antibodies and / or Genetics to improve antibody affinity or other characteristics by generating canine antibodies It may be used for sub-operations. The general principle of humanizing antibodies is the antigen-binding portion of the antibody. The process involves replacing the non-human residue of the antibody with a human antibody sequence while preserving the base sequence.

[0035] Natural antibodies (such as IgG antibodies) consist of two heavy chains and two light chains complexed together. It contains a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain. n (VH), three constant domains (CH1, CH2 and CH3), and CH1 domain It contains a hinge domain located between the CH2 domain and the naturally occurring domain. The basic structural unit of immunoglobulins (e.g., IgG) is typically a glycoside of approximately 150,000 Da. It is a trimer expressed as an protein, having a light chain and two heavy chains. The amino acid of each chain The terminal ("N-terminus") contains approximately 100-110 variable domains that play a major role in antigen recognition. Contains yin. The carboxyl terminus ("C-terminus") of each chain defines a constant region, and the light chain is single. It has a constant domain, and the heavy chain usually has three constant domains and one hinge domain. Therefore, the structure of the light chain of the 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. Variable domes of IgG molecules The in contains multiple complementarity-determining regions (CDRs) that have contact with the epitope, and the f It consists of non-CDR segments called framework segments (FRs), and the above frame The work segment generally maintains the structure of the CDR loop and determines the location of the CDR. This enables such contact (however, certain framework residues also come into contact with the antigen). (Obtained). Therefore, the VL and VH domains have the structure n-FR1-CDR1-FR2-CDR It has 2-FR3-CDR3-FR4-c. The first, second and third CDRs of the antibody light chain Certain polypeptides (or which may function as the first, second, and third CDRs) are used herein. Each is a CDR L 1 domain, CDR L 2 domains and CDR L These are called the 3 domains. Similarly, the first, second, and third CDRs of the antibody heavy chain (or the first, second, and third CDs) Polypeptides that can function as R are referred to herein as CDRs, respectively. H 1 domain, CD R H 2 domains and CDR H It is called the 3 domains. Therefore, CDR L 1 domain, CDR L 2 domains, CDR L 3 domains, CDR H 1 domain, CDR H 2 domains, and CDR H The term "3-domain" refers to a state in which, when incorporated into a protein, the protein becomes lighter. Chains and heavy chains or diabodies or single-chain linking molecules (e.g., scFv, BiTe, etc.) Whether it is an antibody having or another type of protein, the protein This research focuses on polypeptides that can bind to specific epitopes. When used herein, the term "epitope-bin A "ding fragment" is a fragment of an antibody that can bind immunospecifically to a particular epitope. It means a piece, and the term is "epitope-binding site". " refers to the part of a molecule containing an epitope-binding fragment that plays a role in epitope binding. It points to. The epitope binding sites are 1, 2, 3, and 4 of the CDR domain of such antibodies. It may contain all five or six of these epitopes, and may be immune-specifically bound to such epitopes. Furthermore, the immunospecificity and affinity for epitopes different from the epitopes of such antibodies, or The epitope-binding fragment may exhibit selectivity. However, preferably, the epitope-binding fragment is such This will contain all six of the CDR domains of the antibody. The composite fragment may be a single polypeptide chain (e.g., scFv), or each may be an amide. Two or more polypeptide chains having a ∐ terminus and a carboxyl terminus (e.g., diabody, F It may include ab fragments, F(ab')2 fragments, etc.

[0036] In particular, the present invention relates to the single-chain variable domain fragment ("scFv") of the anti-PD-1 antibody of the present invention, and It includes multispecific binding molecules that possess this. The single-chain variable domain fragment has a short binding peptide. It is prepared by joining the light chain and / or variable domain using cydo. Bird et al. al. (1988) ("Single-Chain Antigen-Binding Proteins," Science 242:423-426) The distance between the carboxyl terminus of one variable domain and the amino terminus of the other variable domain is approximately 3.5 Examples of linking peptides that fill nm are described. Linkers for other sequences have also been designed and used. (Bird et al. (1988), "Single-Chain Antigen-Binding Proteins," Science 242) (423-426). The linker further attaches the drug or attachment to a solid support. It can be modified for functional purposes. Single-chain mutants can be produced by recombination or synthesis. scF An automated synthesizer can be used for the synthetic production of v. For the recombinant production of scFv, A suitable plasmid containing polynucleotides encoding SCFv is used in yeast, plants In suitable host cells such as eukaryotic cells like insect or mammalian cells, or prokaryotic cells like E. coli. It can be implemented. The polynucleotide that encodes the scFv of interest is polynucleotide It can be produced by conventional operations such as ligation. The resulting scFv is It can be isolated using standard protein purification techniques known in the technical field.

[0037] In particular, the present invention relates to a humanized mutant of the anti-PD-1 antibody of the present invention, and a multispecific antibody containing the same. It also includes hybrid molecules. The term "humanized" refers to antibodies, which are chimeric molecules. Generally prepared using recombinant technology, the antigen-binding site of immunoglobulins from non-human species And the remaining immunoglobulin structures of molecules based on the structure and / or sequence of human immunoglobulins This refers to a chimeric molecule having a structure. The anti-human PD-1 antibody of the present invention is antibody PD-1 mA 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 includes humanized, chimeric, or canine variants. The variable domain of such antibodies The polynucleotide sequence is used to generate the above derivatives and to enhance the affinity or other characteristics of the above antibody. It can be used for genetic engineering to improve [something]. The general principle in humanizing antibodies is that [something] While preserving the base sequence of the antigen-binding portion of the body, the non-human residue of the antibody is replaced with the human antibody sequence. The process involves four general steps to humanize a monoclonal antibody. There exists. The above steps are as follows: (1) Variable doping of the light chain and heavy chain of the starting antibody (1) A step of determining the nucleotide and predicted amino acid sequence of the gene; (2) a humanized antibody Alternatively, the step of designing canine antibodies, i.e., the antibody used during the humanization or canine process. Steps to determine the framework area; (3) Actual humanization or canine transformation methods / techniques; and (4) Transfection and expression of humanized antibodies. For example, U.S. No. 4,816,56 U.S. Patent No. 7; U.S. Patent No. 5,807,715; U.S. Patent No. 5,866,692; and U.S. Patent No. See License No. 6,331,415.

[0038] The antigen-binding site is either a fully variable domain fused to the constant domain, or an appropriate framework. It possesses only complementarity-determining regions (CDRs) of such variable domains grafted into the region. The antigen-binding site may be wild-type, or may be modified by one or more amino acid substitutions. It can be modified as follows. This eliminates the constant region, which is an immunogen in the individual human, but the foreign The possibility of a variable domain 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 a human-derived constant region. Furthermore, in order to deform the above variable domain to be as close as possible to human form, The focus is also on modifying the variable domains in both the heavy and light chains. The four framework regions change in response to the antigen in question and determine its binding ability. It is known that a region (FR) contains three adjacent complementary determination regions (CDRs), The above framework domain is relatively preserved in a given species, and also for CDR. It is presumed to provide a scaffold for when preparing non-human antibodies against a specific antigen. By transplanting CDRs derived from non-human antibodies to FRs present in the modified human antibody, This allows for the "reshaping" or "humanization" of the variable domain. This approach to various 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 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 -1154 has been reported. In some embodiments, the humanized antibody is used in all CDRs. Save the column (for example, a humanized mouse antibody containing all 6 CDRs from a mouse antibody). In other embodiments, the humanized antibody is modified to have a different sequence from the original antibody. It has one or more CDRs (one, two, three, four, five, or six).

[0039] Numerous "humanized" antigen-binding sites derived from non-human immunoglobulins. d) The antibody molecules are described, and these have a rodent or modified rodent variable domain and It has a complementarity-determining region (CDR) related to these, which is fused to the human constant domain. Includes chimeric antibodies (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 See "Human Chimeric Monoclonal Antibody," Cancer Res. 47:3577–3583. The referenced document describes the human support framework region before fusing with the appropriate human antibody constant domain. (FR) describes rodent CDRs superimposed on graphs (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 veneered by rearrangement. This document describes the rodent CDR supported by the Rodent Framework region. See, for example, European Published Patent No. 519,596. These "humanized" molecules are human. Rodent anti-human antibodies limit the duration and effectiveness of therapeutic applications of these parts of cypient. Designed to minimize undesirable immunological responses to molecules. Humanizing antibodies. Another method that may be used for this purpose is 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. Patent No. 6,180,377; U.S. Patent No. 6,05 U.S. Patent No. 4,297; U.S. Patent No. 5,997,867; and U.S. Patent No. 5,866,692 It is disclosed by [the organization / organization].

[0040] II. Fcγ receptor (FcγR) The CH2 and CH3 domains of the two heavy chains interact to form an Fc region, and this Fc region The region is recognized by cellular Fc receptors, including but not limited to Fcγ receptors (FcγR). It is a recognized domain. When used herein, the term "Fc domain (Fc r The term "(evolution)" is used to define the C-terminal region of the IgG heavy chain. (Example: Human) 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 This is numbered by an EU index, similar to the one used in Kabat. X is either ricin (K) or absent.

[0041] The amino acid sequence of the CH2-CH3 domain of an example 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 This is numbered by an EU index, similar to the one used in Kabat. X is either ricin (K) or absent.

[0042] The amino acid sequence of the CH2-CH3 domain of an example 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 This is numbered by an EU index, similar to the one used in Kabat. X is either ricin (K) or absent.

[0043] The amino acid sequence of the CH2-CH3 domain of an example human IgG4 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 This is numbered by an EU index, similar to the one used in Kabat. X is either ricin (K) or 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 According to MD (1991) ("Kabat," which is expressly incorporated herein by reference) This is the numbering of the EU index in Kabat. "(EU index as in Kabat)" is a numbered representation of human IgG1 EU antibodies. This refers to the amino acids from the variable domains of the mature heavy and light chains of immunoglobulins. It is specified by the position of the amino acids. Kabat uses a large number of amino acid sequences related to antibodies. The amino acid consensus sequences for each subgroup are described, 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 Defined by "the regions of immunoglobrins," J. Mol. Biol. 196:901-917. CDR H (Please understand that 1 starts from 5 residues before.) Kabat numbering ski Mu refers to conserved amino acids to identify the antibody in question, and the consensus in Kabat By aligning with one of the sequences, antibodies not included in the Kabat overview can be added. It is expandable up to [a certain point]. The above method for assigning residue numbers is standard in the art. This is a quasi-identical representation, and includes equivalent positions in different antibodies, including chimeric or humanized variants. It easily identifies amino acids. For example, the amino acid at position 50 of a human antibody light chain is similar to that of a mouse antibody light chain. It occupies a position equivalent to the 50th amino acid.

[0045] Polymorphisms are found in numerous different locations within the antibody constant region (for example, in the EU in Kabat). The numbering by the index includes, but is not limited to, positions 192, 193, and 214. CH1 position that cannot be accessed; as well as positions 270, 272, 312, 315, 356 and 35 This has been observed in Fc positions (including but not limited to the 8th position), and therefore, it is proposed here Slight differences may exist between the shown sequence and the sequence of conventional technology. Human immunoglobulin The polymorphic forms of n have been well characterized. Currently, 18 Gm allotypes are known. ru: 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 Combining any allotype, isoallotype, or haplotype of the immunoglobulin gene It is possible to include allotypes, isoallotypes, or haptic sequences of the sequence presented herein. It is thought that this is not limited to any specific type. Furthermore, depending on the expression system, the C-terminus of the CH3 domain End amino acid residues (shown in bold above) can be removed after translation. Therefore, the C-terminal residue of the CH3 domain... The base is any amino acid residue of the PD-1 binding molecule of the present invention. Specifically according to the present invention This includes PD-1 binding molecules lacking the C-terminal residue of the CH3 domain. The invention specifically encompasses structures that include a C-terminal lysine residue of the CH3 domain. be.

[0046] Activation and inhibition signals affect the ligation of the Fc region of the cellular Fc gamma receptor (FcγR). The trait is introduced by this process. The ability of such ligation to produce diametrically opposed functions. This is due to structural differences between different FcγRs. Immune receptor tyrosine system activation motif ( These are receptor fragments called ITAMs and immune receptor tyrosine system inhibitory motifs (ITIMs). Two distinct domains within the spore-signaling domain are responsible for different responses. Supplementing these structures with different cytoplasmic enzymes determines the outcome of the FcγR-mediated cellular response. . The ITAM-containing FcγR complex contains FcγRI, FcγRIIA, and FcγRIIIA. On the other hand, the ITIM-containing complex contains only FcγRIIB. Human neutrophils contain Fc It expresses the γRIIA gene. Crosslinking of FcγRIIA by immune complexes or specific antibodies. Rastering involves agglutination of ITAM with receptor-associated kinases that promote ITAM phosphorylation. It plays a role in this. ITAM phosphorylation serves as a docking site for Syk kinase. This activation leads to the activation of downstream substrates (e.g., PI3K). Cell activation is associated with inflammation. This leads to the release of stimulating mediators. The FcγRIIB gene is expressed on B lymphocytes. Its extracellular domain is 96% identical to that of FcγRIIA, and it is distinguishable from the IgG complex. They bind in a way that is not possible. The presence of ITIM in the cytoplasmic domain of FcγRIIB is Fc The aforementioned inhibitory subclass of γR is defined. Recently, the molecular basis for this inhibition has been established. When co-ligated with activated FcγR, ITIM in FcγRIIB is phosphorylated, and poly(I) It induces the SH2 domain of inositol acid 5'-phosphatase (SHIP), and this , phosphoino released as a result of ITAM-containing FcγR-mediated tyrosine kinase activation Citol is hydrolyzed, and as a result, intracellular Ca ++ To prevent the inflow of FcγRI IB crosslinking weakens the activation response to FcγR ligation and inhibits cellular responsiveness. In this way, B cell activation, B cell proliferation, and antibody secretion are interrupted.

[0047] III. Bispecific antibodies, multispecific diabodies, and DART® diabodies Di The ability of an antibody to bind to an antigen epitope depends on the presence of the VL and VH domains of the antibody, as well as It depends on the amino acid sequence. The interaction between the antibody light chain and antibody heavy chain, especially between its VL domain and V The interaction with the H domain forms one of the two eptope-binding sites of the natural antibody. Natural antibodies can bind to only one epitope species (i.e., they are monospecific). These can combine with multiple copies of the above species (i.e., exhibiting divalent or polyvalent nature).

[0048] The binding domain of the present invention binds to the epitope in an "immunospecific" manner. When used in this context, antibodies, diabodies, or other epitope-binding molecules may be alternatives. Compared to epitopes, it is more frequent, faster, longer-lasting, and / or has higher affinity. When it reacts with or binds to the epitope, a region of another molecule (i.e., the epitope) It is said that it "binds in an immune-specific manner." For example, it binds to a certain viral epitope in an immune-specific manner. Antibodies that bind specifically are immune-specific to other viral epitopes or non-viral epitopes. Rather than binding, it binds more easily, with higher affinity and binding activity, and for a longer period of time. It is an antibody that binds to the viral epitope in question. Reading this definition, for example, the first target... An antibody (or partial or epitope) that binds immunologically is specific to a second target or It is also understood that preferential binding may or may not occur. Therefore, "immunospecific binding (imm "Unospecific binding" does not necessarily require exclusive binding. (However, it can include). Generally, but not necessarily, references to combinations. This means a "specific" bond. Two molecules, these bonds, these two molecules each When a receptor exhibits specificity in binding to a ligand, it is described as "physiologically specific (phy) It is said that they can be combined with each other in the "siospecific" style.

[0049] The functionality of an antibody depends on two distinct antigens (or different epitopes of the same antigen). By generating a multispecific antibody-based molecule that can simultaneously bind to, and / or the same It has a higher titer (i.e., three or more binding sites) with respect to one epitope and / or antigen. This can be enhanced by generating antibody-based molecules.

[0050] To provide molecules with higher efficacy than natural antibodies, a wide range of recombinant bispecific antibodies are available. A format has been developed (e.g., International Publication No. 2008 / 003116; International Publication) Publication No. 2009 / 132876; International Publication No. 2008 / 003103; International Publication No. 200 Issue No. 7 / 146968; International Publication No. 2009 / 018386; International Publication No. 2012 / 00 (See publication No. 9544; International Publication No. 2013 / 070565), most of them are further epitopes Binding fragments (e.g., scFv, VL, VH, etc.) to antibody cores (IgA, IgD, IgE, Ig To fuse with G or IgM or into the antibody core, or to multiple epitopes To fuse binding fragments (e.g., two Fab fragments or scFv), linkerpe Use petido. Alternative formats include epitope-bound fragments (e.g., scFv, V). (L, VH, etc.) are dimerized into a CH2-CH3 domain or a substitute polypeptide. To fuse with the ym, a linker peptide is used (International Publication No. 2005 / 0709) Issue 66; International Publication No. 2006 / 107786A; International Publication No. 2006 / 107617A (Issue; International Publication No. 2007 / 046893). Typically, such an approach involves compromise. And it involves trade-offs. For example, International Publication No. 2013 / 174873; International Publication No. 20 Publication No. 11 / 133886 and International Publication No. 2010 / 136172 concerning the use of linkers Therefore, it is disclosed that problems may arise in the therapeutic setting, and that it may be linked to two or more antigens. To enable this, the CL and CH1 domains switch from their respective natural positions, and V This describes a triplicate antibody with diversified L and VH domains (International Publication No. 2). (No. 008 / 027236; International Publication No. 2010 / 108127). Therefore, these sentences The molecules disclosed in this book exchange binding specificity for the ability to bind to additional antigen species. International Publication No. 2013 / 163427 and International Publication No. 2013 / 119903 are The CH2 domain is modified to include a fusion protein adduct with a binding domain. The steps are disclosed. This document states that CH2 has the minimum requirements for mediating effector functions. It is stated that it only serves a specific purpose. International Publication No. 2010 / 028797; International Publication No. 2010028796; and International Publication No. 2010 / 028795 are Fc The region is replaced with additional VL and VH domains to form a trivalent binding molecule, recombinant The antibody is disclosed in International Publication No. 2003 / 025018; and International Publication No. 2003 Patent No. 012069 discloses a recombinant diamond body in which each strand contains an scFv domain. International Publication No. 2013 / 006544 describes the synthesis as a single polypeptide chain. After being subjected to proteolysis, a heterodimer structure is obtained, which is a polyvalent fa The b molecule is disclosed. Therefore, the molecules disclosed in these documents have effector functions. It exchanges all or part of its ability to mediate for the ability to bind to additional antigen species. International Publication No. 2014 / 022540; International Publication No. 2013 / 003652; International Publication Publication No. 2012 / 162583; International Publication No. 2012 / 156430; International Publication No. 201 Issue No. 1 / 086091; International Publication No. 2008 / 024188; International Publication No. 2007 / 02 Publication No. 4715; International Publication No. 2007 / 075270; International Publication No. 1998 / 002463 International Publication No. 1992 / 022583 and International Publication No. 1991 / 003493 are , the step of adding an additional binding domain or functional group to the antibody or antibody moiety (for example, the antibody The step of adding a diabody to the light chain, or adding additional VL and VH to the light and heavy chains of the antibody. A step of adding a domain, or a step of adding heterogeneous fusion proteins to each other. Alternatively, it discloses a step of linking multiple Fab domains to each other. The molecules disclosed in these documents bind the naive antibody structure to additional antigen species. They are exchanging their abilities for what they can do.

[0051] This technology can further be used to bind with two or more different epitope species (i.e., bivalent or polyvalent). In addition to sex, it can exhibit bispecificity or multiple specificity, which is why such natural We are focusing on the possibility of producing a diabody different from that of antibodies (for example, 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; International Publication No. 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 See "Cancer Therapy," Cancer Res. 69(12):4941-4944.

[0052] The design of the diabody is based on antibody derivatives known as single-chain variable domain fragments (scFv). Based on this, such molecules use light chain and / or heavy chain variable domains with short chain peptides. It is made by linking them together. Bird et al. (1988) (Single-Chain Antigen-B "Inding Proteins," Science 242:423-426) states that the carboxyl terminus of one variable domain and An example of a linked peptide that fills approximately 3.5 nm between the other variable domain and the amino terminus is described. Other linkers have also been designed and used (Bird et al. (1988) “Singl e-Chain Antigen-Binding Proteins, Science 242:423-426). The linker is responsible for the attachment of the drug. They can be modified for additional functions such as adhesion or attachment to rigid supports. Single-chain mutants can be recombinant It can be produced by or by synthesis. Regarding the synthetic production of scFv, automated A synthesizer can be used. For the recombination production of scFv, encode the scFv. A suitable plasmid containing polynucleotides is used in yeast, plants, insects, or mammals. It can be introduced into suitable host cells, such as eukaryotic cells or prokaryotic cells such as E. coli. The polynucleotides that encode the scFv are polynucleotide ligations, etc. It can be produced by conventional operations. The resulting scFv is a standard known in the art. It can be isolated using protein purification techniques.

