Antigen-binding protein and its use

Abstract

The present application relates to antigen binding proteins and uses thereof that compete with pembrolizumab for binding to the same or overlapping PD-1 epitopes. The antigen binding proteins have an EC of about 2 μg / mL or less. 50 and can effectively inhibit the binding of human PD-L1 to human PD-1.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceuticals, and specifically, to antigen-binding proteins and their use.

Background Art

[0002] Human programmed cell death receptor-1 (PD-1) is known as one of the major immune checkpoint molecules expressed on the surface of activated T lymphocytes. When it binds to ligands PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death-Ligand 2), it can inhibit the activity of T lymphocytes and related cellular immune responses in vivo. PD-L1 is widely expressed in peripheral cells such as B and T lymphocytes and microvascular epithelial cells, and in tissue cells such as the lung, liver, and heart. Many studies have revealed that the interaction between PD-1 and PD-L1 is the main cause of immune surveillance escape by PD-L1-expressing positive tumor cells. By blocking the negative regulation of the PD-1 / PD-L1 signaling pathway by tumor cells and activating the immune system, T cell-related tumor-specific cellular immune responses can be promoted and tumors can be treated. Currently, anti-PD-1 antibodies also have the disadvantages of low selectivity and low affinity. Therefore, it is necessary to develop a novel anti-PD-1 antibody with high affinity and high specificity for PD-1.

Disclosure of the Invention

[0003] The present application provides an isolated antigen-binding protein having one or more of the following characteristics: 1) binding to PD-1 with an EC50 of about 11 μg / mL or less in an ELISA assay; 2) inhibiting the binding of human PD-L1 to human PD-1 with an EC50 of about 2 μg / mL or less in a competitive ELISA assay; and 3) being able to bind to PD-1 of primates (such as humans or monkeys). In some embodiments, the isolated antigen-binding protein binds to the same or overlapping PD-1 epitopes as the reference antibody.

[0004] In some embodiments, the isolated antigen-binding protein competes with the reference antibody for binding to PD-1. In some embodiments, the isolated antigen-binding protein has a binding affinity for PD-1 that is substantially the same as the binding affinity of the reference antibody for PD-1. In some embodiments, the reference antibody comprises an HCDR3 having the amino acid sequence represented by SEQ ID NO: 128. In some embodiments, the reference antibody comprises an HCDR2 having the amino acid sequence represented by SEQ ID NO: 127. In some embodiments, the reference antibody comprises an HCDR1 having the amino acid sequence represented by SEQ ID NO: 126. In some embodiments, the reference antibody comprises a heavy chain variable region VH comprising an HCDR1 having the amino acid sequence represented by SEQ ID NO: 126, an HCDR2 having the amino acid sequence represented by SEQ ID NO: 127, and an HCDR3 having the amino acid sequence represented by SEQ ID NO: 128. In some embodiments, the VH of the reference antibody has the amino acid sequence represented by SEQ ID NO: 132. In some embodiments, the reference antibody comprises an LCDR3 having the amino acid sequence represented by SEQ ID NO: 131. In some embodiments, the reference antibody comprises an LCDR2 having the amino acid sequence represented by SEQ ID NO: 130. In some embodiments, the reference antibody comprises an LCDR1 having the amino acid sequence represented by SEQ ID NO: 129. In some embodiments, the reference antibody comprises a light chain variable region VL comprising an LCDR1 having the amino acid sequence represented by SEQ ID NO: 129, an LCDR2 having the amino acid sequence represented by SEQ ID NO: 130, and an LCDR3 having the amino acid sequence represented by SEQ ID NO: 131. In some embodiments, the VL of the reference antibody has the amino acid sequence represented by SEQ ID NO: 133.

[0005] In some embodiments, the reference antibody includes pembrolizumab or an antigen-binding fragment thereof. In some embodiments, the isolated antigen-binding protein includes an HCDR3 having the amino acid sequence represented by SEQ ID NO: 138. In some embodiments, the isolated antigen-binding protein includes an HCDR3 having the amino acid sequence represented by SEQ ID NO: 142. In some embodiments, the isolated antigen-binding protein includes an HCDR3 having the amino acid sequence represented by any one of SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67. In some embodiments, the isolated antigen-binding protein includes an HCDR2 having the amino acid sequence represented by SEQ ID NO: 137. In some embodiments, the isolated antigen-binding protein includes an HCDR2 having the amino acid sequence represented by SEQ ID NO: 141. In some embodiments, the isolated antigen-binding protein includes an HCDR2 having the amino acid sequence represented by any one of SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54. In some embodiments, the isolated antigen-binding protein includes an HCDR1 having the amino acid sequence represented by SEQ ID NO: 136. In some embodiments, the isolated antigen-binding protein includes an HCDR1 having the amino acid sequence represented by SEQ ID NO: 140. In some embodiments, the isolated antigen-binding protein described above comprises an HCDR1 having an amino acid sequence represented by any one of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41. In some embodiments, the isolated antigen-binding protein described above comprises a heavy chain variable region VH comprising the above HCDR1 having an amino acid sequence represented by SEQ ID NO: 136, the above HCDR2 having an amino acid sequence represented by SEQ ID NO: 137, and the above HCDR3 having an amino acid sequence represented by SEQ ID NO: 138. In some embodiments, the isolated antigen-binding protein described above comprises a heavy chain variable region VH comprising the above HCDR1 having an amino acid sequence represented by SEQ ID NO: 140, the above HCDR2 having an amino acid sequence represented by SEQ ID NO: 141, and the above HCDR3 having an amino acid sequence represented by SEQ ID NO: 142. In some embodiments, the above VH of the isolated antigen-binding protein described above comprises the above HCDR1 having an amino acid sequence represented by any one of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, the above HCDR2 having an amino acid sequence represented by any one of SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and the above HCDR3 having an amino acid sequence represented by any one of SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67.

[0006] In some embodiments, the HCDR1, HCDR2, and HCDR3 of the isolated antigen-binding protein described above are 1) HCDR1: SEQ ID NO: 1, HCDR2: SEQ ID NO: 2, and HCDR3: SEQ ID NO: 3; 2) HCDR1: SEQ ID NO: 1, HCDR2: SEQ ID NO: 42, and HCDR3: SEQ ID NO: 55; 3) HCDR1: SEQ ID NO: 4, HCDR2: SEQ ID NO: 43, and HCDR3: SEQ ID NO: 3; 4) HCDR1: SEQ ID NO: 5, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 3; 5) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 3; 6) HCDR1: SEQ ID NO: 7, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 7) HCDR1: SEQ ID NO: 8, HCDR2: SEQ ID NO: 47, and HCDR3: SEQ ID NO: 3; 8) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 9) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 3; 10) HCDR1: SEQ ID NO: 11, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 3; 11) HCDR1: SEQ ID NO: 12, HCDR2: SEQ ID NO: 43, and HCDR3: SEQ ID NO: 3; 12) HCDR1: SEQ ID NO: 13, HCDR2: SEQ ID NO: 48, and HCDR3: SEQ ID NO: 3; 13) HCDR1: SEQ ID NO: 14, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 3; 14) HCDR1: SEQ ID NO: 15, HCDR2: SEQ ID NO: 47, and HCDR3: SEQ ID NO: 3; 15) HCDR1: SEQ ID NO: 16, HCDR2: SEQ ID NO: 43, and HCDR3: SEQ ID NO: 3; 16) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 49, and HCDR3: SEQ ID NO: 3; 17) HCDR1: SEQ ID NO: 18, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 18) HCDR1: SEQ ID NO: 19, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 19) HCDR1: SEQ ID NO: 20, HCDR2: SEQ ID NO: 50, and HCDR3: SEQ ID NO: 56; 20) HCDR1: SEQ ID NO: 21, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 57; 21) HCDR1: SEQ ID NO: 22, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 22) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 51, and HCDR3: SEQ ID NO: 3; 23) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 58; 24) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 25) HCDR1: SEQ ID NO: 24, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 59; 26) HCDR1: SEQ ID NO: 25, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 60; 27) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 61; 28) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 29) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 3; 30) HCDR1: SEQ ID NO: 29, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 31) HCDR1: SEQ ID NO: 25, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 62; 32) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 50, and HCDR3: SEQ ID NO: 63; 33) HCDR1: SEQ ID NO: 30, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 34) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 35) HCDR1: SEQ ID NO: 32, HCDR2: SEQ ID NO: 53, and HCDR3: SEQ ID NO: 3; 36) HCDR1: SEQ ID NO: 33, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 3; 37) HCDR1: SEQ ID NO: 34, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 64; 38) HCDR1: SEQ ID NO: 35, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 57; 39) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 54, and HCDR3: SEQ ID NO: 3; 40) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 65; 41) HCDR1: SEQ ID NO: 36, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 42) HCDR1: SEQ ID NO: 37, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 3; 43) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 59; 44) HCDR1: SEQ ID NO: 38, HCDR2: SEQ ID NO: 54, and HCDR3: SEQ ID NO: 3; 45) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 46) HCDR1: SEQ ID NO: 40, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 55; 47) HCDR1: SEQ ID NO: 41, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 48) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 66; 49) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 67 and comprises any one group of amino acid sequences selected from

[0007] In some embodiments, the isolated antigen-binding protein described above has its C-terminus directly or indirectly linked to the N-terminus of the above HCDR1 and comprises an H-FR1 having an amino acid sequence represented by SEQ ID NO: 68, 144 - 148, 230, and / or 235. In some embodiments, the isolated antigen-binding protein described above is located between the above HCDR1 and the above HCDR2, and comprises an H-FR2 having an amino acid sequence represented by SEQ ID NO: 69, 149-151 and / or 231. In some embodiments, the isolated antigen-binding protein described above is located between the above HCDR2 and the above HCDR3, and comprises an H-FR3 having an amino acid sequence represented by SEQ ID NO: 70, 152-161 and / or 232. In some embodiments, the isolated antigen-binding protein described above has its N-terminus directly or indirectly linked to the C-terminus of the above HCDR3, and comprises an H-FR4 having an amino acid sequence represented by SEQ ID NO: 71, 162 and / or 233. In some embodiments, the isolated antigen-binding protein described above comprises an H-FR1 having an amino acid sequence represented by SEQ ID NO: 68, 144-148, 230 and / or 235, an H-FR2 having an amino acid sequence represented by SEQ ID NO: 69, 149-151 and / or 231, an H-FR3 having an amino acid sequence represented by SEQ ID NO: 70, 152-161 and / or 232, and an H-FR4 having an amino acid sequence represented by SEQ ID NO: 71, 162 and / or 233. In some embodiments, the isolated antigen-binding protein described above comprises a heavy chain variable region VH having an amino acid sequence represented by SEQ ID NO: 139 and / or 234. In some embodiments, the isolated antigen-binding protein described above comprises a heavy chain variable region VH having an amino acid sequence represented by SEQ ID NO: 143 and / or 236. In some embodiments, the VH of the isolated antigen-binding protein described above has an amino acid sequence represented by any one of SEQ ID NOs: 72-120 and / or 163-227.

[0008] In some embodiments, the isolated antigen-binding protein described above comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the isolated antigen-binding protein described above comprises a single-domain antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment described above is selected from the group consisting of Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and / or dAb. In some embodiments, the antibody described above comprises a chimeric antibody, a humanized antibody and / or a fully human antibody. In some embodiments, the isolated antigen-binding protein described above has an amino acid sequence represented by any one of SEQ ID NOs: 72-120 and / or 163-227. In some embodiments, the isolated antigen-binding protein described above further comprises an immunoglobulin Fc region. In some embodiments, the VH of the antigen-binding protein described above is directly or indirectly linked to the Fc region described above. In some embodiments, the C-terminus of the VH of the antigen-binding protein described above is directly or indirectly linked to the N-terminus of the Fc region described above. In some embodiments, the VH of the antigen-binding protein described above is in-frame fused with the Fc region described above. In some embodiments, the VH of the antigen-binding protein described above is linked to the Fc region described above via a linker.

[0009] In some embodiments, the linker described above comprises a peptide linker. In some embodiments, the linker described above comprises a flexible linker. In some embodiments, the isolated antigen-binding protein described above sequentially comprises, from the N-terminus to the C-terminus, the VH of the antigen-binding protein described above, the linker described above and the Fc region described above.

[0010] In some embodiments, the Fc region described above comprises an Fc derived from IgG1 or an Fc derived from IgG4. In some embodiments, the above Fc region has an amino acid sequence represented by any one of SEQ ID NOs: 121-125.