[0053] The provision of non-single-specific diabolic bodies allows for the co-binding of multiple cells expressing different epitopes. This includes, but is not limited to, the ability to (co-ligate) and coexist. It offers significant advantages over antibodies. Therefore, bispecific diabody is used in therapy and immunology. It has a wide range of applications, including cutting. Its dual specificity is used in the design of diamond bodies in various applications. This allows for greater flexibility in processing, thereby increasing the binding activity of multimeric antigens and different multimeric antigens. Cross-linking of multiple antigens and targeted targeting of specific cell types based on the presence of both target antigens. The present invention provides a diabody molecule known in the art, which is a small (~50kDa or less) (Regarding the size of the diamond body) Its high bonding rate, low dissociation rate and rapid release from circulation By eliminating the need for elimination, it has also demonstrated specific uses 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] Due to the bispecificity of the diabody, co-connection between different cells, such as cytotoxic T cells and tumor cells, is possible. The above-mentioned diabodies can be used for cross-linking with 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 (macol. Sin. 26:649-658). Alternatively, using a dual specificity diabody, different Receptors can be collated on the surface of cells or on a single cell. Different cells and / Alternatively, receptor collision alters effector function and / or immune cell signaling. Useful for adjusting. Multispecific molecules containing epitope binding sites (e.g., bispecific molecules). Diabody) is a T lymphocyte, natural killer (NK) cell, antigen-presenting cell or other B7-H3 (CD276), B7-H4 (VTCN1), and BTL are expressed on mononuclear cells. 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 target any of the surface determinants of immune cells, such as VISTA(PD-1H). It is possible. In particular, cell surface receptors involved in the regulation of immune checkpoints (or their ligands) Epitope binding sites that target the body are used for inhibitory signaling of immune checkpoint molecules. By antagonizing or blocking it, the immune response of the subject is stimulated, upregulated or It is useful in enhancing the generation of bispecific or multispecific binding molecules. Molecules involved in the regulation of the chokepoint include, but are not limited to, B7-H3 and 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 Examples include T, TIM-3, and / or VISTA.

[0055] However, the aforementioned advantages lead to significant costs. Such non-single-specific diamonds Body formation requires a good assembly of two or more distinct polypeptides (i.e.) The above formation occurs when the diabody is formed by heterodimerization of different polypeptide chain species. (This requires that) This fact is due to homodimerization of the same polypeptide chain. This is in contrast to the single-specificity diamond body that is formed. To achieve this, provide at least two different polypeptides (i.e., two polypeptide species). Therefore, homodimerization of such polypeptides is necessary, and inactive molecules Bringing about (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 bonding between polypeptides of the same type. This must be achieved in a way that prevents homodimerization (Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antib (ody) Using A Refolding System,” Protein Eng. 13(8):583-588). Therefore, in this technical field... This teaches non-covalent linking of 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, this technology is a double composed of non-covalently linked polypeptides. Recognizing that the specific diabody is unstable and easily decomposes into non-functional monomers (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, this technology is DART(registered trademark)( D ual A ffi nity R e- T Argeting Reagents) called Diabody, We have successfully developed a defined covalent heterodimeric non-uniform specificity diabody (for example, in the United States). Published Patent No. 2013-0295121; U.S. Published Patent No. 2010-0174053; and U.S. Published Patent No. 2009-0060910; European Published Patent No. 2714079; European State Published Patent No. 2601216; European Published Patent No. 2376109; European Published Patent No. 21 Publication No. 58221; and International Publication No. 2012 / 162068; International Publication No. 2012 / 01 Publication No. 8687; International Publication No. 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 (see). Such a diabody It comprises two or more covalently complexed polypeptides, and one or more cysts The residues can form a disulfide bond, thereby covalently bonding two polypeptide chains. This involves processing into each of the selected polypeptide species. The addition of a cysteine ​​residue to the C-terminus of a structure like this creates disulfide bonds between polypeptide chains. It has been found that this enables bonding, and this does not interfere with the bonding properties of divalent molecules. The resulting heterodimer is stabilized.

[0058] The two polypeptides of the simplest bispecific DART(registered trademark) diabody are Each has three domains. The first polypeptide (from the N-terminus to the C-terminus) (i) The first immunoglobulin having a binding domain for the light chain variable domain (VL1) Domain; (ii) comprising a binding region for the heavy chain variable domain (VH2) of the second immunoglobulin. (iii) a second domain; and a cysteine ​​residue (or cysteine-containing domain) , promotes heterodimerization of the above-mentioned diamond body with the above-mentioned second polypeptide, the die The hete plays a role in covalently bonding the first and second polypeptides of the body to each other. The second polypeptide comprises a third domain containing a dimerization promoting domain. From the N-terminus to the C-terminus: (i) The variable light chain domain (VL) of the second immunoglobulin. (ii) The first domain having a binding region of (2) the first immunoglobulin heavy chain variable domain A second domain comprising a binding region for (VH1); and (iii) a cysteine ​​residue (or (This is a cysteine-containing domain) and the heterodimer-promoting domain of the first polypeptide chain. Complementary hemoglobin that complexes with the first polypeptide chain to promote heterodimerization with the first polypeptide chain. It contains a third domain which contains a telodimer-promoting domain. The cysteine ​​residue (or cysteine-containing domain) of the third domain of the cysteine ​​chain is as described above. Covalent bonding of the second polypeptide chain to the first polypeptide chain of the diamond body It plays a role in promoting this. Such molecules are stable, potent, and also two or more It has the ability to bind antigens simultaneously. In one embodiment, the first and second polypeptides described above Each of the above third domains integrates the above polypeptides by disulfide bonds. It contains cysteine ​​residues that play a role in binding. Figure 1 shows such a diabody. A schematic diagram is provided, and the above-mentioned diamond body has an E-coil / K-coil heterodimer promoting domain and A cysteine-containing linker is used for covalent bonding. (See Figures 2 and 3A-3C) As presented, one or both of the above polypeptides are further CH2-CH It may have a sequence of 3 domains, thereby between the two diabody polypeptides. Through complex formation, cells (e.g., B lymphocytes, dendritic cells, natural killer cells, macromolecules) Fc region capable of binding to Fc receptors of phages, neutrophils, eosinophils, basophils, and mast cells. A CH2 of such polypeptide chain is formed. As will be presented in more detail below, The and / or CH3 domains do not need to have identical sequences, and advantageously, these two positories It is modified to promote complex formation between lipeptide chains.

[0059] Numerous variants of such molecules have been described (for example, U.S. Patent Publication No. 2015 / Patent No. 0175697; U.S. Published Patent No. 2014 / 0255407; U.S. Published Patent No. 201 4 / 0099318; U.S. Published Patent No. 2013 / 0295121; U.S. Published Patent No. 2 Patent No. 010 / 0174053; and U.S. Published Patent No. 2009 / 0060910; European Publication Patent No. 2714079; European Publication Patent No. 2601216; European Publication Patent No. 23761 Patent No. 09; European Publication No. 2158221; and International Publication No. 2012 / 162068; (See International Publication No. 2012 / 018687; International Publication No. 2010 / 080538). These Fc region-containing DART® diabodies contain two pairs of polypeptide chains. That's fine. The first polypeptide chain is (from the N-terminus to the C-terminus): (i) First immunity (ii) A first domain containing a binding region for the globulin light chain variable domain (VL1); The second domain contains a binding region for the heavy chain variable domain (VH2) of the second immunoglobulin. (iii) containing a cysteine ​​residue (or cysteine-containing domain), the above Diabody This promotes heterodimerization of the above second polypeptide with the above second diabody. A third domain that plays a role in covalently bonding polypeptides 1 and 2 to each other; and It has a (iv)CH2-CH3 domain. The second polypeptide is (from the N-terminus to the C-terminus). (i) The binding region of the light chain variable domain (VL2) of the second immunoglobulin (ii) the first domain possessing; (ii) the heavy chain variable domain (VH1) of the first immunoglobulin (iii) a second domain comprising a binding region; and (iii) a cysteine ​​residue (or cysteine-containing) The heterodimerization promoter promotes the heterodimerization of the domain) and the first polypeptide chain described above. It contains a third domain which contains an advanced domain. Here, two first polypeptides The dops complex together to form an Fc region. Figures 3A-3C show different heterodimer formations. We present schematic diagrams of three variants of such a diabody that utilize advanced domains.

[0060] Other Fc region-containing DART(registered trademark) diabodies contain three polypeptide chains. That's fine. The first polypeptide of such a DART(registered trademark) diamond body consists of three Domains: (i) VL1-containing domains; (ii) VH2-containing domains; and (iii) C Contains a domain containing the H2-CH3 sequence. Such DART(registered trademark) Abody's second polypeptide consists of: (i) a VL2-containing domain; (ii) a VH1-containing domain. (iii) heterodimerization of the diabody with the first polypeptide chain and co It contains a domain that promotes bonding. Such DART (trademark registered) diamond bodies The third polypeptide has a CH2-CH3 sequence. Therefore, such DART( (Registered Trademark) The first and second polypeptide chains of the Diabody are related as a whole, epito A VL1 / VH1 binding site that can bind to the second epitope, and a VL2 / binding site that can bind to the second epitope. It forms a VH2 binding site. Such a relatively complex DART(registered trademark) molecule also It has a cysteine-containing domain that functions to form a covalent complex. Therefore, the above Polypeptides 1 and 2 involve cysteine ​​residues within their respective third domains. They are linked to each other via disulfide bonds. In particular, the first and third polypeptide chains They complex with each other to form an Fc region stabilized by disulfide bonds. Figure 4 Figures A-4B present schematic diagrams of such a diabody containing three polypeptide chains.

[0061] Furthermore, other Fc region-containing DART(registered trademark) diabodies can support up to three different immunoglobulins. Robulin's light and heavy chain variable domains (VL1 / VH1, VL2 / VH2, and VL3 / V It may contain five polypeptide chains, each having a binding region derived from H3 (called H3). The first polypeptide chain of such a dia body is: (i) VH1-containing domain; ( ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. It may have. The second and fifth polypeptide chains of such a diamond body are: (i) VL1 (ii) Contains a CL-containing domain; and (ii) may contain a CL-containing domain. Such a diamond body The third polypeptide chain consists of: (i) a VH1-containing domain; (ii) a CH1-containing domain; (iii) Domains containing CH2-CH3 sequences; (iv) Domains containing VL2; (v )VH3-containing domain: and (vi) heterodimer-promoting domain may be included, and the above The telodimerization-promoting domain promotes the dimerization of the third and fourth chains. The fourth polypeptide of the IAbody is: (i) VL3-containing domain; (ii) VH2-containing domain Main; and (iii) Heterodimization of the above diabody with a third polypeptide chain. and may contain domains that promote covalent bonding. Here the first and third peptides The DARTs combine with each other to form an Fc region. Such a relatively complex DART (registration The (trademark) molecule also contains cysteine-containing domains that function to form covalent complexes. Thus, each polypeptide chain is connected by a disulfide bond containing a cysteine ​​residue. It binds to at least one additional polypeptide chain. Preferably, such domains They are aligned in the direction from the N-terminus to the C-terminus. Figure 5 shows such a chain containing five polypeptide chains. A schematic diagram of the earbody is presented.

[0062] For applications where a tetravalent molecule is desirable but Fc is not required, an alternative configuration is available for this technology. This is publicly known in the field, and it involves the VH1, VL2, VH2, and VL2 domains. It is also called "TandAb" because it is formed by the homodimerization of two identical chains that each possesses. This includes, but is not limited to, a quadrivalent tandem antibody that can be detected (e.g., U.S. Published Patent No. 2005). -0079170; U.S. Published Patent No. 2007-0031436; U.S. Published Patent No. 201 U.S. Published Patent No. 0-0099853; U.S. Published Patent No. 2011-020667; U.S. Published Patent No. 201 3-0189263; European Published Patent No. 1078004; European Published Patent No. 237186 No. 6; European Publication No. 2361936; and European Publication No. 1293514; International Publication Publication No. 1999 / 057150; International Publication No. 2003 / 025018; and International Publication No. (See issue 2013 / 013700).

[0063] In recent years, two diabody-type binding domains and one non-diabody-type binding domain, Furthermore, trivalent structures incorporating the Fc region are described (for example, PCT application PCT / US15 / 3 Publication No. 3076, Title: "Tri-Specific Binding Molecules and Methods of Use Thereof", 20 Filing on May 29, 2015; and PCT application No. PCT / US15 / 33081, title "Tri -Specific Binding Molecules That Specifically Bind to Multiple Cancer Antigens See “and Methods of Use Thereof”, application filed May 29, 2015). Such trivalent The offspring may be used to generate single-specific, bispecific, or triplicate specific molecules. (Figures 6A-6) F presents a schematic diagram of such a trivalent molecule containing three or four polypeptide chains.

[0064] IV. The Anti-Human PD-1 Binding Molecule of the Present Invention Preferred PD-1 binding molecules of the present invention include antibodies, diabodies, and BiTE. These are the continuous or discontinuous (e.g., conformational) portions (epi) of human PD-1 (CD279). It can bind to (topes). The PD-1 binding molecule of the present invention is preferably one or more non-human molecules. Ability of species, especially primate species (and especially primate species such as crab-eating macaques), to bind to the PD-1 molecule. It also shows a representative human PD-1 polypeptide (NCBI sequence NP_005009.2;2 The 0-amino acid residue signal sequence (underlined) and the 268-amino acid residue mature protein are , 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 holds.

[0065] In certain embodiments, the PD-1 binding molecule of the present invention is any one of the following criteria ( Characterized by: (or multiple) (1) 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 a M concentration of 40 nM or less; (3) Equilibrium binding constant (K D ) Specifically binds to human PD-1 at a M concentration of 5 nM or less; (4) On speed (K a ) 1.5 × 10 4 M -1 minutes -1 The above describes how it specifically binds to human PD-1. ; (5) On speed (K a ) 90.0 × 10 4 M -1 minutes -1 The above describes how it specifically binds to human PD-1. ru; (6) Off speed (K d )7×10 -4 minutes -1 The following molecules specifically bind to human PD-1: (7) Off speed (K d )2×10 -4 minutes -1 The following molecules specifically bind to human PD-1: (8) Specifically binds to non-human primate PD-1 (e.g., cynomolgus macaque PD-1); (9) Binding / inhibitory activity of PD-1 ligands (PD-L1 / PD-L2) to PD-1 To inhibit (i.e., block or interfere with) sex; (10) Stimulate an immune response; and / or (11) Stimulates antigen-specific T cell response through synergistic effect with anti-human LAG-3 antibody. .

[0066] As used herein, the term "antigen-specific T cell response" "Specific T-cell response" refers to a response in which T cells respond to an antigen that is specific to them. This refers to the response of T cells caused by stimulation of T cells. Non-limiting examples of the answer include proliferation and cytokine production (e.g., TNF-α, IFN-γ). Examples include production. The ability of molecules to stimulate antigen-specific T cell responses is, for example, as specified herein. Staphylococcus aureus enterotoxin type B antigen ("SEB") stimulating PBMCs as described above. This can be determined using an assay.

[0067] The preferred PD-1 binding molecule of the present invention is the mouse anti-human PD-1 monoclonal antibody "P 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 -1 mAb 13", "PD-1 mAb 14", or "PD-1 mAb 15" Having VH and / or VL domains, more preferably such anti-human PD-1 monoc CDR of the VH domain of ronal antibodies HOne, two, or all three of them, and / or VL domain CDR L It possesses one, two, or all three of these. Preferred anti-human PD-1 binding molecules include bispecific (or multispecific) antibodies, chimeric or hi TOR-mediated antibodies, BiTe, Diabody, etc., and such binding molecules having a variant Fc region. Includes.

[0068] The present invention is particularly characterized by the following: (A)(1) Three CDRs of the VH domain of PD-1 mAb 1 H ; (2) Three CDRs of the VL domain of PD-1 mAb 1 L ; (3) Three CDRs of the VH domain of PD-1 mAb 1 H , and PD-1 Three CDRs of the VL domain of mAb 1 L ; (4) VH domain of hPD-1 mAb 1 VH1; (5) VL domain of hPD-1 mAb 1 VL1; (6) VH and VL domains of hPD-1 mAb 1; (B)(1) Three CDRs of the VH domain of PD-1 mAb 2 H ; (2) Three CDRs of the VL domain of PD-1 mAb 2 L ; (3) Three CDRs of the VH domain of PD-1 mAb 2 H , and PD-1 Three CDRs of the VL domain of mAb 2 L ; (4) VH domain of hPD-1 mAb 2 VH1; (5) VL domain of hPD-1 mAb 2 VL1; (6) VH and VL domains of hPD-1 mAb 2; (C)(1) Three CDRs of the VH domain of PD-1 mAb 3 H ; (2) Three CDRs of the VL domain of PD-1 mAb 3 L ; (3) Three CDRs of the VH domain of PD-1 mAb 3 H , and PD-1 Three CDRs of the VL domain of mAb 3 L ; (D)(1) 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) Three CDRs of the VH domain of PD-1 mAb 4 H , and PD-1 Three CDRs of the VL domain of mAb 4 L ; (E)(1) Three CDRs of the VH domain of PD-1 mAb 5 H ; (2) Three CDRs of the VL domain of PD-1 mAb 5 L ; (3) Three CDRs of the VH domain of PD-1 mAb 5 H , and PD-1 Three CDRs of the VL domain of mAb 5 L ; (F)(1) 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) Three CDRs of the VH domain of PD-1 mAb 6 H , and PD-1 Three CDRs of the VL domain of mAb 6 L ; (G)(1) Three CDRs of the VH domain of PD-1 mAb 7 H ; (2) PD-1 mAb 7 or hPD-1 mAb 7 VL2 or Three CDRs of the VL domain of hPD-1 mAb 7 VL3 L ; (3) 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 Three CDRs of the VL3 VL domain L ; (4) hPD-1 mAb 7 VH1 or hPD-1 mAb 7 VH Two 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) 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) Three CDRs of the VH domain of PD-1 mAb 8 H , and PD-1 Three CDRs of the VL domain of mAb 8 L ; (I)(1) PD-1 mAb 9 or hPD-1 mAb 9 VH2 VH The three main CD-Rs H ; (2) PD-1 mAb 9 or hPD-1 mAb 9 VL2 VL The three main CD-Rs L ; (3) PD-1 mAb 9 or hPD-1 mAb 9 VH2 VH The three main CD-Rs H and PD-1 mAb 9 or hPD-1 mAb 9 V Three CDRs of the L2 VL domainL ; (4) hPD-1 mAb 9 VH1 or hPD-1 mAb 9 VH Two VH domains; (5) hPD-1 mAb 9 VL1 or hPD-1 mAb 9 VL Two 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) 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) Three CDRs of the VH domain of PD-1 mAb 10 H , and PD-1 Three CDRs of the VL domain of mAb 10 L ; (K)(1) PD-1 mAb 11 VH domain 3 CDRs 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 Three CDRs of the VL domain of mAb 11 L ; (L)(1) PD-1 mAb 12 VH domain 3 CDRs 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 Three CDRs of the VL domain of mAb 12 L ; (M)(1) PD-1 mAb 13 VH domain 3 CDRsH ; (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 Three CDRs of the VL domain of mAb 13 L ; (N)(1) PD-1 mAb 14 VH domain 3 CDRs 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 Three CDRs of the VL domain of mAb 14 L ; (O)(1) PD-1 mAb 15 VH domain 3 CDR H ; (2) Three CDRs of the VL domain of PD-1 mAb 15 L ; (3) Three CDRs of the VH domain of PD-1 mAb 15 H , and PD-1 Three CDRs of the VL domain of mAb 15 L ; (4) VH domain of hPD-1 mAb 15 VH1; (5) VL domain of hPD-1 mAb 15 VL1; (6) VH and VL domains of hPD-1 mAb 15; It has, 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 mAb 14 or PD-1 mAb 15 binds to the same epitope, or This relates to PD-1 binding molecules possessing 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 indicated by underlining. DVQLQESGPG RVKPSQSLSL TCTVTGFSIT NDYAWN WIRQ FPGNKLEWMG HITYSGSTSY NPSLKS RISI TRDTSKNHFF LQLSSVTPED TATYYCAR DY GSGYPYTLDY WGQGTSVTVS S PD-1 mAb 1 CDR H 1 (Sequence ID 71): NDYAWN PD-1 mAb 1 CDR H 2 (Sequence ID 72): HITYSGSTSYNPSLKS PD-1 mAb 1 CDR H 3 (Sequence ID 73): DYGSGYPYTLDY

[0070] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 1 is: Sequence ID 70 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 AGA gattac ggtagtggct acccctatac tttggactac tggggtcaag gtacctcagt caccgtctcc tcc That is the case.

[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 indicated by underlines): QIVLTQSPAL MSASPGEKVT MTC SATSIVS YVY WYQQKPG SSPQPWIY LT SNLAS GVPAR FSGSGSGTSY SLTISSMEAE DAATYYC QQW SDNPYT FGGG TKLEIK PD-1 mAb 1 CDR L 1 (Sequence ID 76): SATSIVSYVY PD-1 mAb 1 CDR L 2 (Sequence ID 77): LTSNLAS PD-1 mAb 1 CDR L 3 (Sequence ID 78): QQWSDNPYT

[0072] An example polynucleotide encoding the VL domain of PD-1 mAb 1 is: Sequence ID 75 (CDR L The nucleotides encoding the residues are indicated by underlining): caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc atgacctgc a gtgccacctc aattgtaagt tacgtttac t ggtaccagca gaagcctgga tcctcccccc aaccctggat ttat ctcaca touthaacctgg cttct ggagt ccctgctcgc ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa gatgctgcca cttattactg c cagcagtgg agtgataacc cgtacacg tt cggagggggg accaagctgg aaataaaa That is the case.

[0073] 2. Anti-human PD-1 antibody PD-1 mAb 1 for the formation of "hPD-1 mAb 1" Humanization of b 1 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 1 was identified by the antigen epitope. If this occurs, the drug is humanized and further deimmunized to reduce its antigenicity when administered to human recipients. We demonstrated the ability to humanize anti-human PD-1 antibodies for this purpose. Through humanization, here we see "h One humanized VH domain called "PD-1 mAb 1 VH1" and, here, "h A single humanized VL domain called "PD-1 mAb 1 VL1" was obtained. Therefore, an antibody possessing the above-mentioned humanized VL domain paired with the above-mentioned humanized VH domain is "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) (CDR) H Residues are indicated by underlines): 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 is 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 That is the case.

[0076] The amino acid sequence of the VL domain of hPD-1 mAb 1 VL1 (SEQ ID NO: 81) (CDR) L Residues are indicated by underlines): 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 is sequence Number 82 (CDR) L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DVQLVESGGG LVQPGGSRKL SCAASGFVFS SFGMH WVRQA PEKGLEWVA Y ISSGSMSISY ADTVKG RFTV TRDNAKNTLF LQMTSLRSED TAIYYCAS LS DYFDY WGQGT TLTVSS PD-1 mAb 2 CDR H 1 (Sequence ID 85): SFIGMH PD-1 mAb 2 CDRH 2(Sequence ID 86):YISSGSMSISYADTVKG PD-1 mAb 2 CDR H 3 (Sequence ID 87): LSDYFDY

[0079] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 2 is: Sequence ID 84 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 act ac tgggg ccaaggcacc actctcacag tctcctcc That is the case.

[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 indicated by underlines): DVVMSQTPLS LPVSLGDQAS ISC RSSQSLV HSTGNTYLH W YLQKPGQSPK LLIY RVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV FFC SQTTHVP WTFGGGTKLE IK PD-1 mAb 2 CDR L 1 (Sequence ID 90): RSSQSLVHSTGNTYLH PD-1 mAb 2 CDR L 2 (Sequence ID 91): RVSNRFS PD-1 mAb 2 CDR L 3 (Sequence ID 92): SQTTHVPWT

[0081] An example polynucleotide encoding the VL domain of PD-1 mAb 2 is: Sequence ID 89 (CDR L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0082] 2. Anti-human PD-1 antibody PD-1 m for forming "hPD-1 mAb 2" Humanization of Ab 2 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 2 was identified by the antigen epitope. If this occurs, the drug is humanized and further deimmunized to reduce its antigenicity when administered to human recipients. We demonstrated the ability to humanize anti-human PD-1 antibodies for this purpose. Through humanization, here we see "h One humanized VH domain called "PD-1 mAb 2 VH1" and, here, "h A single humanized VL domain called "PD-1 mAb 2 VL1" was obtained. Which antibody possesses the humanized VL domain paired with the humanized VH domain mentioned above? This is called "hPD-1 mAb 2".

[0083] The amino acid sequence of the VH domain of hPD-1 mAb 2 VH1 (SEQ ID NO: 93) (CDR) H Residues are indicated by underlines): 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 is sequence Number 94 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0085] The amino acid sequence of the VL domain of hPD-1 mAb 2 VL1 (SEQ ID NO: 95) (CDR) L Residues are indicated by underlines): DVVMTQSPLS LPVTLGQPAS ISC RSSQSLV HSTGNTYLH W YLQKPGQSPQ LLIY RVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYC SQTTHVP WT FGQGTKLE IK

[0086] An exemplary polynucleotide encoding hPD-1 mAb 2 VL1 is sequence Number 96 (CDR) L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlining. QVQLQQSGAE LVRPGASVTL SCKASGYTFT DYVMH WVKQT PVHGLEWIG T IDPETGGTAY NQKFKG KAIL TADKSSNTAY MELRSLTSED SAVYYFTR EK ITTIVEGTYW YFDV WGTGTT VTVSS PD-1 mAb 3 CDR H 1 (Sequence ID 99): DYVMH PD-1 mAb 3 CDR H 2 (Sequence ID 100): TIDPETGGTAYNQKFKG PD-1 mAb 3 CDR H 3 (Sequence ID 101): EKITTIVEGTYWYFDV

[0088] Exemplary polynucleotides encoding the VH domain of PD-1 mAb 3 are , Sequence ID 98 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DVLLTQTPLS LPVSLGDQAS ISC RSSQNIV HSNGDTYLE W YLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHLP YT FGGGTKLE IK PD-1 mAb 3 CDR L 1 (Sequence ID 104): RSSQNIVHSNGDTYLE PD-1 mAb 3 CDR L 2 (Sequence ID 105): KVSNRFS PD-1 mAb 3 CDR L 3 (Sequence ID 106): FQGSHLPYT

[0090] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 3 is , Sequence ID 103 (CDR L (The nucleotides encoding the residues are indicated by underlining.) : gatgttttgc tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgc a gatctagtca gaacattgta catagtaatg gagacaccta tttggaa tgg tacctgcaga aaccaggcca gtctccaaag ctcctgatct at aaagtttc caaccgattt tct gggg tcc cagacaggtt cagtggcagt gggtcaggga cagattttac actcaaaatc agcagagtgg aggctgagga tctgggagtt tattactgc t ttcaaggttc acatcttccg tacacg ttcg gaggggggac caagctggaa ataaaa That is the case.

[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 indicated by underlines): DVQLVESGGG LVQPGGSRKL SCAASGFVFS SFGMH WVRQA PEKGLEWVA Y ISSGSMSISY ADTVKG RFTV TRDNAKNTLF LQMTSLRSED TAIYYCAS LT DYFDY WGQGT TLTVSS PD-1 mAb 4 CDR L 1 (Sequence ID 109): SFIGMH PD-1 mAb 4 CDR L 2 (Sequence ID 110): YISSGSMSISYADTVKG PD-1 mAb 4 CDR L 3 (Sequence ID 111): LTDYFDY

[0092] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 4 is: Sequence ID 108 (CDR HThe nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DVVMSQTPLS LPVSLGDQAS ISC RSSQSLV HSTGNTYFH W YLQKPGQSPK LLIY RVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQTTHVP WT FGGGTKLE IK PD-1 mAb 4 CDR L 1 (Sequence ID 114): RSSQSLVHSTGNTYFH PD-1 mAb 4 CDR L 2 (Sequence ID 115): RVSNRFS PD-1 mAb 4 CDR L 3 (Sequence ID 116): SQTTHVPWT

[0094] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 4 is: Sequence ID 113 (CDR L The nucleotides encoding the residues are indicated by underlining): 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 tctgggagtt tatttctgc t ctcaaactac acatgttccg tggacg ttcg gtggaggcac caagctggaa atcaaa That is the case.

[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 indicated by underlines): QVQLQQPGVE LVRPGASVKL SCKASGYSFT AYWMN WMKQR PGQGLEWIG V IHPSDSETWL NQKFKD KATL TVDKSSSTAY MQLISPTSED SAVYYCAR EH YGSSPFAY WG QGTLVTVSA PD-1 mAb 5 CDR H 1 (Sequence ID 119): AYWMN PD-1 mAb 5 CDRH 2 (Sequence ID 120): VIHPSDSETWLNQKFKD PD-1 mAb 5 CDR H 3 (Sequence ID 121): EHYGSSPFAY

[0096] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 5 is: Sequence ID 118 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DIVLTQSPAS LAVSLGQRAT ISC RANESVD NYGMSFMN WF QQKPGQPPKL LIY AASNQGS GVPARFSGSG SGTDFSLNIH PMEEDDTAMY FC QQSKEVPY T FGGGTKLEI K PD-1 mAb 5 CDR L 1 (Sequence ID 124): RANESVDNYGMSFMN PD-1 mAb 5 CDR L 2 (Sequence ID 125): AASNQGS PD-1 mAb 5 CDR L 3 (Sequence ID 126): QQSKEVPYT

[0098] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 5 is: Sequence ID 123 (CDR L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): EVKLVESGGG LVNPGGSLKL SCAASGFTFS SYGMSWVRQT PEKRLEWVA T ISGGGSDTYY PDSVKG RFTI SRDNAKNNLY LQMSSLRSED TALYYCAR QK ATTWFAY WGQ GTLVTVST PD-1 mAb 6 CDR H 1 (Sequence ID 129): SYGMS PD-1 mAb 6 CDR H 2 (Sequence ID 130): TISGGGSDTYYPDSVKG PD-1 mAb 6 CDR H 3 (Sequence ID 131): QKATTWFAY

[0100] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 6 is: Sequence ID 128 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

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

[0102] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 6 is: Sequence ID 133 (CDR L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): QVQLQQPGAE LVRPGASVKL SCKASGYSFT SYWMN WVKQR PGQGLEWIG V IHPSDSETWL DQKFKD KATL TVDKSSTTAY MQLISPTSED SAVYYCAR EH YGTSPFAY WG QGTLVTVSS PD-1 mAb 7 CDR H 1 (Sequence ID 139): SYWMN PD-1 mAb 7 CDR H 2 (Sequence ID 140): VIHPSDSETWLDQKFKD PD-1 mAb 7 CDR H 3 (Sequence ID 141): EHYGTSPFAY

[0104] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 7 is: Sequence ID 138 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DIVLTQSPAS LAVSLGQRAT ISC RANESVD NYGMSFMN WF QQKPGQPPKL LIH AASNQGS GVPARFSGSG FGTDFSLNIH PMEEDDAAMY FC QQSKEVPY T FGGGTKLEI K PD-1 mAb 7 CDR L 1 (Sequence ID 144):RANESVDNYGMSFMN PD-1 mAb 7 CDR L 2 (Sequence ID 145):AASNQGS PD-1 mAb 7 CDR L 3 (Sequence ID 146): QQSKEVPYT

[0106] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 7 is: Sequence ID 143 (CDR L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0107] 2. Anti-human PD-1 antibody PD-1 m for forming "hPD-1 mAb 7" Humanization of Ab 7 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 7 was identified by its antigen epitope. If this occurs, the drug is humanized and further deimmunized to reduce its antigenicity when administered to human recipients. We demonstrated the ability to humanize anti-human PD-1 antibodies for this purpose. Through humanization, here we see "h Two types of ions called "PD-1 mAb 7 VH1" and "hPD-1 mAb 7 VH2" Two humanized VH domains, and here, "PD-1 mAb 7 VL1" and "hPD-1 Three human mAb 7 VL2 and hPD-1 mAb 7 VL3 Humanized VL domains were obtained. Which of the humanized VL domains is the humanized VH domain? It can be paired with any of the above humanized VH domains. Therefore, the above humanized VH domain paired with the above humanized VH domain Any antibody possessing one of the VL domains is called "hPD-1 mAb 7". , a specific combination of humanized VH / VL domains, relative to a specific VH / VL domain They are referred to by reference. For example, hPD-1 mAb 7 VH1 and hPD-1 mA The humanized antibody containing b 7 VL2 is specifically "hPD-1 mAb 7(1.2)". It is called [name].

[0108] The amino acid sequence of the VH domain of hPD-1 mAb 7 VH1 (SEQ ID NO: 147) The following (CDR) H Residues are indicated by underlines): 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 is sequence Number 148 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0110] The amino acid sequence of the VH domain of hPD-1 mAb 7 VH2 (SEQ ID NO: 149) The following (CDR) H Residues are indicated by underlines): 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 is sequence Number 150 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0112] The amino acid sequence of the VL domain of hPD-1 mAb 7 VL1 (SEQ ID NO: 151) The following (CDR) L Residues are indicated by underlines): 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 is sequence Number 152 (CDR) L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0114] The amino acid sequence of the VL domain of hPD-1 mAb 7 VL2 (SEQ ID NO: 153) The following (CDR) L Residues are indicated by underlines): 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 is sequence Number 154 (CDR) L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0116] The amino acid sequence of the VL domain of hPD-1 mAb 7 VL3 (SEQ ID NO: 155) The following (CDR) L Residues are indicated by underlines): 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 is sequence Number 156 (CDR) L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0118] Both hPD-1 mAb 7 VL2 and hPD-1 mAb 7 VL3 VL Main CDR L 1 contains an amino acid substitution from asparagine to serine, and the amino acid sequence :RA S It contains ESVDNYGMSFMN (Sequence ID 157, substituted serine is underlined). A similar substitution is the aforementioned PD-1 mAb 7 CDR. L It can be incorporated into any of the domains. It is thought that this is possible.

[0119] Furthermore, the CDR of the VL domain of hPD-1 mAb 7 VL3 L 2 is from glutamine Includes amino acid substitutions for arginine, amino acid sequence: AASN R GS(Sequence ID 158, replaced The arginine that is substituted has (shown with an underline). Similar substitutions are found in the PD-1 mAb mentioned above. 7 CDR L It is thought 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 indicated by underlines): EGQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMN WVKQN HGKSLEWIG D INPKNGDTHY NQKFKG EATL TVDKSSTTAY MELRSLTSED SAVYYCAS DF DY WGQGTTLT VSS PD-1 mAb 8 CDR H 1 (Sequence ID 161): DYYMN PD-1 mAb 8 CDR H 2 (Sequence ID 162): DINPKNGDTHYNQKFKG PD-1 mAb 8 CDR H 3 (Sequence ID 163): DFDY

[0121] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 8 is: Sequence ID 160 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 indicated by underlines): DVVMTQTPLS LPVGLGDQAS ISC RSSQTLV YSNGNTYLN W FLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQSTHVP FT FGSGTKLE IK PD-1 mAb 8 CDR L 1 (Sequence ID 166): RSSQTLVYSNGNTYLN PD-1 mAb 8 CDR L 2 (Sequence ID 167): KVSNRFS PD-1 mAb 8 CDR L 3 (Sequence ID 168): SQSTHVPFT

[0123] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 8 is: Sequence ID 165 (CDR L The nucleotides encoding the residues are indicated by underlining): gatgttgtga tgacccaaac tccactctcc ctgcctgtcg gtcttggaga tcaagcctcc atctcttgc a gatctagtca gacccttgta tatagtaatg gaaacaccta tttaaat tgg ttcctgcaga agccaggcca gtctccaaag ctcctgatct ac aaagtttc caaccgattt tct gggg tcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tatttctgc 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 indicated by underlines): EVMLVESGGG LVKPGGSLKL SCAASGFTFS SYLVS WVRQT PEKRLEWVA T ISGGGGNTYY SDSVKG RFTI SRDNAKNTLY LQISSLRSED TALYYCAR YG FDGAWFAY WG QGTLVTVSS PD-1 mAb 9 CDR H 1 (Sequence ID 171): SYLVS PD-1 mAb 9 CDR H 2 (Sequence ID 172): TISGGGGNTYYSDSVKG PD-1 mAb 9 CDR H 3 (Sequence ID 173): YGFDGAWFAY

[0125] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 9 is: Sequence ID 170 (CDR HThe nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DIQMTQSPAS LSASVGDIVT ITC RASENIY SYLA WYQQKQ EKSPQLLVY N AKTLAA GVPS RFSGSGSGTQ FSLTINSLQP EDFGNYYC QH HYAVPWT FGG GTRLEIT PD-1 mAb 9 CDR L 1 (Sequence ID 176): RASENIYSYLA PD-1 mAb 9 CDR L 2 (Sequence ID 177): NAKTLAA PD-1 mAb 9 CDR L 3 (Sequence ID 178): QHHYAVPWT

[0127] An example polynucleotide encoding the VL domain of PD-1 mAb 9 is: Sequence ID 175 (CDR L The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0128] 2. Anti-human PD-1 antibody PD-1 m for forming "hPD-1 mAb 9" Humanization of Ab 9 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 9 was identified by the antigen epitope. If this occurs, the drug is humanized and further deimmunized to reduce its antigenicity when administered to human recipients. We demonstrated the ability to humanize anti-human PD-1 antibodies for this purpose. Through humanization, here we see "h Two types of ions called "PD-1 mAb 9 VH1" and "hPD-1 mAb 9 VH2" Two humanized VH domains, and here, "hPD-1 mAb 9 VL1" and "hPD Two humanized VL domains, called "-1 mAb 9 VL2", were obtained. Any of the VL domains can pair with any of the humanized VH domains. Therefore, And, comprising one of the humanized VL domains paired with the humanized VH domain mentioned above. The offset antibody is generally called "hPD-1 mAb 9," and has a humanized VH / VL domain. A specific combination is referred to by a reference to a particular VH / VL domain. For example... Humanized with hPD-1 mAb 9 VH1 and hPD-1 mAb 9 VL2 The antibody is specifically called "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) The following (CDR) H Residues are indicated by underlines): 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 is sequence Number 180 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0131] The amino acid sequence of the VH domain of hPD-1 mAb 9 VH2 (SEQ ID NO: 181) The following (CDR) H Residues are indicated by underlines): 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 is sequence Number 182 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[0133] CDR of the VH domain of hPD-1 mAb 9 VH2 H 1 is serine to glycine Includes amino acid substitutions, amino acid sequence: SYLV G (Sequence ID 183, Substituted Glycine) (The underlined part indicates the presence of the following.) A similar substitution is found in the above-mentioned PD-1 mAb 9 CDR. H It is thought that it can be incorporated into any of the domains.

[0134] The amino acid sequence of the VL domain of hPD-1 mAb 9 VL1 (SEQ ID NO: 184) The following (CDR) L Residues are indicated by underlines): 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 is sequence Number 185 (CDR) L The nucleotides encoding the residues are indicated by underlining): gacattcaga tgactcagtc tcccagcagt ctgtccgcat ccgtggggga tcgggtcacc atcacctgc c gtgcctcaga aaacatctat tcatacctcg cc tggtatca acagaaacct ggtaaagccc caaaattgct catttac aac gccaagaccc tcgcagct gg cgtgccaagt aggttctcag gcagcggctc agggagat ttcaccctca ccatatcctc actgcagccc gaggattttg ccacttacta ctgc cagcat cattacgcag tgccctggac c ttcggacaa ggcactaagc tcgagatcaa a That is the case.

[0136] The amino acid sequence of the VL domain of hPD-1 mAb 9 VL2 (SEQ ID NO: 186) The following (CDR) L Residues are indicated by underlines): 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 is the sequence Number 187 (CDR) L The nucleotides encoding the residues are indicated by underlining): gacattcaga tgactcagtc tcccagcagt ctgtccgcat ccgtggggga tcgggtcacc atcacctgc c gtgcctcaga aaacatctat aactacctcg cc tggtatca acagaaacct ggtaaagccc caaaattgct catttac gac gccaagaccc tcgcagct gg cgtgccaagt aggttctcag gcagcggctc agggagat ttcaccctca ccatatcctc actgcagccc gaggattttg ccacttacta ctgc cagcat cattacgcag tgccctggac c ttcggacaa ggcactaagc tcgagatcaa a

[0138] CDR of the VL domain of hPD-1 mAb 9 VL2 L 1 is serine from asparagus Includes amino acid substitutions to gin, amino acid sequence: RASENIY N YLA(sequence number 188, replaced) The asparagine shown is underlined. Similar substitutions are found in the above-mentioned PD-1 mAb. 9 CDR L It is thought that it can be incorporated into any of the domains.