[0011] On the other hand, the present application provides one or more polypeptides containing the above isolated antigen-binding protein. On the other hand, the present application provides one or more immune complexes containing the above isolated antigen-binding protein or the above polypeptide. On the other hand, the present application provides one or more isolated nucleic acid molecules encoding the above isolated antigen-binding protein or the above polypeptide. On the other hand, the present application provides one or more vectors containing the above isolated nucleic acid molecules. On the other hand, the present application provides one or more cells containing and / or expressing the above isolated antigen-binding protein, the above polypeptide, the above immune complex, the above isolated nucleic acid molecule and / or the above vector. On the other hand, the present application provides a method for preparing the above isolated antigen-binding protein and / or the above polypeptide, the method comprising culturing the above cells under conditions that allow expression of the above isolated antigen-binding protein and / or the above polypeptide. On the other hand, the present application provides one or more pharmaceutical compositions containing the above isolated antigen-binding protein, the above polypeptide, the above immune complex, the above isolated nucleic acid molecule, the above vector, the above cell, and optionally a pharmaceutically acceptable adjuvant and / or excipient. On the other hand, the present application provides a method for detecting the presence and / or content of PD-1, the method comprising administering the above isolated antigen-binding protein or the above polypeptide. On the other hand, the present application provides one or more kits containing the above isolated antigen-binding protein or the above polypeptide. In some embodiments, the above kit contains an instruction manual describing a method for detecting the presence and / or content of PD-1. On the one hand, the present application provides the use of the above-mentioned isolated antigen-binding protein or the above-mentioned polypeptide in the preparation of a kit for detecting the presence and / or content of PD-1. On the other hand, the present application provides the use of one or more of the above-mentioned isolated antigen-binding proteins and / or the above-mentioned polypeptides in the preparation of a pharmaceutical for preventing and / or treating a disease or illness. On the one hand, the present application provides one or more of the above-mentioned isolated antigen-binding proteins, the above-mentioned polypeptides, the above-mentioned immune complexes, the above-mentioned isolated nucleic acid molecules, the above-mentioned vectors, the above-mentioned cells, and / or the above-mentioned pharmaceutical compositions for preventing, alleviating, and / or treating a disease or illness.

[0012] In some embodiments, the above-mentioned disease or illness includes a tumor. In some embodiments, the above-mentioned tumor includes a solid tumor. In some embodiments, the above-mentioned tumor includes a hematological tumor. In some embodiments, the above-mentioned tumor includes a tumor associated with the expression of PD-L1. In some embodiments, the above-mentioned tumor is selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma, and breast cancer.

[0013] On the one hand, the present application provides a method for preventing and / or treating a disease or illness, the method including administering to a subject in need of an effective amount of the above-mentioned isolated antigen-binding protein, the above-mentioned polypeptide, the above-mentioned immune complex, the above-mentioned isolated nucleic acid molecule, the above-mentioned vector, and / or the above-mentioned cell. In some embodiments, the above-mentioned disease or illness includes a tumor. In some embodiments, the above-mentioned tumor includes a solid tumor. In some embodiments, the above-mentioned tumor includes a hematological tumor. In some embodiments, the above tumors include tumors associated with the expression of PD-L1. In some embodiments, the above tumors are selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma, and breast cancer.

[0014] On the other hand, the present application provides a method for inhibiting the interaction between PD-1 and PD-L1, which includes administering to a subject in need of an effective amount of the above isolated antigen-binding protein, the above polypeptide, the above immune complex, the above isolated nucleic acid molecule, the above vector, and / or the above cell. On the other hand, the present application provides the above isolated antigen-binding protein, the above polypeptide, the above immune complex, the above isolated nucleic acid molecule, the above vector, the above cell and / or the above pharmaceutical composition for inhibiting the interaction between PD-1 and PD-L1. On the other hand, the present application provides the use of the above isolated antigen-binding protein and / or the above polypeptide in the preparation of a pharmaceutical product for inhibiting the interaction between PD-1 and PD-L1. Those skilled in the art can easily understand other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the content of the present application allows for modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention related to the present application. Correspondingly, the drawings and descriptions in the specification of the present application are not limiting but merely exemplary.

Brief Description of the Drawings

[0015] The specific features of the invention according to the present application are shown in the appended claims. The features and advantages of the invention according to the present application can be better understood by referring to the exemplary embodiments described in detail below and the appended drawings. The brief description of the appended drawings is as follows.

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0016] Hereinafter, the embodiments of the present invention will be described by specific examples. However, other advantages and effects of the present invention can be easily understood by those skilled in the art from the disclosure of this specification.

[0017] Definition of Terms In the present application, the term "isolated" generally refers to being obtained by artificial means from its natural state. An "isolated" substance or component may have its natural environment changed, be isolated from its natural environment, or both, when it exists in nature. For example, if an unisolated polynucleotide or polypeptide naturally exists in a living animal, the same high-purity polynucleotide or polypeptide isolated in this natural state is said to be isolated. The term "isolated" does not exclude the mixing of artificial or synthetic substances or the presence of other impurities that do not affect the activity of the substance.

[0018] In the present application, the term "antigen-binding protein" generally means a polypeptide molecule capable of specifically recognizing and / or neutralizing a specific antigen. In the present application, the term "antigen-binding protein" may include "antibody" or "antigen-binding fragment". For example, the above antibody includes an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, and may include any molecule containing its antigen-binding portion. The term "antibody" includes monoclonal antibodies, antibody fragments or antibody derivatives, and also includes, but is not limited to, camel antibodies, fully human antibodies, chimeric antibodies, single-chain antibodies (e.g., scFv), and antibody fragments bound to antigens (e.g., Fab, Fab’ and (Fab)2 fragments). The term "antibody" may include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and any antigen-binding antibody fragments and their derivatives described herein. In the present application, the above "antibody" may also include single-domain antibodies. In this application, the term "single domain antibody" usually refers to a class of antibodies that lack the light chain of the antibody and are composed only of the heavy chain variable region. According to research, in dromedary camels, llamas, alpacas, and humans, there are heavy chain antibodies (hcAbs) that are composed only of heavy chains but have complete functions, and the molecular weight of the variable domains (simply called VHH) of the heavy chain antibodies is only 1 / 10 of that of conventional antibodies. They are the smallest molecular fragments with complete functions among currently available antibodies and are called single domain antibodies (sdAbs). Single domain antibodies have the advantages of low immunogenicity, small molecular size, and strong permeability compared to other antibodies, and thus are expected to have a wide range of applications in basic research, drug development, disease treatment, etc. For example, the single domain antibody may be derived from alpacas. The single domain antibody may be composed of the heavy chain variable region (VH). The term "heavy chain variable region" usually refers to the amino-terminal domain of the heavy chain of the antigen-binding fragment. The heavy chain variable region can be further distinguished into hypervariable regions called complementarity-determining regions (CDRs) that are interspersed in more conserved regions called framework regions (FRs). Each heavy chain variable region may be composed of three CDRs and four FR regions, with H-FR1, HCDR1, H-FR2, HCDR2, H-FR3, HCDR3, and H-FR4 arranged in this order from the amino terminus to the carboxyl terminus. The heavy chain variable region contains a binding domain that interacts with an antigen (e.g., PD-1). The exact definition of the boundaries of the above CDRs varies depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) provides a clear residue numbering system applicable to any variable region of an antigen-binding protein and also provides the exact residue boundaries for defining CDRs. These CDRs may be called Kabat CDRs.Chothia et al. (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) discovered that although there is significant diversity at the amino acid sequence level, a certain subportion within the Kabat CDRs adopts a conformation of a nearly identical peptide backbone. These subportions are named L1, L2, L3 or H1, H2, H3, where "L" indicates the light chain region and "H" indicates the heavy chain region, respectively. These regions may be referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDRs. Other boundaries for defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5): 732-45 (1996)). Note that other definitions of CDR boundaries may not strictly follow any of the above systems, but can be shortened or extended according to predictions or experimental findings that certain residues, residue groups, or the CDRs as a whole do not have a significant impact on antigen binding, and still overlap with the Kabat CDRs. In the present application, the CDRs can be defined by the IMGT numbering system. In the present application, the term "single domain antibody" is used interchangeably with "nanobody" and "VHH". The claims of the present application are based on the sequences shown in the definition of the IMGT numbering system, but amino acid sequences corresponding to other CDR definition rules are also included in the protection scope of the present application. Exemplary definition rules are shown in the following table.

[0019]

Table 1

[0020] Here, the amino acid ranges in the table refer to the amino acid numbering ranges starting from the N-terminus of the antigen-binding protein of the present application. For example, "26-33" refers to the amino acid sequence from position 26 to position 33 from the N-terminus.

[0021] In the present application, the term "monoclonal antibody" generally refers to a population of antibodies that are substantially homogeneous; that is, the individual antibodies that make up the population are identical except for possible natural mutations that may be present in trace amounts. Monoclonal antibodies are highly specific and directly correspond to a single antigenic site. For example, the above monoclonal antibodies may be prepared by the hybridoma method or may be produced in bacteria, eukaryotes, or plant cells by recombinant DNA methods. Monoclonal antibodies may also be obtained from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0022] In the present application, the term "chimeric antibody" generally means an antibody in which a part of each amino acid sequence of the heavy or light chain is derived from a specific species, or is homologous to the corresponding amino acid sequence of an antibody belonging to a specific class, and the remaining segments of that chain are homologous to the corresponding sequences of other species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of antibodies of one animal species (e.g., mouse, rat, etc.), and the constant part is homologous to the sequence of an antibody derived from another species (e.g., human). For example, to obtain a chimeric antibody, non-human B cells or hybridoma cells can be used to produce the variable regions, and the constant regions that bind to them can be of human origin. The above variable regions have the advantage of being easy to prepare, and their specificity is not affected by the origin of the constant regions to which they are combined. At the same time, since the constant regions of chimeric antibodies can be of human origin, they are less likely to cause an immune response compared to the case of using antibodies with non-human-derived constant regions in the case of injection.

[0023] In the present application, the term "PD-1" or "PD1" generally refers to human programmed cell death receptor-1, a 288 amino acid type I membrane protein first described in 1992 (Ishida et al., EMBO J, 11 (1992), 3887-3895). PD-1 is a member of the extended CD28 / CTLA-4 family of T cell regulators and has two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The structure of this protein includes an extracellular IgV domain, followed by a transmembrane region, and an intracellular tail. The intracellular tail contains two phosphorylation sites located in immunoreceptor tyrosine-based inhibitory motifs and immunoreceptor tyrosine-based switch motifs, suggesting that PD-1 negatively regulates TCR signals. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 upon ligand binding. PD-1 is upregulated following activation mediated by the T cell receptor (TCR) and is observed in both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). PD-1 has a relatively broad expression pattern, but its most important role is likely to be as a co-inhibitory receptor for T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Therefore, in order to improve T cell responses, efforts have focused on treating tumors by blocking the interaction between PD-1 and its ligands. The terms "PD-1 protein", "PD-1", "PD1", "PDCD1", "hPD-1", and "hPD-I" can be used interchangeably and include isoforms, species homologs, functionally active fragments, and analogs of human PD-1 that have at least one common epitope with PD-1. For example, the above-mentioned "functionally active fragment" may include a fragment that retains at least one endogenous function of the naturally occurring protein (e.g., binds to the antigen-binding protein described in the present application). For example, the above-mentioned "functionally active fragment" may include a domain that binds to the antigen-binding protein of the present application.An exemplary amino acid sequence of human PD1 can be shown in UniProt accession number Q15116.

[0024] In this application, the terms "PD-L1" or "PDL1" usually also refer to B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, and refer to programmed cell death receptor-ligand 1, which down-regulates T cell activation and cytokine secretion when binding to PD-1. "PD-L1" includes any natural PD-L1 derived from any vertebrate, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). This term includes "full-length" unprocessed PD-L1 and any form of PD-L1 generated by cellular processing. PD-L1 can exist as a transmembrane protein or a soluble protein. "PD-L1" includes the complete PD-L1 and its fragments, and further includes functional fragments, isoforms, species homologs, derivatives, analogs, and analogs having at least one common epitope with PD-L1. For example, the above "functionally active fragment" may include a fragment that retains at least one endogenous function of a naturally occurring protein (e.g., binding to the antigen-binding protein described in this application). For example, the above "functionally active fragment" may include a domain that binds to the antigen-binding protein of this application. An exemplary amino acid sequence of human PD-L1 can be found in NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7. This application may include, in addition to the specific proteins and nucleotides described herein, their functional variants, derivatives, analogs, homologs and fragments thereof.

[0025] The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially identical to a naturally occurring sequence or is encoded by a substantially identical nucleotide sequence and can have one or more activities of a naturally occurring sequence. In the context of this application, a variant of any given sequence means that a specific sequence of its residues (either amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide retains at least one endogenous functional sequence substantially. Variant sequences can be obtained by addition, deletion, substitution, modification, switching, and / or mutation of at least one amino acid residue and / or nucleotide residue present in a naturally occurring protein and / or polynucleotide, as long as the original functional activity is maintained. In this application, the term "derivative" generally means, in relation to a polypeptide or polynucleotide of this application, any substitution, mutation, modification, switching, deletion, and / or addition from / to one (or more) amino acid residues of the sequence, as long as the resulting polypeptide or polynucleotide substantially retains at least one endogenous function. In this application, the term "analog" generally means, in relation to a polypeptide or polynucleotide, any mimetic of the polypeptide or polynucleotide, i.e., a chemical compound having at least one endogenous function of the polypeptide or polynucleotide mimicked thereby.