[0139] CDR of the VL domain of hPD-1 mAb 9 VL2 L 2 is from asparagine Including an amino acid substitution to spartic acid, amino acid sequence: D AKTLAA(Sequence ID 189, Substitution) The substituted aspartic acid is shown underlined. Similar substitutions are found in the above PD-1 mAb 7 CDR L It is thought 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 indicated by underlines): EVILVESGGG LVKPGGSLKL SCAASGFTFS NYLMSWVRQT PEKRLEWVA S ISGGGSNIYY PDSVKG RFTI SRDNAKNTLY LQMNSLRSED TALYYCAR QE LAFDY WGQGT TLTVSS PD-1 mAb 10 CDR H 1 (Sequence ID 192): NYLMS PD-1 mAb 10 CDR H 2 (Sequence ID 193): SISGGGSNIYYPDSVKG PD-1 mAb 10 CDR H 3 (Sequence ID 194): QELAFDY

[0141] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 10 is , Sequence ID 191 (CDR H (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): DIQMTQTTSS LSASLGDRVT ISC RTSQDIS NFLN WYQQKP DGTIKLLIY Y TSRLHS GVPS RFSGSGSGTD YSLTISNLEQ EDIATYFC QQ GSTLPWT FGG GTKLEII PD-1 mAb 10 CDR L 1 (Sequence ID 197): RTSQDISNFLN PD-1 mAb 10 CDR L 2 (Sequence ID 198): YTSRLHS PD-1 mAb 10 CDR L 3 (Sequence ID 199): QQGSTLPWT

[0143] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 10 is , Sequence ID 196 (CDR L (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): EVQLQQSGTV LARPGASVKM SCKTSGYTFT GYWMH WVKQR PGQGLKWMG A IYPGNSDTHY NQKFKG KAKL TAVTSASTAY MELSSLTNED SAIYYCTT GT YSYFDV WGTG TTVTVSS PD-1 mAb 11 CDR H 1 (Sequence ID 202): GYWMH PD-1 mAb 11 CDR H 2 (Sequence ID 203): AIYPGNSDTHYNQKFKG PD-1 mAb 11 CDR H 3 (Sequence ID 204): GTYSYFDV

[0145] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 11 is , Sequence ID 201 (CDR H (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): DILLTQSPAI LSVSPGERVS FSC RASQSIG TSIH WYQHRT NGSPRLLIK Y ASESIS GIPS RFSGSGSGTD FTLSINSVES EDIADYYC QQ SNSWLT FGAG TKLELK PD-1 mAb 11 CDR L 1 (Sequence ID 207):RASQSIGTSIH PD-1 mAb 11 CDR L 2 (Sequence ID 208): YASESIS PD-1 mAb 11 CDR L 3 (Sequence ID 209): QQSNSWLT

[0147] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 11 is , Sequence ID 206 (CDR L (The nucleotides encoding the residues are indicated by underlining.) : 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 indicated by underlines): QGHLQQSGAE LVRPGASVTL SCKASGFTFT DYEMH WVKQT PVHGLEWIG T IDPETGGTAY NQKFKG KAIL TVDKSSTTTY MELRSLTSED SAVFYCSR ER ITTVVEGAYW YFDV WGTGTT VTVSS PD-1 mAb 12 CDR H 1 (Sequence ID 212): DYEMH PD-1 mAb 12 CDR H 2 (Sequence ID 213): TIDPETGGTAYNQKFKG PD-1 mAb 12 CDR H 3 (Sequence ID 214): ERITTVVEGAYWYFDV

[0149] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 12 is , Sequence ID 211 (CDR H (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): DVLMTQTPLS LPVSLGDQAS ISC RSSQNIV HSNGNTYLE W YLQKPGQSPK LLIC KVSTRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHVP YT FGGGTKLE IK PD-1 mAb 12 CDR L 1 (Sequence ID 217): RSSQNIVHSNGNTYLE PD-1 mAb 12 CDR L 2 (Sequence ID 218): KVSTRFS PD-1 mAb 12 CDR L 3 (Sequence ID 219): FQGSHVPYT

[0151] An example polynucleotide encoding the VL domain of PD-1 mAb 12 is: , Sequence ID 216 (CDR L (The nucleotides encoding the residues are indicated by underlining.) : gatgttttga tgacccagac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc atctcttgc a gatctagtca gaacattgta catagtaatg gaaacaccta tttagaa tgg tacctgcaga aaccaggcca gtctccaaag ctcctgatct gc aaagtttc cacccgattt tct gggg tcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc agcagagtgg aggctgagga tctgggagtt tattattgc t ttcaaggttc acatgttccg tacacg ttcg gaggggggac caagctggaa ataaaa That is the case.

[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 indicated by underlines): EVMLVESGGG LVKPGGSLKL SCAASGFTFS SHTMS WVRQT PEKRLEWVA T ISGGGSNIYY PDSVKG RFTI SRDNAKNTLY LQMSSLRSED TALYYCAR QA YYGNYWYFDV WGTGTTVTVS S PD-1 mAb 13 CDR H 1 (Sequence ID 222): SHTMS PD-1 mAb 13 CDRH 2 (Sequence ID 223): TISGGGSNIYYPDSVKG PD-1 mAb 13 CDR H 3(Sequence ID 224):QAYYGNYWYFDV

[0153] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 13 is , Sequence ID 221 (CDR H (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): DIQMTQSPAT QSASLGESVT ITC LASQTIG TWLA WYQQKP GKSPQLLIY A ATSLADGVPS RFSGSGSGTK FSFKISSLQA EDFVSYYC QQ LDSIPWT FGG GTKLEIK PD-1 mAb 13 CDR L 1 (Sequence ID 227): LASQTIGTWLA PD-1 mAb 13 CDR L 2 (Sequence ID 228): AATSLAD PD-1 mAb 13 CDR L 3 (Sequence ID 229): QQLDSIPWT

[0155] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 13 is , Sequence ID 226 (CDR L (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): QVQLQQPGAE LVKPGASVKM SCKASGYNFI SYWIT WVKQR PGQGLQWIG N IYPGTDGTTY NEKFKS KATL TVDTSSSTAY MHLSRLTSED SAVYYCAT GL HWYFDV WGTG TTVTVSS PD-1 mAb 14 CDR H 1 (Sequence ID 232): SYWIT PD-1 mAb 14 CDR H 2 (Sequence ID 233): NIYPGTDGTTYNEKFKS PD-1 mAb 14 CDR H 3(Sequence ID 234):GLHWYFDV

[0157] An exemplary polynucleotide encoding the VH domain of PD-1 mAb 14 is , Sequence ID 231 (CDR H (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): DIVMTQSQKF MSTSVGDRVS VTC KASQSVG TNVA WYQQKP GQSPKALIY S ASSRFS GVPD RFTGSGSGTD FTLTISNVQS EDLAEYFC QQ YNSYPYT FGG GTKLEIK PD-1 mAb 14 CDR L 1 (Sequence ID 237): KASQSVGTNVA PD-1 mAb 14 CDR L 2 (Sequence ID 238): SASSRFS PD-1 mAb 14 CDR L 3 (Sequence ID 239): QQYNSYPYT

[0159] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 14 is , Sequence ID 236 (CDR L (The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[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 indicated by underlines): EVMLVESGGG LVKPGGSLKL SCAASGFIFS SYLIS WVRQT PEKRLEWVA A ISGGGADTYY ADSVKG RFTI SRDNAKNTLY LQMSSLRSED TALYYCTR RG TYAMDY WGQG TSVTVSS PD-1 mAb 15 CDR H 1 (Sequence ID 242): SYLIS PD-1 mAb 15 CDR H 2 (Sequence ID 243): AISGGGADTYYADSVKG PD-1 mAb 15 CDR H 3 (Sequence ID 244): RGTYAMDY

[0161] An exemplary polynucleotide encoding the PD-1 mAb 15VH domain is: Sequence ID 241 (CDR H The nucleotides encoding the residues are indicated by underlining): 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 That is the case.

[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 indicated by underlines): DIQMTQSPAS QSASLGESVT ITC LASQTIG TWLA WYQQKP GKSPQLLIY A ATSLAD GVPS RFSGSGSGTK FSFKISSLQA EDFVNYYC QQ LIGHTINGWT FGG GTKLEIK PD-1 mAb 15 CDR L 1 (Sequence ID 247): LASQTIGTWLA PD-1 mAb 15 CDR L 2 (Sequence ID 248): AATSLAD PD-1 mAb 15 CDR L 3 (Sequence ID 249): QQLYSIPWT

[0163] An exemplary polynucleotide encoding the VL domain of PD-1 mAb 15 is , Sequence ID 246 (CDR L(The nucleotides encoding the residues are indicated by underlining.) : 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 That is the case.

[0164] 2. Anti-human PD-1 antibody PD-1 for forming "hPD-1 mAb 15" Humanization of mAb 15 The above-mentioned mouse anti-human PD-1 antibody PD-1 mAb 15 was identified by the antigen epitope. If necessary, it is humanized and then deimmunized to reduce its antigenicity when administered to human recipients. We demonstrated the ability to humanize anti-human PD-1 antibodies to reduce the risk of PD-1. Through humanization, we found that " One humanized VH domain called "hPD-1 mAb 2 VH1" and here " A single humanized VL domain called "hPD-1 mAb 1 VL1" was obtained. An antibody comprising the above-mentioned VL domain paired with the above-mentioned humanized VH domain is called "hPD-1 mA It is called "b 15".

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

[0166] Exemplary polynucleotides encoding hPD-1 mAb 15 VH1 are distributed Column number 251 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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) The following is shown (CDR L Residues are indicated by underlines): 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 distributed Column number 253 (CDR) H The nucleotides encoding the residues are indicated by underlining): 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 antibody PD-1 mAb 1-15 and having a modified Fc region its derivatives In conventional immune function, the interaction between antibody-antigen complexes and immune system cells is antibody-dependent. Effector functions such as cytotoxicity, mast cell degranulation, and phagocytosis lead to lymphocyte proliferation and antibody secretion. This results in a wide range of responses, including immunomodulatory signals that control various functions. All applications involve the Fc region of antibodies or immune complexes, specifically specialized cell surface receptors on hematopoietic cells. It is initiated by binding to. Diverse cellular responses triggered by antibodies and immune complexes. Sex is caused by three Fc receptors: FcγRI (CD64), FcγRII (CD32), and Fc It is obtained by the structural heterogeneity of γRIII (CD16). FcγRI (CD64), F cγRIIA (CD32A) and FcγRIII (CD16) are activated (i.e., the immune system is enhanced). ) is a receptor; FcγRIIB(CD32B) is an inhibitory (i.e., immune system attenuation) receptor. Furthermore, interaction with the neonatal Fc receptor (FcRn) is transmitted from endosomes to the cell surface. It mediates the recirculation and release of IgG molecules into the bloodstream. Exemplary wild-type IgG1 (SEQ ID NO: 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 changes in serum half-life, changes in stability, and changes in cells. This can lead to changes in sensitivity to enzymes or changes in effector function. The antibody or other of the present invention Modifying the binding molecule in terms of its effector function, for example, in the treatment of cancer. In some cases, it is desirable to enhance effectiveness. For example, the mechanism of action involves the support of the target antigen. In certain cases, such as when antibodies block or antagonize cells rather than kill them, Therefore, a reduction or elimination of effector functions is desirable. An increase in effector functions is desirable when FcγR is low. Tumor and exogenous cells expressing B at low levels, such as FcγRIIB, which is a tumor-specific B. Cells (e.g., non-Hodgkin lymphoma, CLL, and Burkitt lymphoma), preferably It is generally desirable when targeting cells that do not have this function. In the above embodiment, effector functional activity is The given or modified molecules of the present invention are desirable for enhancing the efficiency of effector functional activity. It is useful for the treatment and / or prevention of diseases, disorders, or infections.

[0171] In certain embodiments, the PD-1 binding molecule of the present invention has a wild-type Fc region (e.g., SEQ ID NO:). 1) Having one or more modifications (e.g., substitution, deletion, or insertion) to the amino acid sequence The molecule comprises an Fc region, and the above modification is one or more of the above Fc region, and therefore of the molecule of the present invention. It reduces the affinity and binding activity of to the FcγR receptor. In other embodiments, the present invention The molecule has an Fc region having one or more modifications to the amino acids in the wild-type Fc region. The above modification affects one or more FcγR receptors in the Fc region, and therefore in the molecule of the present invention. It increases affinity and binding activity to the body. In other embodiments, the above molecule is mutant Fc This region includes, where the above variant does not include the Fc domain, or the wild-type Fc domain Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity against molecules containing the substance, and / or It provides or mediates increased binding to FcγRIIA. In an alternative embodiment, the molecule It includes a variant Fc region, where the variant is one that does not include an Fc region, or a wild-type Fc region. For molecules containing, a decrease in ADCC activity (or other effector function), and / or It provides or mediates increased binding to FcγRIIB. In some embodiments, this The molecule contains a PD-1 binding molecule including a mutant Fc region, and the mutant Fc region is wild-type Compared to equivalent molecules containing an Fc region, it does not show detectable binding to any of the FcγR regions. In other embodiments, the present invention includes a PD-1 binding molecule containing a mutant Fc region, and The variant Fc region is a single FcγR, preferably FcγRIIA, FcγRIIB, or It binds to only one of the FcγRIIIA molecules. Such increased affinity and / or Preferably, the binding activity is such that the parent molecule (without a modified Fc region) is bound within the cell. In cells expressing low levels of FcγR when the binding activity of 3 cannot be detected, or 3 Density of 0000-20000 molecules / cell, density of 20000-10000 molecules / cell, 1 Density of 0000-5000 molecules / cell, density of 5000-1000 molecules / cell, 1000 Density of ~200 molecules / cell, or less than 200 molecules / cell (but at least 10, 5 Cells expressing non-FcγR receptor target antigens at a density of 0, 100, or 150 molecules / cell In cells, the degree of detectable FcγR binding or FcγR-related activity can be measured in vitro. It is evaluated by determining its value.

[0172] The PD-1 binding molecule of the present invention exhibits altered affinity for the activating and / or inhibiting Fcγ receptor. It may have a modified mutant Fc region. In one embodiment, the PD-1 binding molecule is wild-type For equivalent molecules possessing an Fc domain, affinity for FcγRIIB is increased, and F Mutant Fc regions with reduced affinity for cγRIIIA and / or FcγRIIA In another embodiment, the PD-1 binding molecule of the present invention is an equivalent having a wild-type Fc region. For the molecule, the affinity for FcγRIIB decreases, and FcγRIIIA and / Alternatively, it may have a variant Fc region with increased affinity for FcγRIIA. Morphologically, the PD-1 binding molecule of the present invention is similar to an equivalent molecule having a wild-type Fc domain. Affinity for FcγRIIB is reduced, and FcγRIIIA and / or FcγRII It also has a mutant Fc region in which affinity for A is reduced. In yet another embodiment, the present invention The PD-1 binding molecule is equivalent to an equivalent molecule with a wild-type Fc domain in FcγRIIB. The affinity for FcγRIIIA and / or FcγRIIA has not changed, and the affinity for FcγRIIIA and / or FcγRIIA It has a mutant Fc region in which affinity is reduced (or increased).

[0173] In certain embodiments, the PD-1 binding molecule of the present invention enhances immunoglobulins. To have an ejector function, affinity for FcγRIIIA and / or FcγRIIA It includes a mutant Fc region with altered characteristics. Non-limited examples of effector cell function include antibody-dependent persistent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis, phagocytosis, opsonization, opsonophagocy These include action, cell binding, rosette formation, C1q binding, and complementarity-determining cell-mediated cytotoxicity. It can be done.

[0174] In one preferred embodiment, the change in affinity or effector function includes a wild-type Fc region. Compared to an equivalent molecule, it is at least twice, preferably at least four times, at least five times, less At least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, At least 50 times, or at least 100 times. In other embodiments of the present invention, mutant The Fc region is at least 65%, preferably at least, compared to a molecule containing a wild-type Fc region. 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, With 175%, 200%, 225%, or 250% higher affinity, immunity to one or more FcRs. It binds specifically to the disease. Such measurements can be performed as in vivo or in vitro assays. In a preferred embodiment, an in vitro assay such as ELISA or surface plasmon resonance assay can be performed. This is a roassay.

[0175] In various embodiments, the PD-1 binding molecule of the present invention comprises a mutant Fc region, and the above The mutant stimulates the activity of at least one FcγR receptor or the FcγR receptor Antagonizes at least one activity. In a preferred embodiment, the molecule is FcγRIIB One or more of the following activities, e.g., B cell receptor-mediated signaling, B cell activation, B cell proliferation, antibody production, intracellular calcium influx into B cells, cell cycle progression, FcεRI signaling FcγRIIB-mediated inhibition of naring, phosphorylation of FcγRIIB, SHIP mobilization, S HIP phosphorylation and linkage with Shc, or one of the FcγRIIB signaling pathways Or the activity of multiple downstream molecules (e.g., MAP kinase, JNK, p38, or Akt) Includes antagonistic variants. In another embodiment, the PD-1 binding molecule of the present invention is FcεRI One or more activities, e.g., mast cell activation, calcium mobilization, degranulation, cytokine Includes variants that stimulate production or serotonin release.

[0176] In certain embodiments, the molecule may be two or more IgG isotypes (e.g., IgG1, Contains Fc region-containing regions from IgG2, IgG3, and IgG4. When used, the Fc region has an amino acid sequence that is different from other IgG isotypes. If it has the highest homology to a specific IgG isotype, then the IgG isotype It is described as such. The various IgG isotypes mentioned above are, for example, 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 Due to differences in the amino acid sequence of its hinge and / or Fc region, serum half-life, complement binding, Various factors including FcγR binding affinity and effector functional activity (e.g., ADCC, CDC, etc.) It exhibits physical and functional properties. This type of mutant Fc region can be used alone or with amino acids. Used in combination with modifications, it affects Fc-mediated effector function and / or binding activity. It can be done. In combination, amino acid modification and IgG hinge / Fc region, It can exhibit similar functionality (e.g., increased affinity for FcγRIIA), and also phase Acting additively, or more preferably synergistically, compared to the molecule of the present invention containing the wild-type Fc region In all cases, the effector functionality of the molecule of the present invention can be modified. In other embodiments, amino acid modification is used. And the IgG Fc region has opposite functionality (for example, affinity for FcγRIIA). It can show increases and decreases, and also, the same isotype does not include the Fc region. Compared to the molecule of the present invention containing the wild-type Fc region, the specific functionality of the molecule of the present invention is selectively achieved. It can act to regulate or reduce

[0177] In a preferred specific embodiment, the PD-1 binding molecule of the present invention comprises a mutant Fc region, The above mutant Fc region includes at least one amino acid modification compared to the wild-type Fc region. As a result, the above mutant Fc region was found in Sondermann et al. (2000) Nature 406:267-73. Based on crystallographic and structural analysis of Fc-FcR interactions, as shown, If there is no substitution at the position in direct contact with FcγR, the above molecule is a parent of FcR. The compatibility changes. An example of a location within the Fc region that is in direct contact with FcγR is amino acid residue 234. ~239), amino acid residues 265~269 (B / C loop), amino acid residues 297~29 It consists of 9 (C' / E loop) and amino acid residues 327-332 (F / G loop). How many? In that embodiment, the molecule of the present invention is directly reacted with FcγR based on structural and crystallographic analysis. Includes modification of at least one residue that does not come into contact, for example, is not within the Fc-FcγR binding site. It includes a variant Fc region.

[0178] The variant Fc region is known in the art, for example, NK-dependent or macrophages. Fc regions (or one of them) that are functionally assayed in a phase-dependent assay. In order to impart or modify the effector function exhibited by the molecule of the present invention comprising (part), Known variant Fc regions may be used in the present invention. For example, altered effector function The Fc region variant identified as is in International Publication No. 04 / 063351; International Publication No. 06 / 088494; International Publication No. 07 / 024249; International Publication No. 06 / 113665 International Publication No. 07 / 021841; International Publication No. 07 / 106707; and International Publication No. Disclosed in issue 2008 / 140603, any preferred variant disclosed The body may be used in the molecule of the present invention.

[0179] In certain embodiments, the PD-1 binding molecule of the present invention has one or more regions. or comprising a mutant Fc region having multiple amino acid modifications, wherein one or more of the above modifications are mutant Activation F for the affinity of the heteromorphic Fc region to inhibitory FcγR (FcγRIIB, etc.) The affinity ratio for cγR (FcγRIIA or FcγRIIIA, etc.) is (wild-type F Change the c region:

[0180]

number

[0181] A mutant Fc region whose affinity ratio (to wild-type Fc region) exceeds 1. The PD-1 binding molecule of the present invention having a region is particularly preferred. Such molecules are, for example, cancer or Increased efficiency of effector cell functions mediated by FcγR (e.g., ADCC) in infectious diseases. Treatment or preventive measures for diseases, disorders, or infections for which strong treatment is desired, or relief of their symptoms. It has a specific use in providing a solution. In contrast, mutations with an affinity ratio of less than 1. Atypical Fc regions mediate a decrease in the efficiency of effector cell function. Table 1 shows exemplary single regions. Double, triple, quadruple, and quintuple mutations are defined as having an affinity ratio greater than or less than 1. They are listed by [the specified method].

[0182] [Table 1]

[0183] In one specific embodiment, in the mutant Fc region, 235, 240, 241, 24 Among the 3rd, 244th, 247th, 262nd, 263rd, 269th, 298th, 328th, or 330th place Any amino acid modification (e.g., substitution) in any of the following, preferably the following residue: A2 40, I240, L241, L243, H244, N298, I328 or V330 One or more of these. In different specific embodiments, in the variant Fc region, 268, 2 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 the positions 35, 437, 438, or 439 (for example) (By substitution), preferably the following residues: H280, Q280, Y280, G290, S290 , T290, Y290, N294, K295, P296, D298, N298, P298 V298, I300, or L300.

[0184] In one preferred embodiment, a mutant Fc region that binds to FcγR with altered affinity And, 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 of the following positions: 30, 434, 435, 437, 438, or 439 Mino acid modification (e.g., substitution). Preferably, the above mutant Fc region is one of the following residues. It has one of the following: 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 with reduced affinity (via its Fc region). 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 positions 439.

[0186] In a different embodiment, it binds to FcγR with enhanced affinity (via its Fc region). 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 Any amino acid modification (e.g., substitution). In different embodiments, F with enhanced affinity. In the mutant Fc region that binds to cγRIIA, 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 Includes one or more modifications at position 0 or 332.

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

[0189] [Table 2]

[0190] Furthermore, particularly preferred mutants include one or more modifications selected from groups 1 to 105:

[0191] [Table 3] JPEG0007866012000005.jpg83163

[0192] In one embodiment, the polyPD-1 binding molecule of the present invention has at least one in the Fc region It includes a modified mutant Fc region. In certain embodiments, the mutant Fc domain is From L235V, F243L, R292P, Y300L, V305I, and P396L It includes at least one substitution selected from the group.

[0193] In one specific embodiment, the variant Fc region includes: (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 permutations 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; or (E)(1)F243L, R292P, Y300L, V305I and P396L; and to (2) L235V, F243L, R292P, Y300L and P396L At least five permutations selected from the group consisting of the following.

[0194] In another specific embodiment, the variant Fc region includes 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 molecule of the present invention is (wild-type IgG1 Fc region (sequence number) Compared to the binding exhibited by (1), FcγRIA(CD64), FcγRIIA(CD32A ), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRII It possesses a variant Fc region in which binding to IB(CD16b) is reduced (or almost completely absent). In one embodiment, the PD-1 binding molecule of the present invention is FcγR (e.g., FcγRIII). The coupling to A) is reduced (or almost non-existent), and the ADCC effector function is reduced. It comprises a mutant Fc region (or one that does not have the above function). In certain embodiments, the above mutation The Fc region consists of L234A, L235A, D265A, N297Q, and N297G. The group includes at least one substitution selected from the group. In one specific embodiment, the above variant The Fc region is: L234A; L235A; L234A and L235A; D265A; N29 Includes substitution of 7Q or N297G.

[0196] Preferred IgG1 sequences relating to the CH2 and CH3 domains of the PD-1 binding molecule of the present invention This is 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 It has such that X is either ricin (K) or absent.

[0197] In different embodiments, the PD-1 binding molecule of the present invention is (wild-type IgG1 Fc region (distributed Binding to FcγRIIIA(CD16a) is reduced compared to the binding shown in column 1). It has an Fc area that is (or not partially combined) and / or has reduced effector functionality. In one specific embodiment, the PD-1 binding molecule of the present invention has an IgG2 Fc region (SEQ ID NO: 2). ) or comprises an IgG4 Fc region (SEQ ID NO: 4). When using the IgG4 Fc region The present invention relates to the substitution of the IgG4 hinge region S228P (for example) to reduce the occurrence of chain exchange. , Sequence ID 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). By introducing other stabilizing mutations known in the relevant technical field into the IgG4 Fc region... (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; International Publication No. 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 U.S. Patent No. 6,194,551; U.S. Patent No. 7,276,586; and U.S. Patent No. 7,317 ,091; and International Publication No. 00 / 42072 and International Publication No. 99 / 58572 This includes the use of any known variant Fc region, such as those disclosed.

[0199] In some embodiments, the molecule of the present invention further comprises one or more glycosylation sites. This may result in one or more carbohydrate portions being covalently attached to the above molecule. Preferably, the Fc region has one or more glycosylation sites and / or one or more The molecule of the present invention, having multiple modifications, exhibits enhanced antibody-mediated response compared to an unmodified antibody. Effector function, for example, providing or having enhanced ADCC activity. Several implementations In terms of form, the present invention further involves direct or indirect interaction with the carbohydrate portion of the Fc region. It is known that 241, 243, 244, 245, 245, 249, 256, 258 It contains amino acids at positions 260, 262, 264, 265, 296, 299 and 301. Molecules containing one or more modifications of amino acids, not limited to those mentioned above. Carbohydrates in the Fc region Amino acids that directly or indirectly interact with the compound moiety are known in the art. For example, Jefferis et al. (1995) Immunology Letters, 44: 111-7 (this reference is for reference) See (the entire text is incorporated herein by reference).

[0200] In another embodiment, the present invention preferably relates to the functionality of the molecule, for example, a target molecule or Fc Without altering the binding activity to γR, one or more glycosylation sites are modified in the molecule. It includes molecules that have been modified by introducing glycosyl into one or more sites. The compounding site may be introduced into the variable and / or constant region of the molecule of the present invention. When used, the term "glycosylation site" is: Ligosaccharides (i.e., carbohydrates containing two or more monosaccharides linked together) are specifically covalently linked. Therefore, it contains any specific amino acid sequence in the antibody that adheres. Oligosaccharide side chains are typical. It is linked to the antibody backbone via N-bonds or O-bonds. N-bonded glycosylation is This represents the attachment of the oligosaccharide moiety to the side chain of the asparagine residue. O-bond glycosylation is, for example, This represents the attachment of oligosaccharide moieties to hydroxy amino acids such as baserine and threonine. The molecule contains one or more glycosylated sites, including N-bond and O-bond glycosylation sites. It may have a position. For N-bond or O-bond glycosylation known in the art The glycosylated sites of the displacement may be used in accordance with the present invention. An exemplary N-linked glycosylation site is in the amino acid sequence: Asn-X-Thr / Ser Yes, where X can be any amino acid, and Thr / Ser can be threonine or seri. This indicates one or more such parts are known in the art in which the present invention relates. The molecule of the present invention may be introduced using the following method (e.g., IN VITRO MUTAGENESIS, RECOMBINAN). T DNA: A SHORT COURSE, JD Watson, et al. WH Freeman and Company, New York, See 1983, chapter 8, pp. 106-116 (the entire text is incorporated herein by reference). (This refers to) An exemplary method for introducing a glycosylation site into the molecule of the present invention is: the molecular ami Modify or mutate the no-acid sequence to create the desired Asn-X-Thr / Ser sequence. The step of obtaining may include the step of obtaining

[0201] In some embodiments, the present invention can be achieved by adding or deleting glycosylation sites. The present invention includes a method for modifying the carbohydrate content of the molecule. Antibodies (and antibody domains, For example, a method for modifying the carbohydrate content of a molecule (including an Fc region) is publicly known in the relevant field. This is knowledge and is included in the present invention. For example, U.S. Patent No. 6,218,149; European Patent No. 0 359096B1; U.S. Patent Application No. 2002 / 0028486; International Publication No. 03 / 0 Patent No. 35835; U.S. Patent Application No. 2003 / 0115614; U.S. Patent No. 6,218,1 U.S. Patent No. 49; U.S. Patent No. 6,472,511 (these in whole are incorporated herein by reference) See (refer to). In other embodiments, the present invention relates to one or more endogenous molecules This invention relates to a method for modifying the carbohydrate content of a molecule by deleting the carbohydrate portion. Includes. In one specific embodiment, the present invention modifies the position adjacent to position 297. Therefore, it includes a step of shifting the glycosylation site of the Fc region of the antibody. In a specific embodiment, the present invention modifies the 296th position, rather than the 297th position. This includes the step of glycosylation.

[0202] The effector function also involves introducing one or more cysteine ​​residues into the Fc region. This enables interchain disulfide bonding in this region, and as a result, the absorption capacity is improved. Homodimeric antibodies that may improve and / or increase complement-mediated cell killing and ADCC By generating it, it can be modified (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" "Anticlolytic Activity," J. Immunol. 148(9):2918-2922). Homodimeric with enhanced antitumor activity. Somatic antibodies are also discussed in Wolff, EA et al. (1993) "Monoclonal Antibody Homodimers: Enhance d Antitumor Activity In Nude Mice," described in Cancer Research 53:2560-2565. It can be prepared using such a heterobifunctional crosslinker. Alternatively, a double Fc domain It is possible to process antibodies that possess and thereby enhance 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 molecule of the present invention having an Fc region is determined by the binding affinity of the Fc region to FcRn. It can be increased by increasing the properties. The term "half-life" is a measure of the average survival time of a molecule after administration. This refers to the pharmacokinetic properties of a molecule. Half-life refers to the half-life in serum (i.e., circulating half-life) or in other tissues. When measured, 50 percent (50%) of the known amount of molecules is found in the subject's body (for example) The time required for the patient (or other mammal) to be removed from their specific body cavity It can be expressed as follows. Generally, an increase in half-life corresponds to the mean retention of the administered molecule in the circulation. This leads to an increase in time (MRT).

[0204] In some embodiments, the PD-1 binding molecule of the present invention comprises a mutant Fc region, and the above The mutant Fc region contains at least one amino acid modification compared to the wild-type Fc region, and therefore The above molecule has an increased half-life (compared to the wild-type Fc region).

[0205] In some embodiments, the PD-1 binding molecule of the present invention comprises a mutant Fc region, and the above The variant 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 One selected from the group consisting of 424, 428, 433, 434, 435 and 436 It contains amino acid substitutions at multiple positions to extend the half-life. It increases the half-life of molecules containing the Fc region. Numerous specific mutations that can occur are known in the art, for example, M25 Examples include 2Y, S254T, T256E, and combinations thereof. For example: U.S. Patent Patent No. 6,277,375; US Patent No. 7,083,784; US Patent No. 7,217, Patent No. 797; U.S. Patent No. 8,088,376; U.S. Published Patent No. 2002 / 0147311 U.S. Patent Publication No. 2007 / 0148164; and International Publication No. 98 / 23289 International Publication No. 2009 / 058492; and International Publication No. 2010 / 033279 ( These refer to the mutations described herein (which are incorporated in their entirety by reference). Molecules containing Fc regions with enhanced decay time include Fc region residues 250, 252, 254, and 2 56, 257, 288, 307, 308, 309, 311, 378, 428, 433, 4 Substitutions in two or more of 34, 435 and 436, particularly T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428 Having two or more substitutions selected from L, N434A, H435K, and Y436I This can also be mentioned.

[0206] In one specific embodiment, the variant Fc region includes 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: (A) One or more mutations that alter the effector function and / or FcγR; and to (B) One or more mutations that prolong the serum half-life It also includes variant Fc regions, including [the specified region].

[0208] VI. Bispecific anti-human PD-1 binding molecules One embodiment of the present invention can be coupled to a "first epitope" and a "second epitope". Regarding the bispecificity binding molecule, the first epitope mentioned above is the epitope of human PD-1. Therefore, the second epitope mentioned above is the same or a different epitope of PD-1. Alternatively, they may be present on the surface of immune cells (e.g., T lymphocytes) and play a role in regulating immune checkpoints. It is an epitope of another molecule involved. In one embodiment, the second epitope is B7 -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‐ 3 is an epitope of VISTA. In one embodiment, the second epitope is PD- It is not epitope 1. In one specific embodiment, the second epitope is CD13 7. CTLA-4, LAG-3, OX40, TIGIT, or TIM-3. Specific actual In the application form, the bispecific molecule has three or more epitope binding sites. Heavily specific molecules are two or more different epitopes of LAG-3, and components that are not LAG-3. It may bind to at least one of its children's epitopes.

[0209] The present invention relates to PD-1 and the above second epitopes (e.g., B7-H3, B7-H4, BT LA, CD40, CD80, CD86, CD137, CTLA‐4, ICOS, KIR, (LAG-3, MHC Class I or II, OX40, PD-L1, TCR, TIM-3, etc.) It includes bispecific antibodies that can simultaneously bind to PD-1 and The bispecific antibody described above, which can simultaneously bind to the second epitope, is described in International Publication No. 1998 / Publication No. 002463, International Publication No. 2005 / 070966, International Publication No. 2006 / 1077 Issue 86, International Publication No. 2007 / 024715, International Publication No. 2007 / 075270, International Publication No. 2006 / 107617, International Publication No. 2007 / 046893, International Publication Publication No. 2007 / 146968, International Publication No. 2008 / 003103, International Publication No. 200 Issue No. 8 / 003116, International Publication No. 2008 / 027236, International Publication No. 2008 / 02 Publication No. 4188, International Publication No. 2009 / 132876, International Publication No. 2009 / 018386 International Publication No. 2010 / 028797, International Publication No. 2010028796, International Publication Publication No. 2010 / 028795, International Publication No. 2010 / 108127, International Publication No. 201 Issue 0 / 136172, International Publication No. 2011 / 086091, International Publication No. 2011 / 13 Publication No. 3886, International Publication No. 2012 / 009544, International Publication No. 2013 / 003652 International Publication No. 2013 / 070565, International Publication No. 2012 / 162583, International Publication No. 2012 / 156430, International Publication No. 2013 / 174873, and International Publication The above document is produced using any of the methods described in No. 2014 / 022540. Each of these is incorporated herein by reference in its entirety.

[0210] A. Bispecific diabody lacking an Fc region One embodiment of the present invention comprises a first polypeptide chain and a second polypeptide chain, and most Preferably composed of the first polypeptide chain and the second polypeptide chain, Regarding the isomer diabody, its sequence is such that the polypeptide chains are covalently bonded to each other. , the first epitope and the second epitope (these epitopes are not identical to each other) This makes it possible to form a covalent diamond body that can be bonded simultaneously. The dual-specificity diabody is "VL1" / "VH1" which can bind to the first epitope mentioned above. The domain and the "VL2" / "VH2" domains that can bind to the second epitope mentioned above. To be prepared. The notations "VL1" and "VH1" are used to describe such a bispecific diabody. This refers to the variable light chain domain and variable heavy chain domain that bind to the "first" epitope of i. Similarly, the notations "VL2" and "VH2" refer to such a dual-singularity diabody. This refers to the variable light chain domain and variable heavy chain domain that bind to the "second" epitope. Whether a particular epitope is designated as a first epitope or a second epitope. This is irrelevant, and this notation is not related to the domain of the polypeptide chain of the binding molecule of the present invention. It is also related to existence or orientation. In one embodiment, one of such epitopes is It is an epitope of PD-1, and another such epitope is the epitope of PD-1. Not a pitope (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, C) D86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC Class I or (These are epitopes such as II, OX40, PD-L1, TCR, and TIM-3).

[0211] The VL domain of the first polypeptide chain described above is the VH domain of the second polypeptide chain described above. It interacts with the first antigen (i.e., PD-1 or the antigen containing the second epitope mentioned above). ) forms a first functional antigen binding site specific to ). Similarly, the second polypeptide chain The VL domain interacts with the VH domain of the first polypeptide chain to form the second antigen. A second functional antigen specific to (i.e., an antigen containing the second epitope or PD-1) They form a binding site. Therefore, the VL and VH of the first and second polypeptide chains. The main selection is that the two polypeptide chains of the above diamond body together constitute PD-1. It comprises VL and VH domains that can bind to both the epitope and the second epitope described above. (In other words, these are VL PD‐1 / VH PD‐1 and VL2 / VH2 (where PD-1 is "the first (first) (is an epitope), or VL1 / VH1 and VL PD‐1 / VH PD‐1 ( Here, PD-1 is the "second epitope" (including).

[0212] In an embodiment having such a bispecific diabody, the first polypeptide chain is N-terminus From the C-terminus toward the N-terminus; monoclonal capable of binding to the first or second epitope mentioned above. The VL1 domain of the Nal antibody (i.e., VL PD‐1 or VL Epitope 2 ); First intervening spacer Petit (linker 1); (the first polypeptide chain above is VL) PD‐1 (If it contains) The second epitope, or (the first polypeptide chain mentioned above is VL Epitope 2 If it contains (i) The VH2 domain of a monoclonal antibody that can bind to the first epitope; optionally, the system A second intervening spacer peptide (linker 2) containing an in residue; heterodimer-promoting dormant Including the yin and C-terminus (Figure 1).

[0213] The second polypeptide chain of this embodiment of the bispecific diabody is N-terminus to C-terminus In the direction of: N-terminus; monoclonal antibody capable of binding to PD-1 or the second epitope mentioned above. The body's VL2 domain (i.e., VL PD‐1 or VL Epitope 2 ; and the above VL domain is the above da Selected not to be included in the above first polypeptide chain of the earbody); first intervening space - Sapeptide (linker 1); (the second polypeptide chain above is VL PD‐1 If it contains (i) The second epitope described above, or (i) The second polypeptide chain described above is VL Epitope 2 Contains (If applicable) VH1 domain of monoclonal antibody capable of binding to PD-1; optionally cyst A second intervening spacer peptide (linker 2) containing a residue; heterodimer-promoting domain n; and including the C-terminus (Figure 1).

[0214] Most preferably, an intervening linker peptide separating the VL domain and the VH domain as described above. The length of (for example, linker 1) is determined by the relative lengths of the VL and VH domains of the polypeptide chain. The first polyp The VL and VH domains of the ptyde chain can be substantially or completely unable to bind to each other. No. Similarly, the VL and VH domains of the second polypeptide chain bind to each other. This is substantially or completely impossible. A preferred intervening spacer peptide (linker 1) is distributed The column (sequence number 14) contains GGGSGGGG.

[0215] The length and composition of the second intervening linker peptide (linker 2) described above promote heterodimerization. Selected based on domain selection. Typically, the second intervening linker peptide ( Linker 2) contains 3 to 20 amino acid residues, particularly the heterodimer-promoting domain. If it does not contain a cysteine ​​residue, a cysteine-containing second intermediary linker peptide (linker -2) is used. The cysteine-containing second intervening spacer peptide (linker 2) is 1 It contains one, two, three, or four or more cysteine ​​molecules. Preferred cysteine-containing spacer The peptide (linker 2) has the sequence of SEQ ID NO: 15: GGCGGG. Alternatively, linker 2 does not contain cysteine ​​(for example, GGG, GGGS (sequence number 29), LGGGSG (sequence number 26) 1) GGGSGGGSGGG (SEQ ID NO: 262), ASTKG (SEQ ID NO: 30), LEPKSS (SEQ ID NO: 33) ), APSSS (SEQ ID NO: 34), etc., and the cysteine-containing heterodimer-promoting drugs described below The main component is used. Optionally, cysteine-containing linker 2 and cysteine-containing heterodimer are used. Both of the body-promoting domains are used.

[0216] The heterodimer-promoting domain has GVEPKSC (SEQ ID NO: 16) on one of the polypeptide chains or This is VEPKSC (SEQ ID NO: 17) or AEPKSC (SEQ ID NO: 18), and on the other polypeptide chain Includes GFNRGEC (SEQ ID NO: 19) or FNRGEC (SEQ ID NO: 20) (US 2007 / 0 No. 004909).

[0217] However, more preferably, the heterodimer-promoting domain of such a diabody is , containing at least 6, at least 7, or at least 8 amino acid residues, opposite pole It is formed from one, two, three, or four tandem repeat coil domains, thereby on The heterodimer-promoting domain has an effective 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 complete repeats or may have substitutions. For example, the coil domain of the heterodimer-promoting domain of the first polypeptide chain is as described above. Eight amino acid residues selected to give a negative charge to the telodimer-promoting domain The sequence may include the coiled heterodimer-promoting domain of the second polypeptide chain. The main focus is on eight elements selected to impart a positive charge to the heterodimer-promoting domains mentioned above. It may contain a sequence of amino acid residues (or vice versa). When using the yl on the other polypeptide chain, which of the first or second polypeptide chains Which coil is used is not important. Positively charged amino acids are lysine, arginine, and histamine. The thidine may be, and / or the loaded amino acid may be glutamic acid, aspartic acid, etc. It may be. The positively charged amino acid is preferably lysine, and / or the uncharged amino acid is Preferably, it is glutamic acid. (Such domains inhibit homodimerization and thus Therefore, to promote heterodimerization, only a single heterodimer-promoting domain can be employed. However, both the first and second polypeptide chains of the diabody of the present invention promote heterodimerization. It is preferable that it contains a domain.