[0026] Typically, amino acid substitutions, such as at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 or more) amino acid substitutions, can be made as long as the modified sequence retains the substantially required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.

[0027] In the present application, the term "homolog" generally refers to an amino acid sequence or a nucleotide sequence having a specific homology to a naturally occurring nucleotide sequence. The term "homology" may correspond to the "identity" of a sequence. A homologous sequence may include an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the subject sequence. Generally, a homolog will include the same active site and the like as the subject amino acid sequence. Homology can also be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions), or homology can be expressed from the perspective of sequence identity. A sequence having a certain percentage of identity to either the amino acid sequence or the nucleotide sequence of the sequence numbers described in the present application refers to a sequence having that percentage of identity over the entire length of the recited sequence number. To determine sequence identity, sequence comparisons can be performed, which can be carried out using various means well-known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters to use for the comparison, including any algorithms necessary to achieve an optimal comparison in the full-length sequences being compared.

[0028] The proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues that result in silent changes and yield a functionally equivalent protein. As long as the endogenous function is retained, amino acid substitutions can be made intentionally based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with similar hydrophilic values and uncharged polar head groups include asparagine, glutamine, serine, threonine and tyrosine.

[0029] In the present application, the term "tumor" generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumor growth factors. For example, the tumor may include solid tumors. For example, the above-mentioned tumor may include hematological tumors. For example, the above-mentioned tumor may include tumors associated with the expression of PD-L1. The term "tumor associated with the expression of PD-L1" generally refers to a tumor formed by changes in PD-L1 expression that lead to disease progression or avoidance of immune surveillance. For example, the above-mentioned "tumor associated with the expression of PD-L1" may be a tumor formed by upregulation of the expression level of PD-L1 that leads to disease progression or avoidance of immune surveillance. The tumor associated with the protein expression of the above-mentioned PD-L1 may be a PD-L1-positive tumor. In a PD-L1-positive tumor, the protein expression level of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or more higher than that of normal cells.

[0030] In the present application, the term "solid tumor" generally refers to a palpable swelling detected by clinical examination (such as X-ray, CT scan, B-ultrasound or palpation). For example, the above-mentioned solid tumor may be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma, and breast cancer.

[0031] In the present application, the term "hematological tumor" generally refers to a tumor that cannot be seen or felt by X-ray, CT scan, B-ultrasound or palpation. For example, the above-mentioned hematological tumor may include leukemia. For example, the above-mentioned hematological tumor may include lymphoma. For example, the above-mentioned hematological tumor may include multiple myeloma.

[0032] In the present application, the term "immunocomplex" generally refers to a complex formed by complexing the above other reagents (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, cytotoxic drugs) with the above isolated antigen-binding protein (e.g., covalent bonding by a linker molecule). The complex can deliver the above other reagents to target cells (e.g., tumor cells) by specific binding of the above isolated antigen-binding protein to an antigen on the target cells. Subsequently, the above immunocomplex is internalized as described above and finally taken up into the interior of the target cells (e.g., vesicles such as lysosomes). At this time, the linker molecule in the above immunocomplex is lysed to release the above other reagents, thereby exerting a cytotoxic effect. Note that the above antigen may be secreted from the above target cells and located in the space outside the above target cells.

[0033] In the present application, the term "subject" generally refers to a human or a non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

[0034] In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides, ribonucleotides or their analogs of any length that are isolated from their natural environment or artificially synthesized.

[0035] In this application, the term "vector" generally means a nucleic acid molecule capable of transporting another nucleic acid that is ligated. The above-mentioned vector can transfer the inserted nucleic acid molecule into cells and / or between cells. The above-mentioned vectors may mainly include vectors used for inserting DNA or RNA into cells, vectors used mainly for replicating DNA or RNA, and vectors used mainly for expressing transcription and / or translation of DNA or RNA. The above-mentioned vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate cell. Generally, by culturing an appropriate cell containing the above-mentioned vector, the above-mentioned vector can produce a desired expression product. In this application, the above-mentioned vector may include a lentiviral vector.

[0036] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that may or may contain a plasmid or vector having the nucleic acid molecule described in this application, or can express the polypeptide described in this application or the antigen-binding protein described in this application. The above-mentioned cells may include the progeny of a single cell. Progeny cells do not necessarily have to be completely identical to the original parental cells in terms of morphology or genome as long as they can express the polypeptide and antigen-binding protein described in this application due to natural mutations, accidental mutations, or intentional mutations. The above-mentioned cells can be obtained by transfecting cells in vitro using the vector described in this application. The above-mentioned cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some embodiments, the above-mentioned cells may be immune cells. For example, the above-mentioned immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells, and / or peripheral blood mononuclear cells.

[0037] In the present application, the term "treatment" generally refers to (i) preventing the occurrence of a disease, illness and / or medical condition in a patient who is susceptible to but not diagnosed with the disease, illness and / or medical condition; (ii) suppressing the disease, illness or medical condition, i.e., arresting its progression; and (iii) alleviating the disease, illness or medical condition, i.e., causing regression of the disease, illness and / or medical condition and / or the symptoms associated with the disease, illness and / or medical condition.

[0038] In the present application, the terms "polypeptide", "peptide", "protein", "protein" are used interchangeably and generally refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include modified amino acid polymers. These modifications may include the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other operation (e.g., conjugation to a labeling component). The term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine, D and L optical isomers, as well as amino acid analogs and peptidomimetics.

[0039] In this application, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably and generally refer to polymeric forms of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: the coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined according to linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. A polynucleotide can contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If necessary, modifications to the nucleotide structure can be introduced before and after the construction of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide can be further modified, for example, by complexing a labeling component after polymerization.

[0040] In this application, "K" D "(similarly, "K" D " or "KD") usually refers to the "affinity constant" or "equilibrium dissociation constant", a value obtained at equilibrium in a titration measurement, or a value obtained by dividing the dissociation rate constant (k d ) by the association rate constant (k a ). The association rate constant (k a ), the dissociation rate constant (k d ), and the equilibrium dissociation constant (K D) is used to show the binding affinity of a binding protein (e.g., the isolated antigen-binding protein described in this application) for an antigen (e.g., PD-1 protein). Methods for determining the association rate constant and the dissociation rate constant are well known in the art. By using fluorescence-based techniques, high sensitivity can be obtained, and samples in physiological buffer at equilibrium can be examined. For example, the above K D value can be measured by Octet (Sartorius) or by other experimental approaches and instruments such as BIAcore (biomolecular interaction analysis system, GE Healthcare Life Science). Note that the above K D value can be measured by KinExA (Kinetic Exclusion Assay) from Sapidyne Instruments (Boise, Idaho) or by other surface plasmon resonance (SPR) devices. In this application, the above K D value can be measured by BIAcore.

[0041] In this application, the term "and / or" should be understood to mean any one or both of the alternatives. In this application, the term "comprising" usually means including the specifically identified features, but does not exclude other elements. In some cases, "comprising" also covers the case of including only the specified components. In some cases, "comprising" also covers the meaning of "consisting of...". In this application, the term "about" usually refers to a range of plus or minus 0.5% - 10% of the specified value, e.g., plus or minus 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the specified value. In this application, the term "comprising" usually refers to including, having, containing, or possessing. In some cases, it also means "being" or "consisting of".

[0042] Detailed Description of the Invention Isolated antigen-binding proteins described in this application On the other hand, the present application provides an isolated antigen-binding protein that can bind to PD-1 with an EC50 of about 11 μg / mL or less (e.g., the above EC 50 is not higher than about 11 μg / mL, not higher than about 10 μg / mL, not higher than about 9.5 μg / mL, not higher than about 9.0 μg / mL, not higher than about 8.5 μg / mL, not higher than about 8.0 μg / mL, not higher than about 7.5 μg / mL, not higher than about 7.0 μg / mL, not higher than 6.5 μg / mL, not higher than about 6.0 μg / mL, not higher than 5.5 μg / mL, not higher than about 5.0 μg / mL, not higher than about 4.5 μg / mL, not higher than about 4.0 μg / mL, not higher than about 3.5 μg / mL, not higher than about 3.0 μg / mL, not higher than about 2.5 μg / mL, not higher than about 2.0 μg / mL, not higher than about 1.5 μg / mL, not higher than about 1.0 μg / mL, not higher than about 0.5 μg / mL, not higher than about 0.4 μg / mL, not higher than about 0.3 μg / mL, not higher than about 0.2 μg / mL or not higher than about 0.1 μg / mL or less). In a competitive ELISA assay, the isolated antigen-binding protein described in the present application can inhibit the binding of human PD-L1 to human PD-1 with an EC50 of about 2 μg / mL or less (e.g., the above EC 50 is not higher than about 1.9 μg / mL, not higher than about 1.8 μg / mL, not higher than about 1.7 μg / mL, not higher than about 1.5 μg / mL, not higher than about 1.3 μg / mL, not higher than about 1.1 μg / mL, not higher than about 1.0 μg / mL, not higher than about 0.9 μg / mL, not higher than 0.8 μg / mL, not higher than about 0.7 μg / mL, not higher than 0.6 μg / mL, not higher than about 0.5 μg / mL, not higher than about 0.4 μg / mL, not higher than about 0.3 μg / mL, not higher than about 0.2 μg / mL, or not higher than about 0.1 μg / mL or less).

[0043] In the present application, the above-mentioned isolated antigen-binding protein can bind to the same or overlapping PD-1 epitopes as the reference antibody. For example, the above-mentioned isolated antigen-binding protein can compete with the above-mentioned reference antibody for binding to PD-1. In the present application, the binding affinity of the above-mentioned isolated antigen-binding protein for PD-1 may be substantially the same as the binding affinity of the above-mentioned reference antibody for PD-1. For example, the binding affinity can be measured by an ELISA assay. For example, the above-mentioned binding affinity 50 may be characterized by. For example, in an ELISA assay regarding binding affinity, the EC 50 of the above-mentioned isolated antigen-binding protein for PD-1 50 is approximately equal to or less than the EC of the above-mentioned reference antibody for PD-1. In the present application, the above-mentioned reference antibody may include a heavy-chain variable region VH that may include at least one, two, or three of HCDR1, HCDR2, and HCDR3.

[0044] In the present application, the exact boundaries of the above HCDRs may be defined differently in different systems. For example, the HCDRs described in the present application can be defined by the Kabat system (see the system described in Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))), which not only provides a clear residue numbering system applicable to any variable region of an antigen-binding protein, but also the exact residue boundaries defining the CDRs. These CDRs may be referred to as Kabat CDRs. For example, the HCDRs described in the present application can be defined by the Chothia system (see the studies of Chothia et al. (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989))). Chothia et al. discovered that although there is significant diversity at the amino acid sequence level, certain subportions within the Kabat CDRs adopt conformations of nearly identical peptide backbones. These subportions are named L1, L2, L3 or H1, H2, H3, where "L" indicates the light chain region and "H" indicates the heavy chain region, respectively. These regions may be referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDRs. Other boundaries for defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). It should be noted that other definitions of CDR boundaries may not strictly follow any of the above systems, but can be shortened or extended according to predictions or experimental findings that certain residues, residue groups, or entire CDRs do not have a major impact on antigen binding, and still overlap with the Kabat CDRs. In the present application, the HCDRs can be defined by the IMGT numbering system.

[0045] In the present application, the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 138. For example, the sequence of the HCDR3 of the above reference antibody may be determined according to the IMGT definition rules. AADX4DX6X7GFDY (SEQ ID NO: 138), where X4 may be I or L, X6 may be H, R or V, and X7 may be A, D, G, H, L, N, R, S, T or V. In the present application, the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 142. For example, the sequence of the HCDR3 of the above reference antibody may be determined according to the IMGT definition rules. AADX4DX6X7GFDY (SEQ ID NO: 142), where X4 may be I or L, X6 may be H or R, and X7 may be A, H or S.

[0046] In the present application, the HCDR3 of the above reference antibody may have the amino acid sequence represented by any one of SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 and 67. For example, the sequence of the HCDR3 of the above reference antibody may be determined according to the IMGT definition rules. In the present application, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 137. For example, the sequence of the HCDR2 of the above reference antibody may be determined according to the IMGT definition rules. X1GWX4X5X6TX8 (SEQ ID NO: 137), where X1 may be I or L, X4 may be A, N, R or S, X5 may be A, F, W or Y, X6 may be A, G or S, and X8 may be E, Q, S or T.