[0219] In a preferred embodiment, one of the heterodimer-promoting domains is a tandem " E-coil spiral domain (SEQ ID NO: 21) E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E Equipped with K) The glutamate residue forms a negative charge at pH 7, and on the other hand, hetero The other of the dimer-promoting domains consists of four tandem "K-coil" domains (sequence number) No. 22: K VAAL K E- K VAAL K E- K VAAL K E- K VAAL K E) is present, and its lysine residue is at pH 7. This forms a positive charge. Due to the presence of such charged domains, the first polypeptide and Linking with the second polypeptide is promoted, and therefore heterodimer formation is promoted. Of the four tandem "E-coil" helical domains of Sequence 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 (Sequence ID 23) It is a heterodimer-promoting domain that has been modified to do so. Similarly, particularly preferred In sequence number 22, one of the four tandem "K-coil" helical domains is cis Thein residues: K VAA CK E- K VAAL K E- K VAAL K E- K VAAL K Modified to include E (Sequence ID 24) This is the heterodimer-promoting domain, which is being used as a decorative element.

[0220] As disclosed in International Publication No. 2012 / 018687, Diabody's invitation To improve the pharmacokinetic properties, the diabody is modified by connecting one of the ends of the diabody or It may be modified to contain multiple polypeptide portions of serum-binding protein. In other words, the polypeptide portion of such serum-binding proteins is the C-terminus of the diabody. It will be placed there. Albumin is the most abundant protein in plasma, and in humans It has a half-life of 19 days. Albumin has multiple small molecular binding sites, and Albumin can extend its serum half-life by non-covalently binding to other proteins. Streptococcal strains The albumin-binding domain 3 (ABD3) of protein 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). Therefore, to improve the in vivo pharmacokinetic properties of Diabody, serum-binding proteins in particular The preferred polypeptide moiety is the albumin-binding domain (A) from streptococcal protein G. BD), and more preferably, the Streptococcus disgalactie strain G148 Albumin-binding domain 3 of protein G (ABD3) (SEQ ID NO: 25): LAEAKVLANR ELD KYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

[0221] Disclosed in International Publication No. 2012 / 162068 (incorporated herein by reference) As shown, the "deimmunized" variant of sequence number 25 is MHC It has the ability to attenuate or eliminate class II binding. As a result of combined mutations. Based on this, the following combinations of substitutions are favorable for forming such deimmunized ABD. This is considered a reasonable substitution: 66D / 70S+71A; 66S / 70S+71A; 6 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 Having decorative elements L64A, I65A and D79A, or modified elements N66S, T70S and D79A Mutant ABD. Amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI D 66 NAK S 70 A 71 EGVKALIDE ILAALP (Sequence ID 26) Or amino acid sequence: LAEAKVLANR ELDKYGVSDY YKN A 64 A 65 NNAKT VEGVKALI A 79 E ILAALP (Sequence ID 27) Or amino acid sequence: LAEAKVLANR ELDKYGVSDY YKNLI S 66 NAK S 70 VEGVKALI A 79 E ILAALP (Sequence ID 28) Mutant deimmunized ABD is particularly preferred because such deimmunized ABD is M This is because it exhibits nearly wild-type binding while providing attenuation of HC class II binding. The first polypeptide chain of such a diamond body having ABD is preferably Positioned at the C-terminus for the E-coil (or K-coil) domain of a polypeptide chain like the one shown. By doing so, the above E coil (or K coil) domain and ABD (this is preferable) It contains a peptide linker that intervenes between (this is deimmunized ABD). The preferred sequence for the peptide linker is SEQ ID NO: 29:GGGS.

[0222] B.Fc region-containing bispecific diabody One embodiment of the present invention relates to PD-1 and a second epitope (e.g., B7-H3, B7-H3). 4, BTLA, CD40, CD80, CD86, CD137, CTLA‐4, ICOS, KIR, LAG-3, MHC Class I or II, OX40, PD-1, PD-L1, TC A bispecific diabody having an Fc region that can simultaneously bind to R, TIM-3, etc. The IgG CH2-CH3 is added to one or both of the above diabody polypeptide chains. By adding a domain, the Fc region is formed by the complexation of the above diabody chains. This increases the biological half-life of the above-mentioned diabody and / or changes its valency. When the IgG CH2-CH3 domain is incorporated into both of the diabody polypeptides, 2 This enables the formation of a diabody containing a chain-double specific Fc region (Figure 2).

[0223] Alternatively, one of the above diabody polypeptides may contain an IgG CH2-CH3 domer. By incorporating the in, it becomes possible to form more complex 4-chain bispecific Fc region-containing diamond bodies. (Figures 3A-3C). Figure 3C shows the steady-state light chain (CL) domain and the steady-state heavy chain CH1 domain. This shows a typical 4-chain diamond body having such a domain and other poly Peptides may be used instead (for example, Figures 3A and 3B, U.S. Patent Publication No. 2013- U.S. Published Patent No. 0295121; U.S. Published Patent No. 2010-0174053 and U.S. Published Patent No. 20 09-0060910; European Published Patent No. 2714079; European Published Patent No. 26012 No. 16; European Publication No. 2376109; European Publication No. 2158221, and International Publication No. 2012 / 162068; International Publication No. 2012 / 018687; International Publication No. 2 (See issue 010 / 080538). Therefore, for example, instead of the CH1 domain, human Ig The amino acid sequences GVEPKSC (SEQ ID NO: 16) and VEPKSC (SEQ ID NO: 16) are derived from the hinge domain of G. 7) A peptide having AEPKSC (SEQ ID NO: 18) may be used, and the CL domain Instead, the C-terminal 6 amino acids of the human κ light chain, GFNRGEC (Sequence ID 19) or FNRGEC (Sequence ID 19) Particle number 20) may be adopted. A representative peptide containing a 4-chain diabody is shown in Figure 3A. Alternatively, or furthermore, a tandem coil domain of opposing charges, for example, an "E coil". Helical domain (SEQ ID NO: 21) E VAAL E K- E VAAL E K- E VAAL E K- E VAAL E K or Sequence ID 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 Sequence ID 24: K VAA CK E- K VAAL K E- K VAAL K E- K VA AL K A peptide having E) may be used. A typical coiled diabody containing a 4-chain diabody. The main part is shown in Figure 3B.

[0224] The Fc region-containing diabody molecule of the present invention is generally an intervening linker peptide (linker). This includes. Typically, this additional linker contains 3 to 20 amino acid residues. Additional or alternative linkers that can be used in Fc region-containing diabody molecules include: :GGGS (Sequence ID 29), LGGGSG (Sequence ID 261), GGGSGGGSGGG (Sequence ID 262) ASTKG (SEQ ID NO: 30), DKTHTCPPCP (SEQ ID NO: 31), EPKSCDKTHTCPPCP (SEQ ID NO: 30) 2) LEPKSS (SEQ ID NO: 33), APSSS (SEQ ID NO: 34), and APSSSPME (SEQ ID NO: 35 Examples include ), LEPKSADKTHTCPPC (sequence number 36), GGC, and GGG. Cloning is possible. To simplify the process, Sequence ID No. 33 may be used instead of GGG or GGC. Furthermore, amino acid G By following GG or EQUAL SEQ ID NO: 31 immediately after EQUAL SEQ ID NO: 33, an alternative linker: GGGD KTHTCPPCP(sequence code 263); and LEPKSSDKTHTCPPCP(sequence code 37) may be formed. The Fc region-containing diabody molecule of the present invention can be used in addition to or in place of a linker. An IgG hinge region may be incorporated. An example hinge region is: EPKS from IgG1. CDKTHTCPPCP(SEQ ID NO: 32); ERKCCVECPPCP(SEQ ID NO: 11); IgG from IgG2 Includes ESKYGPPCPSCP (SEQ ID NO: 12) from 4 and stabilizing substitutions to reduce chain exchange. One example is ESKYGPPCPPCP (SEQ ID NO: 13), which originates from the IgG4 hinge mutation.

[0225] As shown in Figures 3A-3C, the diamond body of the present invention includes four different chains. That's fine. The first and third polypeptide chains of such a diamond body have four domains: (i) VL1-containing domain; (ii) VH2-containing domain; (iii) heterodimerization promotion Domains; and domains containing (iv)CH2-CH3 sequences. Second and The polypeptide chain of 4 consists of: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) Contains a heterodimer-promoting domain, wherein the heterodimer-promoting domain is the first / Promotes dimerization between the third polypeptide chain and the second / fourth polypeptide chain. and the VL and / or VH domains of the fourth polypeptide chain, and the first and second polypeptides The VL and / or VH domains of the cytoplasmic chain may be the same or different, and so This allows for tetravalent bonding with single, bivalent, or quadruple specificity. Notation: "VL3" And "VH3" are coupled to the "third" epitope of such a diamond body, respectively. This refers to variable light chain domains and variable heavy chain domains. Similarly, the notations "VL4" and "VH4" are used. Each of these is a variable light chain that binds to the "fourth" epitope of such a diabody. This refers to the in and variable heavy chain domains. A typical four-chain Fc region-containing diamond body of the present invention The general structure of the lipeptide chain is shown in Table 2.

[0226] [Table 4]

[0227] In one specific embodiment, the diamond body of the present invention consists of a total of four polypeptide chains. It has bispecificity, is tetravalent (i.e., has four epitope-binding sites), and contains Fc. (Figures 3A-3C) The bispecific, tetravalent, Fc-containing diamond body of the present invention is PD -1 has two immune-specific epitope binding sites (these are the same epitopes of PD-1 or (It can bind to different epitopes of 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 possesses two epitope binding sites specific to CR, TIM-3, etc.

[0228] In a further embodiment, the diabody containing the dual specificity Fc region comprises three polypeptides It may contain a chain. The first polypeptide of such a diamond body has three domains: (i) VL1-containing domain; (ii) VH2-containing domain; and (iii) CH2-CH It contains a domain containing 3 sequences. The second polypeptide of such a diabody is :(i) VL2-containing domain; (ii) VH1-containing domain; and (iii) the above-mentioned domain Domains that promote heterodimerization and covalent bonding with the first polypeptide chain of the abody It contains. The third polypeptide of such a diabody contains a CH2-CH3 sequence. Therefore, the first and second polypeptide chains of such a diamond body are linked together. The VL1 / VH1 binding site that can bind to the first epitope, and the second epitope The first and second polypeptides form a VL2 / VH2 binding site that can bind to the chain. These are connected by disulfide bonds involving cysteine ​​residues in each of the third domains. They bind together. In particular, the first and third polypeptide chains complex together, disulfide It forms an Fc region stabilized by filobonds. Such a diabody has strength This has been enhanced. Figures 4A and 4B show the structure of such a diamond body. The Fc region-containing bispecific diabody has either of the two orientations (Table 3) good.

[0229] [Table 5]

[0230] In one specific embodiment, the diamond body of the present invention consists of a total of three polypeptide chains. It has bispecificity, is divalent (i.e., has two epitope-binding sites), and contains Fc. (Figures 4A-4B) The bispecific, divalent Fc-containing diabody of the present invention is PD- 1. An immunospecific epitope binding site, 2. An 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 possesses a single epitope binding site specific to TIM-3, etc.

[0231] In a further embodiment, the diabody containing the bispecific Fc region has a total of five polyp It may include a butyl chain. In a particular embodiment, two of the five polypeptide chains described above. One has the same amino acid sequence. The first polypeptide chain of such a diabody is (i) VH1-containing domain; (ii) CH1-containing domain; and (iii) CH2-C It contains a domain containing an H3 sequence. The first polypeptide chain described above contains VH1 and a heavy chain. The heavy chain of an antibody containing a constant region may be the second and fifth of such a diabody. The polypeptide chain contains: (i) a VL1-containing domain; and (ii) a CL-containing domain. The second and / or fifth polypeptide chains of such a diamond body are the first / The light chain of the antibody contains VL1, which is complementary to VH1 of the third polypeptide chain. i. The first, second and / or fifth polypeptide chains described above can be isolated from naturally occurring antibodies. These can be constructed by rearranging. The polypeptide chain consists of: (i) VH1-containing domain; (ii) CH1-containing domain; (ii i) Domains containing CH2-CH3 sequences; (iv) Domains containing VL2; (v) VH 3. Containing a heterodimer-promoting domain; and (vi) a heterodimer-promoting domain. The advanced domain promotes the dimerization of the third and fourth chains. The fourth polypeptide of the body consists of: (i) a VL3-containing domain; (ii) a VH2-containing domain. (iii) Heterodimation of the above-mentioned diamond body with the third polypeptide chain and It contains a domain that promotes covalent bonding.

[0232] Therefore, the first and second polypeptide chains of such a diamond body and the third and The fifth polypeptide chain consists of two VLs that can link together and bind to the first epitope. The 1 / VH1 binding site is formed. The third and fourth polypept of such a diamond body The cytoplasmic chains are linked to each other, forming VL2 / VH2 binding sites that can bind to a second epitope, and The first and third epitopes form VL3 / VH3 binding sites. The polypeptide is formed by disulfide bonds involving cysteine ​​residues in each constant region. They then bond to each other. In particular, the first and third polypeptide chains complex together. , forming an Fc region. Such a diamond body has increased strength. Figure 5 shows this The following diamond body structures are shown: VL1 / VH1, VL2 / VH2, and VL3 / VH The three domains may be the same or different, which can result in single specificity or dual specificity. It will be understood that binding with sex or triple specificity is possible. However, As presented in the details, these domains preferably involve PD-1 in the second epidemic. Taupe (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, C) D137, CTLA-4, ICOS, KIR, LAG-3 MHC Class I or II, O Selected to be coupled to X40, PD-L1, TCR, TIM-3, etc.

[0233] The VL and VH domains of the polypeptide chain described above are VL / V specific to the desired epitope. Selected to form an H-binding site. V formed by linking the polypeptide chains described above. The L / VH binding sites may be the same or different, which can result in single specificity, or two specificity. This enables tetravalent bonding with heavier specificity, triplicate specificity, or quadruple specificity. In particular, the above VL and V The H domain has a bispecific diabody with two binding sites for the first epitope and and two binding sites related to the second epitope, or three binding sites related to the first epitope One binding site relating to the site and the second epitope, or (as shown in Figure 5) the first epitope Two binding sites relating to the pitope, one binding site relating to the second epitope and the third It may be selected to have one binding site related to an epitope. A typical 5-chain of the present invention. Table 4 shows the general structure of the polypeptide chain of the Fc region-containing diabody.

[0234] [Table 6]

[0235] In one specific embodiment, the diabody of the present invention has two aspects relating to the first epitope A total of five polypeptides, each having two binding sites related to a binding site and a second epitope. Consists of a chain, bispecific, tetravalent (i.e., has four epitope-binding sites), and contains Fc. It is a diabody. In one embodiment, the present invention is a bispecific, tetravalent, Fc-containing diabody. i is an immuno-specific epitope binding site on PD-1 (these are the same epitope binding sites on PD-1). (May bind to a pitope or a different epitope of 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 to D-L1, TCR, TIM-3, etc. In the embodiment, the bispecific, tetravalent, Fc-containing diabody of the present invention is immunospecific to PD-1. Three distinct epitope binding sites (these are the same epitope of PD-1 or PD-1) (may bind to different epitopes), 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 It has one epitope binding site specific to IM-3, etc. In another embodiment, the present invention The bispecific, quadrivalent, Fc-containing diabody is an immunospecific single epithet for PD-1. The binding site and the second epitope (e.g., B7-H3, B7-H4, BTLA, CD4) 0, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3 3 specific to MHC class I or II, OX40, PD-L1, TCR, TIM-3, etc. It has several epitope-binding sites.

[0236] Bispecific trivalent bond molecule containing a C.Fc region Further embodiments of the present invention include an Fc region comprising a first epitope, a second epitope and Regarding bispecific, trivalent binding molecules that can simultaneously bind to a third epitope, the above At least one of the epitopes is not identical to another. Therefore, such a double The specificity diabody consists of "VL1" / "VH1" domains that can bind to the first epitope. , a "VL2" / "VH2" domain that can bind to a second epitope, and a third epitope It has a "VL3" / "VH3" domain that can bind to the pyto. In one embodiment, the above epitome One or two of the epitopes are PD-1 epitopes, and one of the above epitopes is Those (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 (These are epitopes such as TIM-3). Such bispecific trivalent binding molecules have three ethers. A die equipped with pitope bonding sites, two of which provide bonding site A and bonding site B. It is a diabody-type binding domain, and one of them is a non-diabody-type binding domain that provides binding site C. The main part (for example, Figures 6A-6F, and PCT application No. PCT / US15 / 33081) (See the number and PCT application PCT / US15 / 33076).

[0237] Typically, the trivalent bond molecule of the present invention comprises four different polypeptide chains (Figure 6A~ (See 6B) However, the above molecules, for example, connect these polypeptide chains (for example, by peptide bonds) By fusing them together, or by "dividing" these polypeptides By forming additional polypeptide chains (ing), or by adding fewer or more By linking additional polypeptide chains with disulfide bonds, a smaller number or more It can contain a large number of polypeptide chains. Figures 6B-6F show three polypeptide chains. This aspect of the present invention is illustrated by schematically showing the molecules it contains. Figures 6A-6 As presented in F, the trivalent binding molecule of the present invention has the above-mentioned diabody-type binding domain This is the N-terminus (Figures 6A, 6C, and 6D) or C-terminus (Figures 6B, 6E, and 6F) of the Fc region. They may have alternating orientations such as ).

[0238] In a particular embodiment, the first polypeptide chain of such a trivalent bond molecule of the present invention is: i) VL1-containing domain; (ii) VH2-containing domain; (iii) heterodimer-promoting domain Main; and (iv) Contains a domain containing a CH2-CH3 sequence. The above VL1 and The VL2 domain is as shown in Table 5 (Figures 6A and 6B), the above CH2-CH3 The second polyp of such an embodiment is located at the N-terminus or C-terminus of the containing domain. The ptide chain consists of: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) It contains a heterodimer-promoting domain. The third polypeptide chain of such an embodiment is: (i) VH3-containing domain; (ii) CH1-containing domain; and (iii) CH2-CH It contains a domain containing sequence 3. The above third polypeptide chain contains VH3 and heavy chain The heavy chain of an antibody containing a normal region may be such a fourth polypeptide. (i) Contains a VL3-containing domain; and (ii) Contains a CL-containing domain. The fourth point above The lipeptide chain contains VL3 which is complementary to VH3 of the third polypeptide chain described above. It may be the light chain of an antibody. The third or fourth polypeptide chain described above may be a naturally occurring antibody or They can be isolated by recombinant, synthesis, or other means. It can be structured like this.

[0239] The variable light chain domains of the first and second polypeptide chains described above are provided by an intervening spacer linker. This is separated from the variable heavy chain domain of the polypeptide chain, and the above-mentioned intervening spacer The linker integrates these VL1 / VH2 (or these VL2 / VH1) domains into one unit. This allows for the formation of an epitope-binding site that can bind to a first or second epitope. It has a length that is too short to be effective. For this purpose, a preferred intervening spacer peptide ( Linker 1) has the sequence (SEQ ID NO: 14): GGGSGGGG. Other than the above trivalent bond molecule The main component is one or more intervening spacer peptides, which optionally contain cysteine ​​residues. They may be separated. Exemplary linkers useful for the formation of trivalent bond molecules are presented herein. It has been done, and also PCT application No. PCT / US15 / 33081; and PCT application No. This is also presented in T / US15 / 33076. The second polypeptide chain is linked together and can bind to the first epitope VL1 / It forms a VH1 binding site and a VL2 / VH2 binding site that can bind to a second epitope. The third and fourth polypeptide chains of such a trivalent bond molecule are linked together, forming the third It forms a VL3 / VH3 binding site that can bind to an epitope. VL1 / VH1, VL2 / The VH2 and VL3 / VH3 domains may be the same or different, and this allows for a simple It will be understood that monospecific, bispecific, or triplicate binding is possible.

[0240] As described above, the trivalent bond molecule of the present invention may contain three polypeptides. The trivalent bond molecule containing the lipeptide chain has a fourth polypeptide N-terminal domain connected to the third poly It can be obtained by linking to the VH3-containing domain of the peptide. Alternatively, The following three domains: (i) VL3-containing domain; (ii) VH3-containing domain; and ( iii) The third trivalent bond molecule of the present invention containing a domain containing a CH2-CH3 sequence The polypeptide chain is used, and here VL3 and VH3 are formed by linking these domains. Sufficient length (at least 9 or more meshes) to allow the formation of an epitope binding site. They are separated from each other by intervening spacer peptides that have anoacid residues.

[0241] The VL1 / VH1, VL2 / VH2, and VL3 / VH3 domains are the same but different. This can be done, allowing for single-specific, bispecific, or triplicate binding. It will be understood that this is the case. However, as presented herein, this These domains are preferably PD-1 and the second epitope (or the second and third epitope) Selected to combine (preferably, the epitope is 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 (It is an epitope of IM-3, etc.)

[0242] In particular, the VL and VH domains have a trivalent binding molecule with two bindings related to the first epitope. Site and one binding site relating to the second epitope, or one relating to the first epitope The binding site and two binding sites relating to the second epitope, or relating to the first epitope One binding site, one binding site relating to the second epitope, and one relating to the third epitope. A polyparticle containing one binding site may be selected. A typical trivalent bond molecule of the present invention The general structure of the butyl chain is shown in Figures 6A to 6F and Table 5.

[0243] [Table 7]

[0244] One embodiment of the present invention relates to two epitope-binding sites for PD-1, and PD-1 External molecules (e.g., B7-H3, B7-H4, BTLA, CD40, CD80, CD86, CD137, CTLA-4, ICOS, KIR, LAG-3, MHC Class I or II, Regarding the second epitope present on OX40, PD-L1, TCR, TIM-3, etc. This relates to a bispecific trivalent binding molecule possessing a single epitope binding site, concerning PD-1. The two epitope-binding sites may bind to the same epitope or to different epitopes. Another embodiment of the present invention relates to one epitope binding site for PD-1, and 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 Two findings concerning the second antigen present on (OX40, PD-L1, TCR, TIM-3, etc.) This relates to a bispecific trivalent binding molecule having an epitope binding site. The two epitope binding sites are either the same epitope or different epitopes of the above antigen. For example, it may bind to the same or different epitopes of LAG-3. As mentioned above, this Such bispecific trivalent bond molecules may contain three or four polypeptide chains.

[0245] VII. Steady Domains and Fc Regions Presented here are the PD-1 binding molecules of the present invention (e.g., antibodies, diabodies, 3 This is an antibody constant domain useful for generating valence-binding molecules, etc.

[0246] The preferred CL domain is the human IgGCLκ domain. An example is the human IgG C The amino acid sequence of the Lκ domain is (SEQ ID NO: 8): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC That is the case.

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

[0248] As presented herein, the PD-1 binding molecule of the present invention comprises an Fc region. Good. The Fc region of such a molecule of the present invention may be any isotype (e.g., IgG1 The PD-1 binding molecule of the present invention may be IgG2, IgG3, or IgG4. Furthermore, it may include a CH1 domain and / or a hinge region. If a hinge region exists, the above CH1 domain and / or hinge region are either It may be an isotype (e.g., IgG1, IgG2, IgG3, or IgG4). Preferably, it is of the same isotype as the desired Fc region.

[0249] An exemplary CH1 domain is the 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 That is the case.

[0250] An exemplary CH1 domain is the 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 That is the case.

[0251] The exemplary CH1 domain is the 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 That is the case.

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

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

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

[0255] The Fc region of the Fc region-containing molecule of the present invention (e.g., antibody, diabody, and trivalent molecule) is Even if it is a complete Fc region (e.g., a complete IgG Fc region), or even if it is only a fragment of an Fc region It may be present. Optionally, the Fc region of the Fc region-containing molecule of the present invention may be a C-terminal lysine amino acid. It does not contain residues. In particular, the Fc region of the Fc region-containing molecule of the present invention is a modified mutant Fc region. It may be a region. The Fc region of the bispecific Fc region-containing molecule of the present invention is one or more F It may have the ability to bind to c receptors (e.g., one or more FcγRs), but more preferably Alternatively, the above mutant Fc region (in contrast to the binding exhibited by the wild-type Fc region) FcγRIA (CD64), FcγRIIA(CD32A), FcγRIIB(CD32B), Fcγ The binding to RIIIA (CD16a) or FcγRIIIB (CD16b) changes. They are either dolphins, or have reduced ability to bind to one or more inhibitory receptors or It does not possess the above capabilities. Therefore, the Fc region of the bispecific Fc region-containing molecule of the present invention is complete Some or all of the CH2 domains and / or the CH3 domains in the Fc region This may include some or all of the following, or (for example, CH2 in the complete Fc region) This involves one or more insertions and / or one or more deletions into the CH3 domain. It may include mutant CH2 and / or mutant CH3 sequences (which may contain mutations). The Fc region may comprise a non-Fc polypeptide portion, or a complete Fc region that does not occur naturally. It may have a portion of the region, or a non-spontaneous distribution of the CH2 and / or CH3 domains. It may have directions (for example, two CH2 domains or two CH3 regions, or N-terminus). In the direction from the end to the C-terminus, there is the CH3 domain and the CH2 domain which is linked to it, etc. ).

[0256] Fc domain modification, which is expressed as a modified effector function, is known in the art. This involves modifications that increase binding to the activating receptor (e.g., FcγRIIA(CD1 6A)), and modifications that reduce binding to inhibitory receptors (e.g., FcγRIIB(C) D32B)) is included (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,” (see Cancer Res. 57(18):8882-8890). Reduced binding to CD32B, and / Alternatively, an exemplary mutant of the human IgG1 Fc region with increased binding to CD16A is F2. It contains substitutions of 43L, R292P, Y300L, V305I or P296L. Amino acid substitutions, in any combination or partial combination, occur within the human IgG1 Fc region. It may be present in one embodiment. In one embodiment, the above human IgG1 Fc region mutant is F243L, It contains R292P and Y300L substitutions. In another embodiment, the above human IgG1 Fc Regional variants involve the substitutions F243L, R292P, Y300L, V305I, and P296L. 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 (For the binding exhibited by the G1 Fc region (SEQ ID NO: 1) FcγRIA(CD64), Fc γRIIA(CD32A), FcγRIIB(CD32B), FcγRIIIA(CD1 6a) Binding to FcγRIIIB(CD16b) is reduced (or partially bound to these) (Not matching) is preferable. A variant Fc region and a mediating entity for such altered binding. The form of mutation is described above. In one specific embodiment, the Fc region of the present invention The region-containing molecule has an IgG Fc region with reduced ADCC effector function. In a more accurate embodiment, the first and / or third polypeptide chains of such an Fc region-containing molecule The CH2-CH3 domain is substituted with the following: L234A, L235A, N297Q, and It includes one, two, or three of the N297G. In another embodiment, the above human I gG Fc region variants include N297Q substitution, N297G substitution, L234A, and L235A. It contains substitutions or D265A substitutions, but these mutations cause the loss of FcR binding. This is because of the binding exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 1). (and) its binding to FcγRIIIA(CD16a) is inherently low (or hardly binding at all), And / or utilize the CH2-CH3 domains in the Fc region, which inherently have low effector functionality. In a specific embodiment, the Fc region-containing molecule of the present invention is an IgG2 Fc region (SEQ ID NO: 2). ) or comprises an IgG4 Fc region (SEQ ID NO: 4). When using the IgG4 Fc region The present invention relates to stabilization features such as the replacement of the hinge region S228P (see, for example, Sequence ID No. 13). This also includes the introduction of natural mutations: N297G, N297Q, L234A, L235A, and D26. 5A substitution eliminates the effector function, therefore, in situations where the effector function is desired, It is preferable not to employ 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, (corresponding field It is preferable that the serum half-life increases (relative to the half-life of the biotype Fc region). The extended period of the variant Fc region and the mutation morphology have been described above. In one embodiment, the first and / or third polypeptide chains of such an Fc region-containing molecule The CH2-CH3 domain is one of the following substitutions: M252Y, S254T, and T256E The present invention further includes one, two, or three of the following: (A) One or more mutations that alter the effector function and / or FcγR; Bini (B) One or more mutations that prolong the serum half-life The present invention includes an Fc region-containing molecule comprising a variant Fc region.

[0259] Preferred IgG1 sequences relating to the CH2 and CH3 domains of the Fc region-containing molecule of the present invention This is the substitution for L234A / L235A / M252Y / S254T / T256E (Sequence ID 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 This includes, where X is either lysine (K) or absent.

[0260] Preferred IgG4 sequences relating to the CH2 and CH3 domains of the Fc region-containing molecule of the present invention This is the substitution for M252Y / S254T / T256E (sequence number 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 This includes, where X is either lysine (K) or absent.

[0261] Regarding diabodies and trivalent bond molecules whose first and third polypeptide chains are not identical , between the CH2-CH3 domains of two first polypeptide chains, or between two third polypeptide chains The ability to reduce or prevent homodimerization between the CH2-CH3 domains of the butyl chain is to reduce or prevent the occurrence of homodimerization between the CH2-CH3 domains of the butyl chain. Desirable. The CH2 and / or CH3 domains of such polypeptide chains are in sequence. They do not need to be identical, and advantageously, to promote complex formation between the two polypeptide chains. Modified. For example, an amino acid substitution (preferably a "k" knob) in the CH2 or CH3 domain. (A bulky side chain group that forms "nob," for example, substituted with an amino acid containing tryptophan.) By introducing this, interaction with similarly mutated domains is prevented due to steric hindrance, and the above mutation The modified domain is subjected to complementary or adaptive mutations (e.g., substitution with glycine). This can be paired with the domain, namely "hole". Mutations occur in any of the polypeptides containing the CH2-CH3 domains that form the Fc region. It can be applied to pairs. A protein that suppresses homodimerization and promotes heterodimerization. The method of processing the material is publicly known in the art, particularly with respect to the processing of immunoglobulin-like molecules. , as included herein (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 See 296:95-101 (each of which is incorporated herein by reference in its entirety). Preferably, the "knob" is processed into the CH2-CH3 domain of the first polypeptide chain, The "hole" is the CH of the third polypeptide chain of the diamond body containing these polypeptide chains. It is processed into a 2-CH3 domain. Therefore, the "knob" is formed when the first polypeptide chain has its CH3 domain. This plays a role in preventing homodimerization via the 2 and / or CH3 domain. Therefore, the third polypeptide chain preferably contains a "hole" substituent, so the first poly It heterodimerizes with the peptide chain and homodimerizes with itself. This strategy is the same as described above. This can be used with respect to diabodies and trivalent bond 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 modified into the CH2-CH3 domain of the third polypeptide chain.

[0262] A preferred knob is formed by modifying the IgG Fc region to include the modifying group T366W. This is how it is generated. The preferred hole modifies the IgG Fc region with the modifying group T366S, L It is produced by incorporating 368A and Y407V. Hole-supported third polyp This assists in the purification of butyl chain homodimers from molecules containing a bispecific heterodimer Fc region. To do this, preferably the hole-supported CH2 and CH3 domains of the third polypeptide chain The protein A binding site is modified by an amino acid substitution at position 435 (H435R). This causes the third polypeptide chain homodimer carrying the whole to be converted to protein A. It does not bind, while the bispecific monovalent Fc diabody is a tangent of the first polypeptide chain. It retains the ability to bind to protein A via the protein A binding site. (Alternative implementation) In this state, the third polypeptide chain supporting the whole has amino acid substitutions at positions 434 and 435. It is acceptable to include it (N434A / N435K).

[0263] The present invention relates to the CH2 and CH3 domains of the first polypeptide chain of the Fc region-containing molecule. The preferred IgG1 amino acid sequence is the "nob-supported" 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 It has such that X is either ricin (K) or absent.

[0264] Fc region having two polypeptide chains (or three, four, or five polypeptide chains) The second poly of the Fc region-containing molecule of the present invention, having a third polypeptide chain of the region-containing molecule. The preferred IgG1 amino acid sequence for the CH2 and CH3 domains of the peptide chain is " "Role-carrying" sequence (sequence number 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 It has such that X is either ricin (K) or absent.

[0265] As mentioned above, the CH2-CH3 domains of SEQ ID NOs. 6 and 7 are due to alanine. This includes a substitution at position 234 and a substitution at position 235 by alanine, and therefore, (wild-type Fc region) (Compared to the binding shown in Sequence ID 1) FcγRIA(CD64), FcγRIIA(CD 32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or Fcγ Fc regions showing reduced (or virtually no) binding to RIIIB(CD16b) The present invention also modifies the effector function and / or FγR binding activity of the Fc region. This also includes such CH2-CH3 domains, including substitutions as alternatives and / or additions. The present invention also further comprises one or more half-life-extending amino acid substitutions, such as C The present invention also includes the H2-CH3 domain. This further includes such hole-supported and knob-supported CH2-CH3 domains.

[0266] The first polypeptide chain has a "knob-supported" CH2-CH3 sequence, such as that of sequence number 6. It is preferable to do so. However, as can be understood, in the first polypeptide chain, "ho A "role-supporting" CH2-CH3 domain (e.g., sequence number 7) can be adopted, in which case "role-supporting" The CH2-CH3 domain (e.g., SEQ ID NO: 6) is a molecule that has two polypeptide chains. In the second polypeptide chain of the Fc region-containing molecule of the invention (or three, four, or five poly It is used in the third polypeptide chain of an Fc region-containing molecule that has a peptide chain.

[0267] As described above, the present invention relates to having wild-type CH2 and CH3 domains, or the substitutions described above. Fc region-containing molecules having CH2 and CH3 domains including a combination of these (e.g., antibodies) It includes the diabody containing the Fc region. Such variants include IgG1 C An example 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 And here: (a) Both X1 and X2 are L (wild type), or both are A (low FcγR binding). (Below) and; (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 (holes); (d) X9 and X 10 These are either N and H (wild type), or N and R (protein type) (without protein A binding), or A and K (without protein A binding); (e)X 11 It is either K or absent.

[0268] In other embodiments, the present invention is described in International Publication No. 2007 / 110205; International Publication No. 20 Issue No. 11 / 143545; International Publication No. 2012 / 058768; International Publication No. 2013 / 0 Disclosed in Patent No. 6867 (all of which are incorporated herein by reference in their entirety) Using mutations known in the relevant technical field, such as those that have been observed, heterodimerization is preferred over homodimerization. PD-1 molecule comprising CH2 and / or CH3 domains manipulated to promote merization It includes compound molecules.

[0269] VIII.PD-1×LAG-3 bispecific binding molecule In particular, the present invention relates to anti-PD-1 antibodies, preferably novel anti-human antibodies provided herein. One epitope-binding fragment of a PD-1 antibody and an anti-human LAG-3 antibody, preferably One epitope conjugate of a novel anti-human LAG-3 antibody provided herein. A PD-1×LAG-3 bispecificity binding molecule containing a fragment (e.g., bispecificity antibody, bi Regarding specificity diabody etc., the present invention prefers PD-1×LAG-3 bispecificity diabody. The combined molecule binds this PD-1×LAG-3 bispecificity molecule to two different epitopes: By coordinating binding to the PD-1 epitope and the LAG-3 epitope, This has an antibody epitope-binding fragment that can attenuate the inhibitory activity of the above molecule. When used in a specification, such attenuation is detectable. At least 20% reduction in 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. Anti-human PD-1 antibodies and The selection of the epitope-binding fragment (e.g., VL and VH domains) of the anti-LAG-3 antibody is crucial. Polypeptide chains constituting such PD-1×LAG-3 bispecificity binding molecules associate together, At least one functional antibody specific to the 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 what the first antigen is). To form at least one functional antigen binding site specific to G-3), To be arranged.

[0270] In certain embodiments, the PD-1×LAG-3 bispecific binding molecule of the present invention is bispecific It is an heterogeneous diamond body, which is preferably two, three as described herein. , comprising 4 or 5 polypeptide chains. In another specific embodiment, the present invention is PD-1×L The AG-3 bispecificity conjugate molecule is a bispecificity antibody, which is preferably described herein. As described, it contains two, three, or four polypeptide chains (for example, International Publication No. 200) Issue No. 7 / 024715; International Publication No. 2007 / 110205; International Publication No. 2009 / 08 Publication No. 0251; International Publication No. 2009 / 080254; International Publication No. 2009 / 089004 International Publication No. 2011 / 069104; International Publication No. 2011 / 117329; International Publication No. 2011 / 131746; International Publication No. 2011 / 133886; International Publication No. 2 Publication No. 011 / 143545; International Publication No. 2012 / 023053; International Publication No. 2013 / See also No. 060867 (all of these documents are incorporated in their entirety by reference in this application). see).

[0271] A. Anti-human LAG-3 antibody The following are exemplary antibodies that are immune-specific to human LAG-3. Further desired The antibody is an antibody secretion hybrid induced using LAG-3 or its peptide fragment. By isolating the γ, or in relation to binding to LAG-3 or its peptide fragments, This can be produced by screening recombinant antibody libraries. (28Ami It contains an acid-free 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 holds.

[0272] 1. LAG-3 mAb A In this specification, the anti-human LAG-3 antibody BMS-9 is referred to as "LAG-3 mAb A". 86016 (25F7; Medarex / BMS) and its variants have already been explained. (See, for example, International Publication No. 2014 / 008218). LAG-3 mAb The amino acid sequence of the heavy chain variable domain of A is amino acid sequence (SEQ ID NO: 39) (CDR is below) (Indicated by a line): QVQLQQWGAG LLKPSETLSL TCAVYGGSFS DYYWN WIRQP PGKGLEWIG E INHNGNTNSN PSLKS RVTLS LDTSKNQFSL KLRSVTAADT AVYYCAF GYS DYEYNWFDP W GQGTLVTVSS It holds.

[0273] The amino acid sequence of the light chain variable domain of LAG-3 mAb A is the amino acid sequence (sequence number) Issue 40) (CDRs are indicated with an underline): EIVLTQSPAT LSLSPGERAT LSC RASQSIS SYLA WYQQKP GQAPRLLIY D ASNRAT GIPA RFSGSGSGTD FTLTISSLEP EDFAVYYC QQ RSNWPLT FGQ GTNLEIK It holds.

[0274] Additional mouse anti-human LAG-3 antibodies with unique binding properties have recently been identified (USA). (See Patent Application No. 62 / 172,277). Preferred PD-1×LAG-3 of the present invention The highly specific binding molecule is either 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 LAG-3 binding. In terms of compatibility, it does not compete with BMS-986016. LAG-3 mAb is particularly preferred. The present invention has humanized VH and / or VL domains of 1 or LAG-3 mAb 6. 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 indicated by underlines): QIQLVQSGPE LKKPGETVKI SCKASGYTFR NYGMN WVKQA PGKVLKWMG W INTYTGESTY ADDFEG RFAF SLGTSASTAY LQINILKNED TATYFCAR ES LYDYYSMDY W GQGTSVTVSS LAG-3 mAb 1 CDR H 1 (Sequence ID 42): RNYGMN LAG-3 mAb 1 CDR H 2 (Sequence ID 43): WINTYTGESTYADDFEG LAG-3 mAb 1 CDR H 3 (Sequence ID 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 indicated by underlines): DVVVTQTPLT LSVTIGQPAS ISC KSSQSLL HSDGKTYLN W LLQRPGQSPE RLIY LVSELD S GVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYC WQGTHFP YT FGGGTKLE IK LAG-3 mAb 1 CDR L 1 (Sequence ID 46): KSSQSLLHSDGKTYLN LAG-3 mAb 1 CDR L 2 (Sequence ID 47): LVSELDS LAG-3 mAb 1 CDR L 3 (Sequence ID 48): WQGTHFPYT

[0277] In this specification, "hLAG-3 mAb 1 VH1" and "hLAG-3 mAb 1 Two exemplary humanized VH domains of LAG-3 mAb 1, referred to as "VH2", Furthermore, 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 following are "1 VL3" and "hLAG-3 mAb 1 VL4". Pair any of the humanized VL domains with any of the humanized VH domains to bind to LAG-3. A domain can be generated. Therefore, the above humanized VL domain paired with the above humanized VH domain Any antibody containing one of the 'in' genes is generally called "hLAG-3 mAb 1". , a specific combination of humanized VH / VL domains, relative to a specific VH / VL domain They are referred to by reference. For example, hLAG-3 mAb 1 VH1 and hLAG-3 Humanized antibodies containing mAb 1 VL2 specifically include "hLAG-3 mAb 1(1. 2) is called "2)".