[0047] In the present application, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 141. For example, the sequence of the HCDR2 of the above reference antibody may be determined according to the IMGT definition rules. IGWX4X5X6TX8 (SEQ ID NO: 141), where X4 may be A or N, X5 may be F or Y, X6 may be A or S, and X8 may be E, Q, or T. In the present application, the HCDR2 of the above reference antibody may have an amino acid sequence represented by any one of SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54. For example, the sequence of the HCDR2 of the above reference antibody may be determined according to the IMGT definition rules. In the present application, the HCDR1 of the above reference antibody may have an amino acid sequence represented by SEQ ID NO: 136. For example, the sequence of the HCDR1 of the above reference antibody may be determined according to the IMGT definition rules. X1X2X3X4X5IYA (SEQ ID NO: 136), where X1 may be A, G, H, K, S, or V, X2 may be A, G, H, L, N, P, R, S, T, or V, X3 may be D, E, F, G, I, K, Q, R, S, or V, X4 may be D, G, H, K, L, P, Q, R, S, T, V, or Y, and X5 may be A, F, N, Q, R, S, or T. In the present application, the HCDR1 of the above reference antibody may have an amino acid sequence represented by SEQ ID NO: 140. For example, the sequence of the HCDR1 of the above reference antibody may be determined according to the IMGT definition rules. X1X2X3X4X5IYA (SEQ ID NO: 140), where X1 may be G, H, or K, X2 may be A, G, L, N, S, or V, X3 may be D, R, or V, X4 may be G, H, P, Q, R, or T, and X5 may be R, S, or T.

[0048] In the present application, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by any one of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. For example, the sequence of HCDR1 of the above-mentioned reference antibody can be determined according to the IMGT definition rules.

[0049] For example, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 138, the HCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 137, and the HCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 136. For example, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 142, the HCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 141, and the HCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 140. For example, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 1, the HCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 2, and the HCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 3. For example, the above-mentioned reference antibody may include a single-domain antibody MHPD1-A or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 1, the HCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 42, and the HCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 55. For example, the above-mentioned reference antibody may include a single-domain antibody MHPD1-A-1 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0050] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 4, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 43, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-2 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 5, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 44, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-3 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 6, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 45, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-4 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 7, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-5 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0051] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 8, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 47, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-6 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-7 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 10, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 45, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-8 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 11, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 44, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-9 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0052] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 12, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 43, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-10 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 13, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 48, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-11 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 14, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 45, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-12 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 15, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 47, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-13 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0053] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 16, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 43, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-14 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 17, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 49, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-15 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 18, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-16 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 19, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-17 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0054] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 20, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 50, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 56. For example, the above reference antibody may include a single-domain antibody MHPD1-A-18 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 21, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 57. For example, the above reference antibody may include a single-domain antibody MHPD1-A-19 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 22, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-20 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 51, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-21 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0055] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 58. For example, the above reference antibody may include a single-domain antibody MHPD1-A-22 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 23, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-23 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 24, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 59. For example, the above reference antibody may include a single-domain antibody MHPD1-A-24 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 25, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 60. For example, the above reference antibody may include a single-domain antibody MHPD1-A-25 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0056] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 26, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 61. For example, the above reference antibody may include a single-domain antibody MHPD1-A-26 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 27, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-27 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 28, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-28 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 29, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-29 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0057] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 25, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 62. For example, the above reference antibody may include a single-domain antibody MHPD1-A-30 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 50, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 63. For example, the above reference antibody may include a single-domain antibody MHPD1-A-31 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 30, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-32 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 31, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-33 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0058] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 32, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 53, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-35 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 33, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-36 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 34, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 64. For example, the above reference antibody may include a single-domain antibody MHPD1-A-37 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 35, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 57. For example, the above reference antibody may include a single-domain antibody MHPD1-A-38 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0059] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 54, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-39 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 65. For example, the above reference antibody may include a single-domain antibody MHPD1-A-40 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 36, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-41 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 37, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-42 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0060] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 59. For example, the above reference antibody may include a single-domain antibody MHPD1-A-43 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 38, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 54, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-44 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 39, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-45 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 40, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 55. For example, the above reference antibody may include a single-domain antibody MHPD1-A-46 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it.

[0061] For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 41, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above reference antibody may include a single-domain antibody MHPD1-A-47 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 66. For example, the above reference antibody may include a single-domain antibody MHPD1-A-48 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 67. For example, the above reference antibody may include a single-domain antibody MHPD1-A-49 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. For example, the VH of the above reference antibody may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0062] In the present application, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, 144-148, 230, and / or 235. In the present application, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, 149-151, and / or 231. In the present application, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, 152-161, and / or 232. In the present application, the above H-FR4 may have an amino acid sequence represented by SEQ ID NO: 71, 162, and / or 233.

[0063] In the present application, the H-FR1 of the above reference antibody may have an amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have an amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have an amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have an amino acid sequence represented by SEQ ID NO: 71. For example, the above reference antibody may include a single domain antibody MHPD1-A or an antibody having the same H-FR1-4 as it.

[0064] In the present application, the above reference antibody may include a heavy chain variable region that may have an amino acid sequence represented by SEQ ID NO: 139. QVQLVESGGGSVQVGGSLTLSCAASX 26 X 27 X 28 X 29 X 30 IYAMGWFRQAPGKEHEFVAGX 51 GWX 54 X 55 X 56 TX 58 YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100 DX 102 X 103 GFDYWGQGTQVTVSS (SEQ ID NO: 139), where X 26 may be A, G, H, K, S or V, and X 27 may be A, G, H, L, N, P, R, S, T or V, and X 28 may be D, E, F, G, I, K, Q, R, S or V, and X 29 may be D, G, H, K, L, P, Q, R, S, T, V or Y, and X 30 may be A, F, N, Q, R, S or T, and X 51 may be I or L, and X 54 may be A, N, R or S, and X 55 may be A, F, W or Y, and X56 may be A, G or S, and X 58 may be E, Q, S or T, and X 100 may be I or L, and X 102 may be H, R or V, and X 103 may be A, D, G, H, L, N, R, S, T or V.

[0065] In the present application, the above reference antibody may include a heavy chain variable region having an amino acid sequence represented by SEQ ID NO: 143. QVQLVESGGGSVQVGGSLTLSCAASX 26 X 27 X 28 X 29 X 30 IYAMGWFRQAPGKEHEFVAGIGWX 54 X 55 X 56 TX 58 YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100 DX 102 X 103 is GFDYWGQGTQVTVSS (SEQ ID NO: 143), where X 26 may be G, H or K, and X 27 may be A, G, L, N, S or V, and X 28 may be D, R or V, and X 29 may be G, H, P, Q, R or T, and X 30 may be R, S or T, and X 54 may be A or N, and X 55 may be F or Y, and X 56 may be A or S, and X 58 may be E, Q or T, and X 100 may be I or L, and X 102 may be H or R, and X 103 may be A, H or S.

[0066] In this application, the heavy chain variable region of the above-mentioned reference antibody may have an amino acid sequence represented by any one of SEQ ID NOs: 72-120 and / or 163-227. In this application, the HCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 128. In this application, the HCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 127.

[0067] In this application, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 126. For example, the HCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 126, the HCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 127, and the HCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 128. For example, the above-mentioned reference antibody may include pembrolizumab or an antibody having the same HCDR1-3.

[0068] In this application, the above-mentioned reference antibody may include a light chain variable region VL that may include LCDR1, LCDR2, and LCDR3. In this application, the LCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 131. In this application, the LCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 130.

[0069] In this application, the LCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 129. For example, the LCDR1 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 129, the LCDR2 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 130, and the LCDR3 of the above-mentioned reference antibody may have an amino acid sequence represented by SEQ ID NO: 131. For example, the above-mentioned reference antibody may include pembrolizumab or an antibody having the same LCDR1-3. For example, the HCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 126, the HCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 127, the HCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 128, the LCDR1 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 129, the LCDR2 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 130, and the LCDR3 of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 131. For example, the above reference antibody may include pembrolizumab or an antigen-binding protein having the same HCDR1-3 and LCDR1-3. For example, the VH of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 132. For example, the VL of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 133. For example, the above reference antibody may include pembrolizumab or an antigen-binding protein having the same VH and VL.

[0070] In the present application, the above reference antibody may include a heavy chain and a light chain that may have the amino acid sequence represented by SEQ ID NO: 134, for example. For example, the light chain of the above reference antibody may have the amino acid sequence represented by SEQ ID NO: 135. For example, the above reference antibody may include pembrolizumab or an antigen-binding protein having the same heavy chain and light chain. In the present application, the above antigen-binding protein may include a heavy chain variable region VH that may include at least one, two, or three of HCDR1, HCDR2, and HCDR3.

[0071] In the present application, the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 138. For example, the sequence of the HCDR3 of the above antigen-binding protein may be determined according to the IMGT definition rules. It is AADX4DX6X7GFDY (SEQ ID NO: 138), where X4 may be I or L, X6 may be H, R, or V, and X7 may be A, D, G, H, L, N, R, S, T, or V.

[0072] In the present application, the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 142. For example, the sequence of the HCDR3 of the above antigen-binding protein may be determined according to the IMGT definition rules. It is AADX4DX6X7GFDY (SEQ ID NO: 142), where X4 may be I or L, X6 may be H or R, and X7 may be A, H, or S. In the present application, the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by any one of SEQ ID NOs: 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, and 67. For example, the sequence of the HCDR3 of the above antigen-binding protein may be determined according to the IMGT definition rules.

[0073] In the present application, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 137. For example, the sequence of the HCDR2 of the above antigen-binding protein may be determined according to the IMGT definition rules. It is X1GWX4X5X6TX8 (SEQ ID NO: 137), where X1 may be I or L, X4 may be A, N, R, or S, X5 may be A, F, W, or Y, X6 may be A, G, or S, and X8 may be E, Q, S, or T.

[0074] In the present application, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 141. For example, the sequence of the HCDR2 of the above antigen-binding protein may be determined according to the IMGT definition rules. It is IGWX4X5X6TX8 (SEQ ID NO: 141), where X4 may be A or N, X5 may be F or Y, X6 may be A or S, and X8 may be E, Q, or T. In the present application, the HCDR2 of the above antigen-binding protein may have an amino acid sequence represented by any one of SEQ ID NOs: 2, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54. For example, the sequence of the HCDR2 of the above antigen-binding protein can be determined according to the IMGT definition rules.

[0075] In the present application, the HCDR1 of the above antigen-binding protein may have an amino acid sequence represented by SEQ ID NO: 136. For example, the sequence of the HCDR1 of the above antigen-binding protein can be determined according to the IMGT definition rules. X1X2X3X4X5IYA (SEQ ID NO: 136), where X1 may be A, G, H, K, S, or V, X2 may be A, G, H, L, N, P, R, S, T, or V, X3 may be D, E, F, G, I, K, Q, R, S, or V, X4 may be D, G, H, K, L, P, Q, R, S, T, V, or Y, and X5 may be A, F, N, Q, R, S, or T.

[0076] In the present application, the HCDR1 of the above antigen-binding protein may have an amino acid sequence represented by SEQ ID NO: 140. For example, the sequence of the HCDR1 of the above antigen-binding protein can be determined according to the IMGT definition rules. X1X2X3X4X5IYA (SEQ ID NO: 140), where X1 may be G, H, or K, X2 may be A, G, L, N, S, or V, X3 may be D, R, or V, X4 may be G, H, P, Q, R, or T, and X5 may be R, S, or T.

[0077] In the present application, the HCDR1 of the above antigen-binding protein may have an amino acid sequence represented by any one of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and 41. For example, the sequence of the HCDR1 of the above antigen-binding protein can be determined according to the IMGT definition rules.

[0078] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 138, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 137, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 136. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 142, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 141, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 140. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 1, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 2, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 1, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 42, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 55. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-1 or an antibody having the same HCDR1-3 as it.

[0079] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 4, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 43, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-2 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 5, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 44, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-3 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 6, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 45, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-4 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 7, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-5 or an antibody having the same HCDR1-3 as it.

[0080] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 8, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 47, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-6 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-7 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 10, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 45, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-8 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 11, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 44, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-9 or an antibody having the same HCDR1-3 as it.

[0081] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 12, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 43, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-10 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 13, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 48, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-11 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 14, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 45, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-12 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 15, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 47, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-13 or an antibody having the same HCDR1-3 as it.

[0082] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 16, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 43, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-14 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 17, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 49, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-15 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 18, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-16 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 19, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-17 or an antibody having the same HCDR1-3 as it.

[0083] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 20, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 50, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 56. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-18 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 21, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 57. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-19 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 22, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-20 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 51, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-21 or an antibody having the same HCDR1-3 as it.

[0084] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 58. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-22 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 23, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-23 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 24, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 59. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-24 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 25, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 60. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-25 or an antibody having the same HCDR1-3 as it.

[0085] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 26, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 61. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-26 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 27, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-27 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 28, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-28 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 29, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-29 or an antibody having the same HCDR1-3 as it.

[0086] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 25, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 62. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-30 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 50, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 63. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-31 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 30, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-32 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 31, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-33 or an antibody having the same HCDR1-3 as it.

[0087] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 32, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 53, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-35 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 33, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-36 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 34, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 64. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-37 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 35, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 57. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-38 or an antibody having the same HCDR1-3 as it.

[0088] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 54, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-39 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 65. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-40 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 36, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-41 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 37, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 52, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-42 or an antibody having the same HCDR1-3 as it.

[0089] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 59. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-43 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 38, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 54, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-44 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 39, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-45 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 40, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 55. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-46 or an antibody having the same HCDR1-3 as it.

[0090] For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 41, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 3. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-47 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 66. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-48 or an antibody having the same HCDR1-3 as it. For example, the HCDR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 9, the HCDR2 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 46, and the HCDR3 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 67. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-49 or an antibody having the same HCDR1-3 as it. For example, the VH of the above antigen-binding protein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0091] In the present application, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 230 and / or 235. In the present application, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 231. In the present application, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 232. In the present application, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 233.

[0092] In the present application, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, 144-148, and / or 230. In the present application, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69 and / or 149-151. In the present application, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70 and / or 152-161. In the present application, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71 and / or 162.

[0093] In the present application, the H-FR1 of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A or an antibody having the same H-FR1-4 as it.

[0094] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may have the amino acid sequence represented by SEQ ID NO: 234. QVQLVESGGGX1VQX2GGSLX3LSCAX4SX5X6X7X8X9IYAX 10 X 11 WFRQAPGKX 12 X 13 EFVAGX 14 GWX 15 X 16 X 17 TX 18 YYADSVKGRFX 19 ISRDNX 20 KNTX 21 X 22 LX 23 MNSLX 24 X 25 EDTAX 26 YYCAADX 27 DX 28 X 29 GFDYWGQGTX 30 is VTVSS (SEQ ID NO: 234), where X1 may be L or S, X2 may be P or V, X3 may be R or T, X4 may be A or T, X5 may be A, G, H, K, S, or V, X6 may be A, G, H, L, N, P, R, S, T, or V, X7 may be D, E, F, G, I, K, Q, R, S, or V, X8 may be D, G, H, K, L, P, Q, R, S, T, V, or Y, X9 may be A, F, N, Q, R, S, or T, X 10may be A or M, X 11 may be G or M, X 12 may be E or G, X 13 may be H or L, X 14 may be I or L, X 15 may be A, N, R, or S, X 16 may be A, F, W, or Y, X 17 may be A, G, or S, X 18 may be E, Q, S, or T, X 19 may be G or T, X 20 may be S or T, X 21 may be L or V, X 22 may be A or Y, X 23 may be L or Q, X 24 may be K or R, X 25 may be A or P, X 26 may be I or V, X 27 may be I or L, X 28 may be H, R, or V, X 29 may be A, D, G, H, L, N, R, S, T, or V, X 30 may be L or Q.

[0095] In this application, the above antigen-binding protein may include a heavy-chain variable region having the amino acid sequence represented by SEQ ID NO: 236. QVQLVESGGGLVQPGGSLRLSCAX1SX2X3X4X5X6IYAMGWFRQAPGKGLEFVAGIGWX7X8X9TX 10 YYADSVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAADX 11 DX 12 X 13GFDYWGQGTLVTVSS (SEQ ID NO: 236), where X1 may be A or T, X2 may be G, H, or K, X3 may be A, G, L, N, S, or V, X4 may be D, R, or V, X5 may be G, H, P, Q, R, or T, X6 may be R, S, or T, X7 may be A or N, X8 may be F or Y, X9 may be A or S, X 10 may be E, Q, or T, X 11 may be I or L, X 12 may be H or R, X 13 may be A, H, or S.

[0096] In the present application, the above antigen-binding protein may include a heavy-chain variable region having the amino acid sequence represented by SEQ ID NO: 139. QVQLVESGGGSVQVGGSLTLSCAASX 26 X 27 X 28 X 29 X 30 IYAMGWFRQAPGKEHEFVAGX 51 GWX 54 X 55 X 56 TX 58 YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100 DX 102 X 103 GFDYWGQGTQVTVSS (SEQ ID NO: 139), where X 26 may be A, G, H, K, S or V, X 27 may be A, G, H, L, N, P, R, S, T or V, X 28 may be D, E, F, G, I, K, Q, R, S or V, X 29 may be D, G, H, K, L, P, Q, R, S, T, V or Y, X 30 may be A, F, N, Q, R, S or T, X 51 may be I or L, X 54 may be A, N, R or S, X55 may be A, F, W or Y, X 56 may be A, G or S, X 58 may be E, Q, S or T, X 100 may be I or L, X 102 may be H, R or V, X 103 may be A, D, G, H, L, N, R, S, T or V.

[0097] In the present application, the above antigen-binding protein may contain a heavy-chain variable region having the amino acid sequence represented by SEQ ID NO: 143. QVQLVESGGGSVQVGGSLTLSCAASX 26 X 27 X 28 X 29 X 30 IYAMGWFRQAPGKEHEFVAGIGWX 54 X 55 X 56 TX 58 YYADSVKGRFGISRDNTKNTVALLMNSLKPEDTAIYYCAADX 100 DX 102 X 103 GFDYWGQGTQVTVSS (SEQ ID NO: 143), where X 26 may be G, H or K, X 27 may be A, G, L, N, S or V, X 28 may be D, R or V, X 29 may be G, H, P, Q, R or T, X 30 may be R, S or T, X 54 may be A or N, X 55 may be F or Y, X 56 may be A or S, X 58 may be E, Q or T, X 100 may be I or L, X 102 may be H or R, X 103 may be A, H or S.

[0098] In the present application, the heavy-chain variable region may have an amino acid sequence represented by any one of SEQ ID NOs: 72-120 and / or 163-227. In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have an amino acid sequence represented by SEQ ID NO: 1, the above-mentioned HCDR2 may have an amino acid sequence represented by SEQ ID NO: 2, the above-mentioned HCDR3 may have an amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have an amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have an amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have an amino acid sequence represented by SEQ ID NO: 70, the above-mentioned H-FR4 may have an amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have an amino acid sequence represented by SEQ ID NO: 72. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A.

[0099] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 1, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 42, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 55, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-1 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 73. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-1 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-1.

[0100] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 4, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 43, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-2 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 74. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-2 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-2.

[0101] In the present application, the above antigen-binding protein may include a heavy chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 5, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 44, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-3 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 75. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-3 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above MHPD1-A-3.

[0102] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 6, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 45, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-4 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 76. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-4 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above MHPD1-A-4.

[0103] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 6, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 45, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 147, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-4 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 182. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-4 H or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above MHPD1-A-4H.

[0104] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 7, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-5 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 77. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-5 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above MHPD1-A.

[0105] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 8, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 47, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-6 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 78. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-6 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above MHPD1-A-6.

[0106] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-7 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 79. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-7 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-7.

[0107] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-7 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 185. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-7 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-7 H.

[0108] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 10, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 45, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-8 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 80. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-8 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-8.

[0109] In this application, the above antigen-binding protein may include a heavy chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 10, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 45, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 147, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-8 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 186. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-8 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-8 H.

[0110] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 11, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 44, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-9 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 81. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-9 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-9.

[0111] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 12, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 43, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-10 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 82. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-10 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-10.

[0112] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 13, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 48, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-11 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 83. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-11 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-11.

[0113] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 14, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 45, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-12 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 84. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-12 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-12.

[0114] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 15, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 47, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-13 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 85. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-13 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-13.

[0115] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 15, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 47, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 147, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-13 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 191. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-13 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-13 H.

[0116] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 16, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 43, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-14 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 86. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-14 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-14.

[0117] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 17, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 49, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-15 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 87. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-15 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-15.

[0118] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 17, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 49, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-15 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 193. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-15 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-15 H.

[0119] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 18, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-16 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 88. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-16 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-16.

[0120] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 19, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-17 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 89. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-17 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-17.

[0121] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 20, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 50, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 56, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-18 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 90. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-18 or an antigen-binding protein (such as a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (such as a single-domain antibody) having the same VH as the above MHPD1-A-18.

[0122] In the present application, the above antigen-binding protein may include a heavy chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 21, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 57, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-19 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 91. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-19 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-19.

[0123] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 22, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-20 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 92. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-20 or an antigen-binding protein (such as a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (such as a single-domain antibody) having the same VH as the above MHPD1-A-20.

[0124] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 51, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-21 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 93. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-21 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-21.

[0125] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 58, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-22 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 94. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-22 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-22.

[0126] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 23, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-23 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 95. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-23 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-23.

[0127] In the present application, the antigen-binding protein described above may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 24, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 59, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the antigen-binding protein described above may include the single-domain antibody MHPD1-A-24 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the antigen-binding protein described above may have the amino acid sequence represented by SEQ ID NO: 96. For example, the antigen-binding protein described above may include the single-domain antibody MHPD1-A-24 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the antigen-binding protein described above may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-24.

[0128] In the present application, the above antigen-binding protein may include a heavy chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 24, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 59, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-24 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 202. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-24 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-24 H.

[0129] In the present application, the antigen-binding protein described above may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 25, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 60, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include the single-domain antibody MHPD1-A-25 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as that. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 97. For example, the above-mentioned antigen-binding protein may include the single-domain antibody MHPD1-A-25 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as that. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-25.

[0130] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 25, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 60, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-25 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 203. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-25 H or an antigen-binding protein (such as a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (such as a single-domain antibody) having the same VH as the above MHPD1-A-25 H.

[0131] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 26, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 61, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-26 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 98. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-26 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-26.

[0132] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 27, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-27 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 99. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-27 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-27.

[0133] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 28, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 52, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-28 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 100. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-28 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-28.

[0134] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 29, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-29 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 101. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-29 or an antigen-binding protein (such as a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (such as a single-domain antibody) having the same VH as the above MHPD1-A-29.

[0135] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 25, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 52, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 62, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-30 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 102. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-30 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-30.

[0136] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 25, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 52, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 62, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-30 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 208. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-30 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-30 H.

[0137] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 50, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 63, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-31 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 103. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-31 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-31.

[0138] In the present application, the antigen-binding protein described above may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 30, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-32 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 104. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-32 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-32.

[0139] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 30, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-32 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 210. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-32 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-32 H.

[0140] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 31, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-33 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 105. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-33 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-33.

[0141] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 32, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 53, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-35 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 106. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-35 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-35.

[0142] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 33, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 52, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-36 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 107. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-36 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-36.

[0143] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 34, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 64, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-37 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 108. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-37 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-37.

[0144] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 35, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 57, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-38 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 109. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-38 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above MHPD1-A-38.

[0145] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 54, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-39 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 110. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-39 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-39.

[0146] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 54, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-39 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 217. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-39 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-39 H.

[0147] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 65, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-40 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 111. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-40 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-40.

[0148] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 36, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-41 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 112. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-41 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-41.

[0149] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 37, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 52, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-42 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as that. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 113. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-42 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as that. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-42.

[0150] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 37, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 52, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-42 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 223. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-42 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-42 H.

[0151] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 59, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-43 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 114. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-43 or an antigen-binding protein (such as a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (such as a single-domain antibody) having the same VH as the above MHPD1-A-43.

[0152] In the present application, the antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 38, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 54, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-44 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 115. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-44 or an antigen-binding protein (for example, a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (for example, a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-44.

[0153] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 38, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 54, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-44 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 222. For example, the above antigen-binding protein may include the single-domain antibody MHPD1-A-44 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-44 H.

[0154] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 39, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-45 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 116. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-45 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-45.

[0155] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 39, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 148, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 150, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 161, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 162. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-45 H or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 223. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-45 H or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-45 H.

[0156] In the present application, the antigen-binding protein described above may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 40, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 55, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include the single-domain antibody MHPD1-A-46 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as that. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 117. For example, the above-mentioned antigen-binding protein may include the single-domain antibody MHPD1-A-46 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as that. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-46.

[0157] In this application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 41, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 3, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-47 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 118. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-47 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-47.

[0158] In the present application, the above antigen-binding protein may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above HCDR3 may have the amino acid sequence represented by SEQ ID NO: 66, the above H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, the above H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-48 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 119. For example, the above antigen-binding protein may include a single-domain antibody MHPD1-A-48 or an antigen-binding protein (e.g., a single-domain antibody) having the same HCDR3 (e.g., the same HCDR1-3) as it. In some cases, the above antigen-binding protein may include an antigen-binding protein (e.g., a single-domain antibody) having the same VH as the above MHPD1-A-48.

[0159] In the present application, the antigen-binding protein described above may include a heavy-chain variable region that may include HCDR1-3 and H-FR1-4. For example, the above-mentioned HCDR1 may have the amino acid sequence represented by SEQ ID NO: 9, the above-mentioned HCDR2 may have the amino acid sequence represented by SEQ ID NO: 46, the above-mentioned HCDR3 may have the amino acid sequence represented by SEQ ID NO: 67, the above-mentioned H-FR1 may have the amino acid sequence represented by SEQ ID NO: 68, the above-mentioned H-FR2 may have the amino acid sequence represented by SEQ ID NO: 69, the above-mentioned H-FR3 may have the amino acid sequence represented by SEQ ID NO: 70, and the above-mentioned H-FR4 may have the amino acid sequence represented by SEQ ID NO: 71. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-49 or an antigen-binding protein having the same HCDR1-3 and H-FR1-4 as it. For example, the heavy-chain variable region of the above-mentioned antigen-binding protein may have the amino acid sequence represented by SEQ ID NO: 120. For example, the above-mentioned antigen-binding protein may include a single-domain antibody MHPD1-A-49 or an antigen-binding protein (such as a single-domain antibody) having the same HCDR3 (for example, the same HCDR1-3) as it. In some cases, the above-mentioned antigen-binding protein may include an antigen-binding protein (such as a single-domain antibody) having the same VH as the above-mentioned MHPD1-A-49.