[0278] The amino acid sequence of the VH domain of hLAG-3 mAb 1 VH1 (SEQ ID NO: 49) The following (CDR) H Residues are indicated by underlines): 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) The following (CDR) H Residues are indicated by underlines): 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) The following (CDR) L Residues are indicated by underlines): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQKPGQSPE RLIY LVSELD S GVPDRFSGSGSGTDFTLKI 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) The following (CDR) L Residues are indicated by underlines): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQRPGQSPE RLIY LVSELD S GVPDRFSGS GSGTDFTLKI 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) The following (CDR) L Residues are indicated by underlines): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDGKTYLN W LLQKPGQPPE RLIY LVSELD S GVPDRFSGS GSGTDFTLKI 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) The following (CDR) L Residues are indicated by underlines): DIVMTQTPLS LSVTPGQPAS ISC KSSQSLL HSDAKTYLN W LLQKPGQPPE RLIY LVSELD S GVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYC WQGTHFP YT FGGGTKVE IK

[0284] CDR of the VL domain of hLAG-3 mAb 1 VL4 L 1 is derived from glycine. Includes amino acid substitution in the nin, amino acid sequence: KSSQSLLHSD A KTYLN (Sequence number 55, replaced) The alanine that is substituted has (shown with an underline). Similar substitutions are found in the above-mentioned LAG-3 mAb. 1 CD-R L It is thought that it can be incorporated into any of the 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 indicated by underlines): EVLLQQSGPE LVKPGASVKI PCKASGYTFT DYNMD WVKQS HGESLEWIG D INPDNGVTIY NQKFEGKATL TVDKSSSTAY MELRSLTSED TAVYYCAR EA DYFYFDY WGQ GTTLTVSS LAG-3 mAb 6 CDR H 1 (Sequence ID 57): DYNMD LAG-3 mAb 6 CDR H 2(Sequence ID 58):DINPDNGVTIYNQKFEG LAG-3 mAb 6 CDR H 3 (Sequence ID 59): EADYFYFDY

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

[0287] In this specification, "hLAG-3 mAb 6 VH1" and "hLAG-3 mAb 6 Two exemplary humanized VH domains of LAG-3 mAb 6, referred to as "VH2", Furthermore, 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. By pairing any of the humanized VL domains with any of the humanized VH domains, LAG-3 A binding domain can be generated. Therefore, the above humanized VL can be paired with the above humanized VH domain. Any antibody possessing one of the domains is generally referred to as "hLAG-3 mAb 6". This refers to a specific combination of humanized VH / VL domains, and then to a specific VH / VL domain. They are named by their corresponding reference. For example, hLAG-3 mAb 6 VH1 and hLAG- Humanized antibodies containing 3 mAb 6 VL2 specifically include "hLAG-3 mAb 6( It is called "1.2)".

[0288] Amino acid sequence of the VH domain of hLAG-3 mAb 6 VH1 (SEQ ID NO: 294) The following is shown (CDR H Residues are indicated by underlines): 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) The following is shown (CDR H Residues are indicated by underlines): 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) The following is shown (CDR L Residues are indicated by underlines): 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) The following is shown (CDR L Residues are indicated by underlines): DIVMTQSPSS LSASVGDRVT ITC RASQDVS SVVA WYQQKP GKAPKLLIY S ASYRYT GVPD RFSGSGSGTD FTFTISSLQP EDIAVYYCQQ HYSTPWT FGG GTKLEIK

[0292] hLAG-3 mAb 6 VL1 and VL2 VL domain CDR L 1 is ricin The amino acid sequence includes an amino acid substitution from to arginine: R ASQDVSSVVA (Sequence ID 298) (The substituted arginine is shown underlined). Similar substitutions are found in the above LAG- 3 mAb 6 CDR L It is thought that it can be incorporated into any of the domains.

[0293] Exemplary 4-chain Fc region-containing diamond body with BE / K coil (referred to as "DART A", "DART B", "DART C", and "DART I") (It will be revealed) E / K coil heterodimer promoting domain, PD-1×LAG-3 dual special We generated isomer tetra-strand Fc region-containing diabodies. The structure of these Fc region-containing diabodies The construction is described in detail below. These exemplary PD-1×LAG-3 diamond bodies are the present invention. This is intended to illustrate the scope, not to limit it.

[0294] 1. DART A DART A has two binding sites specific to PD-1 and specific to LAG-3. Two specific binding sites, a mutant IgG4 Fc region manipulated for extended half-life, and A bispecific 4-chain Fc region having a cysteine-containing E / K coil heterodimer-promoting domain. It is a region-containing diamond body. The first and third polypeptide chains of DART A are N-terminal or VL domain monoclonal antibodies that can bind to LAG-3 in the direction of the C-terminus:N-terminus n (VL LAG‐3 hLAG-3 mAb 1 VL4) (SEQ ID NO: 54); Intervening linker Peptide (linker 1: GGGSGGGG (SEQ ID NO: 14)); monoclonal molecule capable of binding to PD-1. VH domain of the Nal antibody (VH PD‐1 hPD-1 mAb 7 VH1) (Sequence ID 1) 47); Cysteine-containing intermediary 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); Substitute M2 A mutant IgG4 CH2 containing 52Y / S254T / T256E and lacking the C-terminal residue. - Includes the CH3 domain (sequence number 259) and the C-terminus.

[0295] The amino acid sequences of the first and third polypeptide chains of DART A are the variant of 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 Here, X1, X2, X3 and X4 are independently selected, and X1 is A or G; X2 is Y or M; X3 is T or S; X4 is E or T.

[0296] The amino acid sequences of the first and third polypeptide chains of DART A are shown in SEQ ID NO: 267. Here, 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 direction from the N-terminus to the C-terminus: N-terminus VL domain of monoclonal antibody capable of binding to PD-1 (VL PD‐1 hPD-1 mAb 7 VL2) (SEQ ID NO: 153); Intermediate linker peptide (Linker 1: GGGS) GGGG (SEQ ID NO: 14)); VH domain of a monoclonal antibody that can bind to LAG-3 ( VH LAG‐3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49); cysteine-containing Linker peptide (linker 2: GGCGGG (SEQ ID NO: 15)); cysteine-containing heterozygote Dimer-promoting (K-coil) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE(SEQ ID NO: 24) ); and including the C-terminus.

[0298] The amino acid sequences of the second and fourth polypeptide chains of DART A are (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 That is the case.

[0299] 2. DART B DART B is identical to DART A, but DART B's first and third polyp The cytoplasmic chain contains the VL domain (SEQ ID NO: 53) of hLAG-3 mAb 1 VL3. This is a CD-R L The only difference is that it includes amino acid substitutions in 1. Therefore, DART B The first and third polypeptide chains are arranged in the direction from the N-terminus to the C-terminus: N-terminus; LAG-3 The VL domain of the monoclonal antibody that can bind (VL LAG‐3 hLAG-3 mAb 1 VL3)(SEQ ID NO: 53); Intermediate linker peptide (Linker 1: GGGSGGGG(SEQ ID NO: 53); 14)) VH domain of monoclonal antibodies that can bind to PD-1 (VH PD‐1 hP D-1 mAb 7 VH1) (SEQ ID NO: 147); Intermediate 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 ( Sequence ID 13); Contains substitutions M252Y / S254T / T256E and does not contain a C-terminal residue. , including a variant of the IgG4 CH2-CH3 domain (SEQ ID NO: 259); and the C-terminus.

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

[0301] The amino acid sequences of the second and fourth polypeptide chains of DART B are shown in SEQ ID NO: 268. ru.

[0302] 3. DART C DART C is identical to DART B, but DART C's first and third polyp The cytoplasmic chain is a wild-type IgG4 CH2-CH3 domain (SEQ ID NO: 4) that does not contain a C-terminal residue. The only difference is that it has ). Therefore, the first and third polypeptide chains of DART C are From the N-terminus to the C-terminus: 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 that can bind to PD-1. VH domain of noclonal antibodies (VH PD‐1 hPD-1 mAb 7 VH1) Column number 147); Intermediate linker peptide (linker 2: GGCGGG (sequence number 15)); cis Thein-containing heterodimer-promoting (E-coil) domain (EVAACEK-EVAALEK-EVAALEK-EVAAL EK (SEQ ID NO: 23); Stabilized IgG4 hinge region (SEQ ID NO: 13); Contains C-terminal residues It does not contain the IgG4 CH2-CH3 domain (SEQ ID NO: 4); and the C-terminus.

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

[0304] The amino acid sequences of the second and fourth polypeptide chains of DART C are shown in SEQ ID NO: 268. ru.

[0305] 4. DART I DART I has two binding sites specific to PD-1 and specific to LAG-3. Two specific binding sites, a mutant IgG4 Fc region manipulated for extended half-life, and A bispecific 4-chain Fc region having a cysteine-containing E / K coil heterodimer-promoting domain. It is a region-containing diabody. The first and third polypeptide chains of DART I are N-terminal or VL domain monoclonal antibodies that can bind to LAG-3 in the direction of the C-terminus:N-terminus n (VL LAG‐3 hLAG‐3 mAb 6 VL1) (SEQ ID NO: 296); intervening linker - Peptide (linker 1: GGGSGGGG (SEQ ID NO: 14)); monochromatic, capable of binding to PD-1. VH domain of non-alcoholic antibody (VH PD‐1 hPD-1 mAb 7 VH1) (Sequence ID) 147); Cysteine-containing intermediary 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); Substitute M2 A mutant IgG4 CH2 containing 52Y / S254T / T256E and lacking the C-terminal residue. - Includes the CH3 domain (sequence number 259) and the C-terminus.

[0306] The amino acid sequences of the first and third polypeptide chains of DART I are (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 That is the case.

[0307] The second and fourth polypeptide chains of DART I are arranged in the direction from the N-terminus to the C-terminus: N-terminus End; VL domain of monoclonal antibody capable of binding to PD-1 (VL PD‐1 hPD- 1 mAb 7 VL2) (SEQ ID NO: 153); Intermediate 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); contains cysteine. Intermediate linker peptide (linker 2: GGCGGG (SEQ ID NO: 15)); cysteine-containing hete Lonimer-promoting (K-coil) domain (KVAACKE-KVAALKE-KVAALKE-KVAALKE(SEQ ID NO: 2) 4); and including the C-terminus.

[0308] The amino acid sequences of the second and fourth polypeptide chains of DART I are (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 That is the case.

[0309] Exemplary diabody containing a 4-chain Fc region with a C.CL / CH1 domain These are called "DART D", "DART E", "DART J", and "DART 1". It contains a PD-1×LAG-3 bispecific 4-chain Fc region equipped with a CL / CH1 domain. Diabodies were generated. The structure of these Fc region-containing diabodies is described in detail below. These exemplary PD-1×LAG-3 diamond bodies exemplify the scope of the present invention. This is intentional, not intended to be restrictive.

[0310] 1. DART D DART D has two binding sites specific to PD-1 and specific to LAG-3. Two specific binding sites, the CL / CH1 domain, and mutations manipulated to extend the half-life. This is a bispecific 4-chain Fc region-containing diabody having a type IgG4 Fc region. The first and third polypeptide chains of RT D are arranged in the direction from the N-terminus to the C-terminus: N-terminus; P VL domain of monoclonal antibody that can bind to D-1 (VL PD‐1 hPD-1 mA b 7 VL2) (Sequence ID 153); Intermediate linker peptide (Linker 1: GGGSGGGG( SEQ ID NO: 14)) VH domain of monoclonal antibody capable of binding to LAG-3 (VH LA G‐3 hLAG-3 mAb 1 VH1) (SEQ ID NO: 49); Intermediate linker peptide ( Linker 2:LGGGSG (SEQ ID NO: 261); IgG4 CH1 domain (SEQ ID NO: 254) ); Stabilized IgG4 hinge region (SEQ ID NO: 13); Substitute M252Y / S254T / T25 A variant of the IgG4 CH2-CH3 domain that contains 6E and does not contain the C-terminal residue (sequence number) No. 259); and including the C-terminus.

[0311] The amino acid sequences of the first and third polypeptide chains of DART D are (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 That is the case.

[0312] The second and fourth polypeptide chains of DART D are arranged in the direction from the N-terminus to the C-terminus: N-terminus End; VL domain of a monoclonal antibody that can bind to LAG-3 (VL LAG‐3 hLAG -3 mAb 1 VL4) (SEQ ID NO: 54); Intermediate linker peptide (Linker 1: GG) GSGGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody that can bind to PD-1 ( VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); Intermediate linker peptide Tide (linker 2:LGGGSG (sequence number 261)); κCL domain (sequence number 8); and Includes the C-terminus.

[0313] The amino acid sequences of the second and fourth polypeptide chains of DART D are (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 That is the case.

[0314] 2. DART E DART E has two specific binding sites for PD-1 and specific binding sites for LAG-3. Two specific binding sites, the CL / CH1 domain, and mutations manipulated to extend the half-life. This is another bispecific 4-chain Fc region-containing diabody that has a type IgG4 Fc region. The positions of the PD-1 and LAG-3 binding sites in DART E are inverted compared to DART D. It is being done.

[0315] The first and third polypeptide chains of DART E are arranged in the direction from the N-terminus to the C-terminus: N-terminus End; VL domain of a monoclonal antibody that can bind to LAG-3 (VL LAG‐3 hLAG -3 mAb 1 VL4) (SEQ ID NO: 54); Intermediate linker peptide (Linker 1: GG) GSGGGG (SEQ ID NO: 14); VH domain of a monoclonal antibody that can bind to PD-1 ( VH PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); Intermediate linker peptide Tide (linker 2:LGGGSG (sequence number 261)); IgG4 CH1 domain (sequence number 254); Stabilized IgG4 hinge region (SEQ ID NO: 13); Substitution M252Y / S254T / A mutant of the IgG4 CH2-CH3 domain that contains T256E and does not contain the C-terminal residue. Includes sequence number 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 That is the case.

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

[0318] The amino acid sequences of the second and fourth polypeptide chains of DART E are (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 That is the case.

[0319] 3. DART J DART J has two specific binding sites for PD-1 and specific binding sites for LAG-3. Two specific binding sites, the CL / CH1 domain, and mutations manipulated to extend the half-life. This is a bispecific 4-chain Fc region-containing diabody having a type IgG4 Fc region. The first and third polypeptide chains of RT J are arranged in the direction from the N-terminus to the C-terminus: N-terminus; L VL domain of monoclonal antibody capable of binding to AG-3 (VL LAG‐3 hLAG-3 mAb 6 VL1) (SEQ ID NO: 296); Intermediate linker peptide (Linker 1: GGGSGG) GG (SEQ ID NO: 14)); VL domain (VH) of a monoclonal antibody capable of binding to PD-1 PD‐1 hPD-1 mAb 7 VH1) (SEQ ID NO: 147); Intermediate linker peptide (Linker 2:LGGGSG (Sequence ID 261)); IgG4 CH1 domain (Sequence ID 2 54); Stabilized IgG4 hinge region (SEQ ID NO: 13); Substitution M252Y / S254T / T A mutant of the IgG4 CH2-CH3 domain that contains 256E and does not contain the C-terminal residue ( Includes column number 259 and the C-terminus.

[0320] The amino acid sequences of the first and third polypeptide chains of DART J are (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 That is the case.

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

Claims

1. A combination used to stimulate the T cell-mediated immune response of a subject who requires stimulation of the T cell-mediated immune response, (a) An anti-human PD-1 conjugated monospecific monoclonal antibody comprising a variable heavy chain domain and a variable light chain domain, wherein the variable heavy chain domain comprises the amino acid sequence of SEQ ID NO: 147 and the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 151: and (b) One or more additional molecules that are effective in stimulating an immune response A combination that includes this.

2. A combination used for the treatment of cancer, (a) An anti-human PD-1 conjugated monospecific monoclonal antibody comprising a variable heavy chain domain and a variable light chain domain, wherein the variable heavy chain domain comprises the amino acid sequence of SEQ ID NO: 147 and the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 151: and (b) One or more additional molecules that specifically bind to cancer antigens, and / or chemotherapeutic agents. A combination that includes this.

3. The combination according to claim 1 or 2, wherein the antibody is a chimeric antibody or a humanized antibody.

4. The combination according to any one of claims 1 to 3, wherein the antibody includes an Fc region.

5. The combination according to claim 4, wherein the Fc region is of the IgG1, IgG2, IgG3, or IgG4 isotype.

6. The combination according to claim 5, wherein the antibody further comprises a hinge domain.

7. The Fc region and the hinge domain are of the IgG4 isotype. The combination according to claim 6, wherein the hinge domain includes a stabilizing mutation.

8. The Fc region is a variant Fc region, The aforementioned variant Fc region is: (a) One or more amino acid modifications that reduce the affinity of the mutant Fc region to FcγR; and / or (b) One or more amino acid modifications that increase the serum half-life of the mutant Fc region. A combination according to any one of claims 5 to 7, including the following:

9. The combination according to claim 8, wherein the modification for reducing the affinity of the mutant Fc region to FcγR includes substitutions of L234A;L235A; or L234A and L235A, and the numbering of the substitutions is an EU index numbering, as in Kabat.

10. The combination according to claim 8 or 9, wherein the modification that increases the serum half-life of the variant Fc region includes substitutions of M252Y; M252Y and S254T; M252Y and T256E; M252Y, S254T and T256E; or K288D and H435K, and the numbering of the substitutions is the numbering of the EU index as in Kabat.

11. A combination of the claims according to claim 1 and any one of the claims 3 to 10 incorporating claim 1, wherein one or more additional molecules effective in stimulating the immune response are an anti-CD137 antibody, an anti-CTLA-4 antibody, an anti-OX40 antibody, an anti-LAG-3 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, and / or a cancer vaccine.

12. A combination according to claim 2 and any one of claims 3 to 10 incorporating claim 2, wherein the cancer antigen is 5T4, B7H3, CD19, CD20, CD51, CD123, DR5, EGFR, EpCam, GD2, gpA33, HER2, ROR-1, TAG-72, VEGF-A, and / or VEGF-2.

13. A combination according to any one of claims 1 to 12, used for the treatment of cancer.

14. The aforementioned cancers include: adrenal carcinoma; AIDS-related cancer; alveolar soft part sarcoma; astrocytic tumor; bladder cancer; Bone cancer; brain and spinal cord cancer; metastatic brain tumors; breast cancer; carotid bulb tumors; cervical cancer; chondrosarcoma; chordoma; chromogenic renal cell carcinoma; clear cell carcinoma; colorectal cancer; colorectal cancer; benign fibrous histiocytoma; fibrinogenic small round cell tumor; ependymoma; Ewing's tumor; extraskeletal myxoid chondrosarcoma; osteogenic fibrodysplasia; fibrous dysplasia; gallbladder or bile duct cancer; gastrointestinal cancer; gestational trophoblastic disease; germ cell tumors; head and neck cancer; hepatocellular carcinoma; pancreatic islet cell tumors; Kaposi's sarcoma; kidney cancer, leukemia, lipoma / benign liposomal tumors, liposarcoma / malignant liposomal tumors, liver cancer; lymphoma; Lung cancer; medulloblastoma; melanoma; meningioma; multiple endocrine neoplasms; multiple myeloma; myelodysplastic syndrome; neuroblastoma; neuroendocrine neoplasms; ovarian cancer; The combination according to claim 13, characterized by the presence of cancer cells selected from the group consisting of pancreatic cancer; papillary thyroid carcinoma; parathyroid tumor; childhood cancer; peripheral nerve sheath tumor; pheochromocytoma; pituitary tumor; prostate cancer; posterior uveal melanoma; rare hematological disorders; renal metastasis; rhabdoid tumor; rhabdomyosarcoma; sarcoma; skin cancer; soft tissue sarcoma; squamous cell carcinoma; gastric cancer; synovial sarcoma; testicular cancer; thymic carcinoma; thymoma; thyroid metastasis; and uterine cancer cells.

15. The combination according to claim 13, wherein the cancers are: colorectal cancer; hepatocellular carcinoma; glioma; renal cancer; breast cancer; multiple myeloma; bladder cancer; neuroblastoma; sarcoma; non-Hodgkin 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); pilocytic cell leukemia (HCL); blastic plasmacytoid dendritic cell neoplasm (BPDCN); non-Hodgkin lymphoma (NHL), including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL); Hodgkin lymphoma; systemic mastocytosis; or Burkitt lymphoma.

16. The combination according to claim 13, wherein the cancer is uterine cancer.

17. The combination according to claim 13, wherein the cancer is squamous cell carcinoma.

18. The combination according to claim 13, wherein the cancer is a glioma.

19. The combination according to claim 13, wherein the cancer is cervical cancer.

20. The combination according to claim 13, wherein the cancer is kidney cancer.

21. The combination according to claim 13, wherein the cancer is lung cancer.

22. The combination according to claim 13, wherein the cancer is non-small cell lung cancer.

23. The combination according to claim 13, wherein the cancer is head and neck cancer.

24. The combination according to claim 13, wherein the cancer is a metastatic cancer of the kidney.

25. The combination according to claim 13, wherein the cancer is chromogenic renal cell carcinoma.

26. The combination according to claim 13, wherein the cancer is skin cancer.

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