[0160] In the present application, the above-mentioned isolated antigen-binding protein further includes an immunoglobulin Fc region. For example, the present application provides a polypeptide including the antigen-binding protein of the present application and the Fc region of an immunoglobulin.

[0161] In the present application, the above-mentioned VH of the above-mentioned antigen-binding protein is directly or indirectly linked to the above-mentioned Fc region. In the present application, in the above-mentioned antigen-binding protein, the C-terminus of the above-mentioned VH is directly or indirectly linked to the N-terminus of the above-mentioned Fc region. In the present application, in the above-mentioned antigen-binding protein, the above-mentioned VH is fused in-frame with the above-mentioned Fc region.

[0162] In the present application, the above antigen-binding protein has the above VH linked to the above Fc region via a linker. For example, the above linker includes a peptide linker. For example, the above linker includes a flexible linker.

[0163] In the present application, the above isolated antigen-binding protein sequentially includes the above VH, the above linker, and the above Fc region of the above antigen-binding protein from the N-terminus to the C-terminus.

[0164] In the present application, the above Fc region includes an Fc derived from IgG1 or an Fc derived from IgG4. For example, the above Fc region may have an amino acid sequence represented by any one of SEQ ID NOs: 121-125.

[0165] Polypeptides and immune complexes On the other hand, the present application provides one or more polypeptides that may include the isolated antigen-binding protein of the present application. For example, the above polypeptide may include a fusion protein. On the other hand, the present application provides one or more immune complexes that may include the isolated antigen-binding protein of the present application. In some embodiments, the above immune complex may also include a pharmaceutically acceptable therapeutic agent.

[0166] Nucleic acids, vectors, cells and pharmaceutical compositions On the other hand, the present application further provides one or more isolated nucleic acid molecules capable of encoding the isolated antigen-binding protein described in the present application. For example, each of the above one or more nucleic acid molecules can encode the whole or a part of the above antigen-binding protein (for example, one or more of HCDR1-3, the heavy chain variable region).

[0167] The nucleic acid molecules described in this application may be isolated. For example, they can be produced or synthesized by a method consisting of (i) being amplified in vitro, for example by polymerase chain reaction (PCR), (ii) being recombinantly produced by cloning, (iii) being purified, for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) being synthesized, for example by chemical synthesis. For example, the isolated nucleic acid described above may be a nucleic acid molecule prepared by recombinant DNA technology.

[0168] In this application, the nucleic acid encoding the isolated antigen-binding protein described above can be prepared by various methods known in the art, including but not limited to using reverse transcription PCR and PCR to obtain the nucleic acid molecule of the isolated antigen-binding protein described in this application.

[0169] On the one hand, the present application provides one or more vectors comprising one or more nucleic acid molecules described in the present application. Each vector may contain one or more of the above nucleic acid molecules. The above vectors may also contain other genes such as a marker gene that enables the selection of the vector in a suitable host cell and under suitable conditions. The above vectors may further contain an expression control element that enables the accurate expression of the coding region in a suitable host. Such control elements are known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that control gene transcription or mRNA translation. In some embodiments, the above expression control sequences are elements that can be regulated. The specific structure of the above expression control sequences may vary depending on the species or cell type function, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences that are involved in the initiation of transcription and translation, such as TATA boxes, cap sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region containing a promoter sequence for the transcriptional control of a functionally linked nucleic acid. The above expression control sequences may also include enhancer sequences or upstream activation sequences. In the present application, suitable promoters include, for example, promoters for SP6, T3, and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and those artificial hybrid promoters (e.g., CMV) in which a part is fused to a part of the gene promoter of another cellular protein (e.g., human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) and may or may not contain additional introns. The one or more nucleic acid molecules described in the present application may be operably linked to the above expression control elements.

[0170] Examples of the above vectors include plasmids, cosmids, viruses, bacteriophages, or other vectors conventionally used in genetic engineering, for example. For example, the above vector may be an expression vector. For example, the above vector may be a viral vector. Viral vectors can be administered directly to a patient (in vivo) or indirectly, such as by treating cells with the virus in vitro and administering the treated cells to the patient (ex vivo). Viral vector technology is known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other manuals of virology and molecular biology. Conventional virus-based systems may include retroviral vectors for gene transfer, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and herpes simplex viral vectors. In some cases, genes can be introduced and integrated into the host genome using methods involving retroviruses, lentiviruses, or adeno-associated viruses, enabling long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Lentiviral vectors may contain long terminal repeat sequences 5'LTR and shortened 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS), and / or post-transcriptional regulatory element (WPRE). The vectors described in this application can be introduced into cells.

[0171] On the one hand, the present application provides cells. The above cells may contain the isolated antigen-binding protein described in the present application, the above polypeptide, the above immune complex, one or more nucleic acid molecules and / or one or more vectors described in the present application. For example, each type of cell or each cell may contain one or one type of nucleic acid molecule or vector described in the present application. For example, each type of cell or each cell may contain a plurality (e.g., two or more) or a plurality of types (e.g., two or more) of nucleic acid molecules or vectors described in the present application. For example, the vectors described in the present application can be introduced into the above host cells such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS / 0 cells, HEK293T cells, 293F cells or HEK293A cells, or other eukaryotic cells such as plant-derived cells, fungi or yeast cells. The vectors described in the present application can be introduced into the above host cells by methods known in the art such as electroporation, lipofectine transfection, lipofectamin transfection, etc. For example, the above cells may include yeast cells. For example, the above cells may include Escherichia coli cells. For example, the above cells may include mammalian cells. For example, the above cells may include immune cells.

[0172] The above cells may include immune cells. In some cases, the above cells may include immune cells. For example, the above cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells and / or peripheral blood mononuclear cells.

[0173] On the one hand, the present application provides a pharmaceutical composition. The above-mentioned pharmaceutical composition may include the isolated antigen-binding protein described in the present application, the above-mentioned polypeptide, the above-mentioned immune complex, the above-mentioned isolated nucleic acid molecule, the above-mentioned vector, the above-mentioned cell, and / or a pharmaceutically acceptable adjuvant and / or excipient. In the present application, the above-mentioned pharmaceutically acceptable adjuvant may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a carbohydrate, a chelating agent, a counter ion, a metal complex and / or a nonionic surfactant. Any conventional medium or reagent can also be considered for use in the pharmaceutical composition of the present application, provided that it is not incompatible with the cells described in the present application. In the present application, the above-mentioned pharmaceutically acceptable excipient may include additives other than the main drug of the pharmaceutical preparation, and is also called an auxiliary. For example, the above-mentioned excipient may include a binder, a filler, a disintegrant, and a lubricant for tablets. For example, the above-mentioned excipient may include wine, vinegar, medicinal juice, etc. of traditional Chinese medicine pills. For example, the above-mentioned excipient may include the matrix part in semi-solid preparations such as ointments and creams. For example, the above-mentioned excipients include preservatives, antioxidants, flavoring agents, fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, coloring agents, etc. for liquid preparations.

[0174] Detection methods, kits, uses and methods On the other hand, the present application provides a method for detecting the presence and / or content of PD-1, which includes administering the above-mentioned isolated antigen-binding protein or the above-mentioned polypeptide.

[0175] In the present application, the above-mentioned method may include an in vitro method, a non-diagnostic or non-therapeutic method. For example, the above-mentioned method includes 1) a step of contacting a sample with the antigen-binding protein of the present application; and 2) a step of determining the presence and / or expression level of PD-1 in a sample obtained from a subject by detecting the presence and / or content of the above-mentioned antigen-binding protein bound to the sample It may include a non-diagnostic method for detecting the presence and / or content of PD-1 that may be included. On the other hand, this application provided a kit that may include the above-mentioned isolated antigen-binding protein or the above-mentioned polypeptide.

[0176] In this application, the above-mentioned kit may also include an instruction manual describing a method for detecting the presence and / or content of PD-1. For example, the above-mentioned method may include an in vitro method, an ex vivo method, a non-diagnostic or non-therapeutic method. On the other hand, this application provided the use of the above-mentioned isolated antigen-binding protein or the above-mentioned polypeptide in the preparation of a kit used in a method for detecting the presence and / or content of PD-1. For example, the above-mentioned method may include an in vitro method, an ex vivo method, a non-diagnostic or non-therapeutic method. On the other hand, this application provided the use of the above-mentioned isolated antigen-binding protein and / or the above-mentioned polypeptide in the preparation of a pharmaceutical for preventing and / or treating a disease or illness.

[0177] On the other hand, this application provided the above-mentioned isolated antigen-binding protein, the above-mentioned polypeptide, the above-mentioned immune complex, the above-mentioned isolated nucleic acid molecule, the above-mentioned vector, the above-mentioned cell and / or the above-mentioned pharmaceutical composition for preventing, alleviating and / or treating a disease or illness. For example, the above-mentioned disease or illness may include a tumor. For example, the above-mentioned tumor may include a solid tumor. For example, the above-mentioned tumor may include a hematological tumor. For example, the above-mentioned tumor may include a tumor related to the expression of PD-L1. For example, the above-mentioned heavy chain may include a tumor related to the upregulation of PD-L1 expression. For example, the above-mentioned tumor may be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma, and breast cancer.

[0178] In one aspect, the present application provides a method for preventing and / or treating a disease or disorder, the method comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the immune complex, the isolated nucleic acid molecule, the vector, and / or the cell as described above. For example, the disease or disorder may include a tumor. For example, the tumor may include a solid tumor. For example, the tumor may include a hematological tumor. For example, the tumor may include a tumor associated with the expression of PD-L1. For example, the heavy chain may include a tumor associated with the upregulation of PD-L1 expression. For example, the tumor may be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma, and breast cancer.

[0179] In one aspect, the present application provides a method for inhibiting the interaction between PD-1 and PD-L1, the method comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the immune complex, the isolated nucleic acid molecule, the vector, and / or the cell as described above. The method may be an ex vivo or in vitro method. In the present application, the method may include contacting a biological sample with the antigen-binding protein and / or PD-L1 described in the present application under conditions that allow binding of the antigen-binding protein to PD-1 and / or PD-L1, and detecting whether a complex is formed between the antigen-binding protein and PD-1 and whether a complex is formed between PD-1 and PD-L1.

[0180] In one aspect, the present application provides the isolated antigen-binding protein, the polypeptide, the immune complex, the isolated nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition as described above for inhibiting the interaction between PD-1 and PD-L1. On the one hand, the present application provides the use of the above-mentioned isolated antigen-binding protein and / or the above-mentioned polypeptide in the preparation of a pharmaceutical for inhibiting the interaction between PD-1 and PD-L1.

[0181] The following embodiments are only intended to interpret the antigen-binding protein, preparation method and use of the present application without intending to be limited by any theory, and are not intended to limit the inventive scope of the present application.

[0182] Examples Example 1 Immunization of Alpaca One healthy adult female alpaca was immunized with recombinant human PD-1 protein. In the first immunization, 1 mg of recombinant human PD-1 protein was emulsified with an equal volume of Freund's complete adjuvant and then subjected to multiple subcutaneous injections. In the booster immunizations, 0.5 mg of recombinant human PD-1 protein and an equal volume of Freund's incomplete adjuvant were emulsified and then subjected to multiple subcutaneous injections, and a total of 3 booster immunizations were performed.

[0183] Example 2 Construction of Alpaca Immunization Library 2.1 After the immunization was completed, the peripheral blood of the alpaca was collected, lymphocytes were separated using lymphocyte separation medium (Solarbio, Cat. P8900), and total RNA was extracted using TRIzol TM Reagent (Thermo Fisher Scientific, Cat. 15596018). 2.2 cDNA was obtained by reverse transcription using PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, Code No. 6210B), and the VHH gene was amplified by nested PCR. After recovering the 2.3 VHH gene fragment using a gel purification kit (TAKARA, Code No. 9761), digesting it with the restriction endonuclease sfil (TAKARA, Code No. 1244A), cloning it into the phagemid vector pComb 3XSS, and transforming the constructed clone into E. coli TG1 electrocompetent cells, a PD-1 single-domain antibody library was obtained. 2.4 Using the plate gradient dilution method, the library capacity was measured to be 3.2×10 8 It was shown from the results of colony PCR that the cloning rate of the library was 98%. An appropriate amount of the library bacterial solution (bacterial count approximately 3.2×10 9 ) was taken, inoculated into 100 mL of LB medium (containing ampicillin), cultured until the mid-logarithmic phase, an appropriate amount of helper phage M13KO7 was added, and after infection for 60 minutes, the medium was replaced with 200 mL of 2×YT (containing ampicillin and kanamycin), cultured at 30°C with vigorous shaking for 12 hours, the supernatant was obtained by centrifugation, an appropriate amount of 20% PEG-NaCl solution was added to precipitate the phage, and after resuspending with an appropriate amount of PBS and then precipitating, a phage display library that could be directly used for subsequent screening was obtained.

[0184] Example 3 Screening of Nanobodies The PD-1 nanobody was subjected to a total of 3 screening rounds using the solid-phase affinity screening method. 3.1 Coating: The microtiter plate was coated with recombinant human PD-1 protein at a coating concentration of 100 μg / mL in the first round, 50 μg / mL in the second round, and 20 μg / mL in the third round overnight at 4°C. 3.2 Blocking: The first and third rounds were blocked with 3% ovalbumin, and the second round was blocked with 3% bovine serum albumin at 37°C for 1 hour. 3.3 Binding and washing: In the first round of screening, after removing the blocking solution, 100 μL of the phage display library of the nanobody constructed in Example 2 was added to the microplate, incubated at 37 °C for 1 hour for binding, and then the unbound library was removed. The microplate was washed with PBST and PBS (5 times each) to remove unbound or weakly binding phages; in the second round, it was washed 10 times each with PBST and PBS, and in the third round, it was washed 20 times each with PBST and PBS. 3.4 Elution of specific phages: 100 μL of glycine hydrochloride buffer (pH 2.2) was added to the microplate to dissociate the specific phages. After gently shaking for 10 minutes, the eluate was collected. 3.5 Titer measurement and amplification of the eluate: E. coli TG1 in the logarithmic growth phase was infected with the eluate. A small amount of infected E. coli TG1 was serially diluted, and an appropriate amount of the serially diluted solution was uniformly spread on LB solid medium (plate dilution method). After 12 - 14 hours, the plates were counted to calculate the elution amount of phages, and the remaining phages were amplified to obtain a specific phage enrichment library for the next round of screening. 3.6 Identification: 93 clones were randomly picked from the plates for measuring the eluate titers in the second and third rounds described above, and phages were obtained by amplification and identified by ELISA. The method was as follows: The plate was coated with recombinant human PD - 1 protein (coating concentration 2 μg / mL), blocked with 5% skim milk, then the phages to be identified were added and allowed to bind at 37 °C for 1 hour. After washing, Goat anti - Llama IgG (H + L) Secondary Antibody [HRP] was added and allowed to bind at 37 °C for 1 hour. After washing, TMB substrate solution was added to develop color, and the OD was measured with a microplate reader. 450It was read. Positive control wells (enrichment library for each round), negative control wells (helper phage M13KO7), and blank wells (PBS) were set up. When the OD value of the well to be measured was 3 times or more that of the negative control, it was determined to be positive. The corresponding clones of the positive wells were preserved, plasmids were extracted, sequenced, and the DNA sequences were translated into amino acid sequences for comparison. Clones with all the same HCDR1, HCDR2, and HCDR3 sequences were regarded as the same antibody strain, and clones with different HCDR sequences were regarded as different antibody strains.

[0185] Example 4 Expression and Purification of VHH The positive clones screened in Example 3 were transformed into Top10F', and the expression of VHH was induced by IPTG. Cell pellets were collected by centrifugation, the supernatant was taken after sonication, purified by TALON (registered trademark) Metal Affinity Resins, and the buffer was replaced with phosphate buffer. The purified VHH was quantified by measuring the OD280 value and stored at 4°C for future use. Here, SDS PAGE showing the screening of VHH with high affinity for the antigen (human recombinant PD-1) and capable of competing with human recombinant PD-L1 for binding to human recombinant PD-1 is shown in Figure 1, and the obtained VHH was named MHPD1-A.

[0186] Example 5 ELISA Experiment on the Competition of VHH with Recombinant Human PD-L1 in Binding to Antigen Protein Recombinant human PD-1 antigen (2 μg / mL) was added to a 96-well plate and coated overnight at 4°C. It was blocked with 1% casein. The MHPD1-A sample was serially diluted 2-fold with 0.1% BSA / PBS starting from a concentration of 20 μg / mL. 4 μg / mL of recombinant human PD-L1-Fc and different concentrations of VHH were added to a 96-well plate coated with the antigen (recombinant human PD-1 protein) and incubated at room temperature for 1 hour. The well plates were washed with PBST during different incubations. Detection was performed with a horseradish peroxidase-labeled anti-human IgG secondary antibody (1:5000, containing 1% casein), and curves were fitted using Graphpad Prism software to calculate the EC 50 values, and the results are shown in Table 1. From the results, it was revealed that MHPD1-A could effectively inhibit the binding of human recombinant PD-L1 and human recombinant PD-1.

[0187]

Table 1

[0188] Example 6 Expression and Purification of Fusion Protein A pTT5 recombinant vector having nucleic acid sequences encoding MHPD1-A and the human Fc fragment (the C-terminus of VHH is directly linked to the N-terminus of the human Fc hinge region) was synthesized. As follows, 15 μg of plasmid DNA was diluted with Opti-MEM® I until the total volume reached 0.5 mL, and 30 μL of 293fectin TM reagent was diluted with Opti-MEM® I until the total volume reached 0.5 mL; a liposome-DNA complex was prepared by gently mixing and incubating at room temperature for 5 minutes. The diluted pTT5 recombinant vector was added to the diluted 293fectin TMIn addition to the reagent, it was gently and uniformly mixed and incubated at room temperature for 30 minutes. The above mixture was added to a shake flask containing 1 × 106 cells / mL cells and placed in a cell incubator containing 8% CO2 at 37°C, and incubated with an orbital shaker rotating at 125 rpm. On the 4th day after transfection, the supernatant was collected, purified with PierceTM Protein A agarose gel, the buffer was replaced with phosphate buffer, and the resulting fusion protein was named MHPD1-A Fc.

[0189] Example 7 Interspecies Cross-validation of Fusion Proteins with Human, Monkey, and Mouse PD-1 A 96-well Greiner plate was coated overnight at 4°C with 2 μg / mL of recombinant human PD-1-his protein, recombinant monkey PD-1-his protein, and recombinant mouse PD-1-his protein, respectively. On the second day, after blocking with 1% casein, 5 μg / mL and 1.67 μg / mL of nanobody-Fc fusion protein samples were added to the antigen-coated plate and incubated for 2 hours. Then, the plate was washed with PBST, anti-human IgG-HRP (diluted 1:5000 with 1% casein) was added and incubated for 1 hour to perform a binding assay. As shown in Figure 2, MHPD1-A Fc could bind to recombinant human PD-1-his and recombinant monkey PD-1-his, but did not show binding activity to recombinant mouse PD-1-his.

[0190] Example 8 Detection of Affinity of Fusion Protein for Recombinant Human PD-1 Using an amine coupling kit (GE Healthcare, Cat No. BR100050), approximately 250 RU of recombinant human PD-1-his protein was coupled to a CM5 chip at 25 °C according to standard amine coupling procedures. Serial two-fold diluted MHPD1-A Fc samples (5 concentration detections) were sequentially injected at a flow rate of 30 μL / min at 25 °C with a binding time of 120 s and a dissociation time of 600 s. Using Biacore® 8K analysis software (BIAevaluation), the curve was fitted to a 1:1 Langmuir binding model to determine kinetic constants such as the association rate constant, dissociation rate constant, and dissociation constant of binding. The results revealed that MHPD1-A Fc has high affinity for recombinant human PD-1, and the kinetic constants of the binding of MHPD1-A Fc to recombinant human PD1-his are shown in Table 2.

[0191] [Table 2]

[0192] Example 9 ELISA Experiment on Competition between Recombinant Human PD-L1 and Fusion Protein in Binding to Recombinant Human PD-1 Recombinant human PD-L1 (C-Fc, 0.2 μg / well) was added to a 96-well plate and coated overnight at 4 °C. Blocking was performed with 1% casein. MHPD1-A Fc was serially diluted two-fold with 0.1% BSA / PBS at an initial concentration of 10 μg / mL. 0.5 μg / mL recombinant human PD-1 (C-Fc & C-Avi & C-6×His) and different concentrations of MHPD1-A Fc were added to the 96-well plate coated with the antigen and incubated at room temperature for 1 hour. The well plates were washed with PBST during different incubations. Detection was performed with horseradish peroxidase-labeled streptavidin (1:10000), and the curve was fitted using Graphpad Prism software to calculate the EC 50 value, and the results are shown in Table 3. From the results, it was revealed that the tested MHPD1-A Fc could competitively inhibit the binding of recombinant human PD-L1 and recombinant human PD-1, and its activity was equal to or higher than that of the positive control Nivolumab.

[0193]

Table 3

[0194] Example 10 Binding Activity of the Fusion Protein to HEK293 Cells Overexpressing Human PD-1 HEK293 cells overexpressing human PD-1 were cultured in DMEM medium containing 10% FBS in a CO2 incubator (37 °C, 5% CO2). The cells were collected and washed once with the test buffer. The cells were counted to detect the viability, and 3 × 105 live cells were added to each test tube. 100 μL of MHPD1-A Fc with different concentrations (3000 ng / mL, 1000 ng / mL, 300 ng / mL, 100 ng / mL, 30 ng / mL, 10 ng / mL, 3 ng / mL, 1 ng / mL, and 0.3 ng / mL) was added and incubated at 4 °C for 1 hour. After washing the cells with the washing buffer, 100 μL of PE-anti-human IgG was added according to the instructions and incubated in the dark at 4 °C for 1 hour. The cells were washed three times with the washing buffer, and each sample was resuspended in 200 μL of PBS and detected by a flow cytometer. The data were analyzed using FCS Express 6 Plus and GraphPad Prism 5 software. From the results, it was revealed that MHPD1-A Fc was capable of binding to HEK293 cells overexpressing human PD-1, and the EC of the binding of MHPD1-A Fc to HEK293 cells overexpressing human PD-1 50 is shown in Table 4.

[0195]

Table 4

[0196] Example 11 Competition of the fusion protein with human PD-L1 in binding to HEK293 cells overexpressing human PD-1 HEK293 cells overexpressing human PD-1 were cultured in a CO2 incubator (37 °C, 5% CO2) in DMEM medium containing 10% FBS and 1 mg / mL G418. The cells were collected and washed once with the test buffer. The cells were counted and the viability was detected, and 3 × 105 live cells (cell viability ≧ 95%) were added to each test tube. 1.0 μg / mL biotinylated human PD-L1 protein and serially diluted MHPD1-A Fc (concentration 0.03 μg / mL to 10 μg / mL) were mixed at room temperature and incubated for 30 minutes, and the mixtures were added to centrifuge tubes containing 3 × 105 live cells respectively and incubated at 4 °C for 1 hour. After the cells were washed with the wash buffer, 100 μL of PE-SA was added according to the instructions and incubated at 4 °C for 1 hour. The cells were washed 3 times with the wash buffer, each sample was resuspended in 100 μL of PBS, and detected with a flow cytometer. The data were analyzed using FCS Express 6 Plus and GraphPad Prism 5 software. The results revealed that MHPD1-A Fc could competitively inhibit the binding of recombinant human PD-L1 to HEK293 cells overexpressing hPD-1, and its competitive binding activity was slightly superior to that of the positive control Nivolumab. The EC 50 value of the competition of MHPD1-A Fc with human PD-L1 in binding to HEK293 / hPD1 is shown in Table 5.

[0197] [Table 5]

[0198] Example 12 Detection of the affinity kinetics of the fusion protein and pembrolizumab for the antigen protein Detection of affinity kinetics by Biacore® 8K (GE Healthcare): Using an amine coupling kit (GE Healthcare, Cat No. BR100050), recombinant human PD-1-his protein was coupled to a CM5 chip (GE Healthcare, Cat No. BR100530) at 25 °C according to standard amine coupling procedures. In one experiment, Pembrolizumab (100 nM) and MHPD1-A Fc (2 μM), or Pembrolizumab (100 nM) and buffer, or buffer and MHPD1-A Fc (2 μM) were sequentially injected at a flow rate of 30 μL / min at 25 °C for a binding time of 300 s for each sample, and the affinity kinetics curve is shown in Figure 3A. In another experiment, MHPD1-A Fc (2 μM) and Pembrolizumab (100 nM), or MHPD1-A Fc (2 μM) and buffer, or buffer and Pembrolizumab (100 nM) were sequentially injected at a flow rate of 30 μL / min at 25 °C for a binding time of 300 s for each sample, and the affinity kinetics curve is shown in Figure 3B. Summarizing the above detection results, it is presumed that the epitopes of the binding of MHPD1-A Fc and Pembrolizumab to the PD1-His protein are identical or overlapping.

[0199] Example 13 Construction of VHH variants Based on the structural prediction and experience by performing database sequence analysis on the sequence of MHPD1-A, a series of VHH mutant sequences with some amino acid sites of HCDR1-3 mutated were designed. According to the designed VHH mutant sequences, full gene synthesis (His-tag) was performed respectively. The pcDNA3.4 plasmid was digested with NotI / XbaI (NEB, Cat. R0189L, R0145L), recombined with the gene sequences of VHH mutants, and the recombinant plasmid was transformed into TOP10 competent cells. The bacterial solution was spread and cultured at 37°C for 16 - 20 hours. Single colonies were selected from the plate for colony PCR, positive clones were identified by electrophoresis, positive clones were selected for sequencing, the correctly sequenced clones were subjected to large-scale culture, and the plasmids were extracted. HEK293 cells were transiently transfected with a liposome transfection reagent and placed in a cell incubator containing 5% CO2 at 37°C and incubated on an orbital shaker rotating at 125 rpm. On the 5th day, the supernatant was collected by centrifugation and purified by Ni affinity chromatography ((Ni Smart Beads 6FF, GE #17531802), and the buffer was replaced with phosphate buffer by dialysis to obtain a series of VHH mutants named MHPD1-A-4, MHPD1-A-7, MHPD1-A-8, MHPD1-A-13, MHPD1-A-15, MHPD1-A-24, MHPD1-A-25, MHPD1-A-30, MHPD1-A-32, MHPD1-A-39, MHPD1-A-42, MHPD1-A-44, and MHPD1-A-45 respectively.

[0200] Example 14 ELISA Experiment on the Competition of Recombinant Human PD-L1 with VHH Mutants in Binding to Antigen Protein Recombinant human PD-1 protein (2 μg / mL) was added to a 96-well plate and coated overnight at 4°C. Blocking was performed with 1% casein. VHH mutants were serially diluted 2-fold at an initial concentration of 20 μg / mL using 0.1% BSA / PBS. 4 μg / mL recombinant human PD-L1-Fc and different concentrations of VHH mutants were added to a 96-well plate coated with the antigen (recombinant human PD-1 protein) and incubated at room temperature for 1 hour. The well plates were washed with PBST during different incubations. Detection was performed with a horseradish peroxidase-labeled anti-human IgG secondary antibody (1:5000, containing 1% casein). From the results of the analysis, it was revealed that the VHH mutants could effectively inhibit the binding of recombinant human PD-L1 and recombinant human PD-1.

[0201] Example 15 Construction of a fusion protein containing a VHH mutant included in VHH mutants with high affinity were selected, and their gene sequences and Fc sequences were synthesized into full-length sequences. The pcDNA3.4 plasmid was digested with NotI / XbaI (NEB, Cat. R0189L, R0145L), recombined with the above full-length sequences, the recombinant plasmid was transformed into TOP10 competent cells, the bacterial solution was spread, and cultured at 37°C for 16 - 20 hours. Single colonies were selected from the plate for colony PCR, positive clones were identified by electrophoresis, positive clones were selected for sequencing, the correctly sequenced clones were subjected to large-scale culture, and plasmids were extracted. HEK293 cells were transiently transfected with a liposome transfection reagent, placed in a cell incubator containing 5% CO2 at 37°C, and incubated with an orbital shaker rotating at 125 rpm. On the 5th day, the supernatant was collected by centrifugation, purified by Protein A affinity chromatography, and the buffer was replaced with phosphate buffer by dialysis to obtain a fusion protein containing the VHH mutant.

[0202] Example 16 ELISA experiment on the competition of a fusion protein containing a VHH mutant with recombinant human PD-L1 in binding to an antigen protein Recombinant human PD-L1 (C-Fc, 0.2 μg / well) was added to a 96-well plate and coated overnight at 4°C. It was blocked with 1% casein. The VHH mutant fusion protein was serially diluted 2-fold at an initial concentration of 10 μg / mL using 0.1% BSA / PBS. 0.5 μg / mL recombinant human PD-1 (C-Fc & C-Avi & C-6×His) and VHH mutant fusion proteins at different concentrations were added to the antigen-coated 96-well plate and incubated at room temperature for 1 hour. The well plates were washed with PBST during different incubations. Detection was performed with horseradish peroxidase-labeled streptavidin (1:10,000). From the results of the analysis, it was revealed that the VHH mutant fusion protein could effectively inhibit the binding of recombinant human PD-L1 and recombinant human PD-1.

[0203] Example 17 Detection of Humanization of Nanobody and Binding Activity of Humanized Nanobody-Fc Fusion Protein to Antigen Protein By performing database sequence analysis on the sequence of the original camel antibody MHPD1-A, the reference human sequence Germeline was identified. After humanization in the first round, the degree of humanization was 86.44% to 90.68%. They were constructed into nanobody-Fc fusion proteins and transiently expressed in ExpiCHO-s. Based on the degree of humanization and affinity, the second round of humanization was performed. After humanization in the second round, the degree of humanization was all above 90%. They were constructed into nanobody-Fc fusion proteins and transiently expressed in ExpiCHO-s. The humanized sequences were designed to verify the binding activity. The binding activities of nanobody-Fc fusion proteins with different degrees of humanization to the antigen protein (human PD-1) were verified by ELISA method respectively after two rounds of humanization. 2 μg / mL of recombinant human PD-1-his antigen protein was added to a 96-well ELISA plate at 30 μL / well and coated overnight at 4°C. The plate was washed three times with PBST. 5% PBS-Milk was added and incubated at room temperature for 2 hours. The plate was washed three times with PBST. All nanobody-Fc fusion proteins were serially diluted 3-fold starting from an initial concentration of 10 μg / mL, added to each well at 30 μL / well, and incubated at room temperature for 1 hour. The plate was washed three times with PBST. The secondary antibody Goat-Anti-Human-IgG-Fc-HRP (purchased from Sigma) was diluted 1:8000, added to each well at 30 μL / well, and incubated at room temperature for 50 minutes. The plate was washed three times with PBST. TMB (purchased from SurModics) was added to each well at 30 μL / well and allowed to develop color for 1 to 5 minutes at room temperature in the dark. 2M hydrochloric acid was added at 30 μL / well to stop the reaction. The absorbance value at OD450 was read using a microplate reader. The curve was fitted using Graphpad Prism software to calculate the EC50 value and shown in Table 6. From the results, it was revealed that the humanized nanobody-Fc fusion protein could still effectively bind to the antigen protein, and some of the humanized nanobody-Fc fusion proteins had an affinity for the antigen protein equal to or higher than that of the parental antibody.

[0204]

Table 6

[0205] Example 18 Affinity Maturation of Humanized Nanobody and Detection of Binding Activity of Nanobody-Fc Fusion Protein to Antigen (First Round) The VHH of the parental antibody MHPD1-A-H19 was cloned into a phage display vector of a third party. A series of primers were designed to perform single-point mutations or three consecutive mutations on its HCDR respectively to construct an affinity mature phage display library. Using immunotube screening (i.e., solid-phase screening), the immunotubes were coated with the antigen protein hPD-1-His, the affinity mature phage display library was added, incubated, washed, and eluted. After 2-3 rounds of panning, highly affinity specific monoclonal antibodies were enriched. After affinity maturation screening, candidate antibodies with high affinity for the hPD-1-His antigen were screened at the ELISA level. Based on the affinity and sequence at the VHH level, 14 candidate molecules were selected and nanobody-Fc fusion proteins were constructed. Verification of the binding activity of affinity - matured nanobody - Fc fusion proteins to the antigen protein (hPD - 1 - his) by ELISA: Recombinant human PD - 1 - his antigen protein at 2 μg / mL was added to each well of a 96 - well ELISA plate at 30 μL / well and coated overnight at 4°C. The plate was washed 3 times with PBST. 5% PBS - Milk was added and incubated at room temperature for 2 hours. The plate was washed 3 times with PBST. All nanobody - Fc fusion proteins were serially diluted 3 - fold starting from an initial concentration of 10 μg / mL and added to each well at 30 μL / well, then incubated at room temperature for 1 hour. The plate was washed 3 times with PBST. The secondary antibody Goat - Anti - Human - IgG - Fc - HRP was diluted 1:8000 and added to each well at 30 μL / well, then incubated at room temperature for 50 minutes. The plate was washed 9 times with PBST. TMB was added to each well at 30 μL / well and allowed to develop color at room temperature in the dark for 1 - 5 minutes. 2M hydrochloric acid was added to each well at 30 μL / well to stop the reaction. The absorbance value at OD450 was read using a microplate reader. The curve was fitted using Graphpad Prism software to calculate the EC50 value (see Table 7). From the results, it was revealed that multiple nanobody - Fc fusion proteins had better binding activity to the antigen protein human PD - 1 than the parental MHPD1 - A - H19 Fc after affinity maturation.

[0206]

Table 7

[0207] Example 19 ELISA Experiment on the Competition of Affinity - Matured Nanobody - Fc Fusion Proteins with Recombinant Human PD - L1 in Binding to Recombinant Human PD - 1 Recombinant human PD-L1-mFc (8 μg / mL) was added to a 96-well plate and coated overnight at 4°C. The plate was washed three times with PBST. 5% PBS-Milk was added and incubated at room temperature for 1 hour. All nanobody-Fc fusion proteins were serially diluted 3-fold starting at a concentration of 10 μg / mL and added to each well at 30 μL / well and incubated at room temperature for 1 hour. 0.5 μg / mL recombinant human PD-1-Fc-Biotin was added to the antigen-coated 96-well plate at 30 μL / well and incubated at room temperature for 1 hour. The well plates were washed with PBST during different incubations. Detection was performed with horseradish peroxidase-labeled streptavidin (1:10000), and curves were fitted using Graphpad Prism software to calculate the IC50 values, which are shown in Table 8. From the results, it was revealed that multiple nanobody-Fc fusion proteins had increased competitive inhibitory activity against the antigen protein human PD-1 that binds to the ligand human PD-L1 after affinity maturation compared to before affinity maturation and were superior to the positive control.

[0208]

Table 8

[0209] Example 20 Detection of Biological Activity of Nanobody-Fc Fusion Protein Using Jurkat-NFAT-PD1 genetically engineered cells as effector cells, Hep3B-OS8-PDL1 as target cells, the biological activity of a nanobody-Fc fusion protein that blocks the binding of PD1 / PDL1 was detected by a luciferase assay system. Hep3B-OS8-PDL1 cells were resuspended in PRMI1640 medium supplemented with 10% FBS, seeded into a 96-well cell culture plate, and placed in a cell incubator for overnight culture. On the second day, the medium was removed from the cell culture plate, 50 μL of a pre-prepared medium containing nanobody-Fc fusion proteins with different concentrations and 50 μL of Jurkat-NFAT-PD1 cell suspension were added to each well, and then placed in a carbon dioxide incubator (37 °C, 5% CO2) for 6 hours of incubation. The cell culture plate was taken out and equilibrated at room temperature for 5 - 10 minutes. 100 μL of a luciferase reporter gene detection reagent (One-Glo TM Luciferase assay system, Promega) was added to each well. After incubation for at least 3 minutes, the fluorescence signal was measured with a microplate reader (see Table 9). From the results, it was revealed that the affinity-matured antibodies A-3 H Fc, A-4 H Fc, and A-7 H Fc could effectively block the inhibition of the binding of PD-1 and PD-L1 to the fluorescence signal of the downstream reporter gene, and their biological activity was equal to or higher than that of the positive control Pembrolizumab.

[0210]

Table 9

[0211] Example 21 Affinity Maturation of Nanobodies, Binding of Nanobody-Fc Fusion Proteins to Human PD-1, and Competitive Binding Activity with Human PD-L1 in Binding to Human PD-1 (Round 2) The VHH of the parent MHPD1-A-7 H was cloned into a phage display vector of a third party. A series of primers were designed to introduce single mutations or three consecutive mutations into its HCDR respectively to construct an affinity mature phage display library. Using immunotube screening (i.e., solid-phase screening), the immunotubes were coated with the antigen protein hPD-1-His, and the affinity mature phage display library was added for incubation, washing, and elution. After 2-3 rounds of panning, high-affinity specific monoclonal antibodies were enriched. After affinity maturation screening, candidate antibodies with high affinity for the hPD-1-His antigen were screened at the ELISA level, and 29 candidate molecules were selected based on the affinity and sequence at the VHH level to construct nanobody-Fc fusion proteins. Verification of the binding activity of affinity matured nanobody-Fc fusion proteins to the antigen protein (hPD-1-his) by ELISA method: 30 μL / well of 2 μg / mL recombinant human PD-1-his antigen protein was added to each well of a 96-well ELISA plate and coated overnight at 4°C. The plate was washed three times with PBST. 5% PBS-Milk was added and incubated at room temperature for 2 hours. The plate was washed three times with PBST. All nanobody-Fc fusion proteins were serially diluted 3-fold starting from an initial concentration of 10 μg / mL and added to each well at 30 μL / well and incubated at room temperature for 1 hour. The plate was washed three times with PBST. The secondary antibody Goat-Anti-Human-IgG-Fc-HRP was diluted 1:8000 and added to each well at 30 μL / well and incubated at room temperature for 50 minutes. The plate was washed nine times with PBST. 30 μL / well of TMB was added to each well and allowed to develop color at room temperature in the dark for 1-5 minutes. 30 μL / well of 2M hydrochloric acid was added to stop the reaction. The absorbance value at OD450 was read using a microplate reader. Using Graphpad Prism software, the curve was fitted to calculate the EC50 value and shown in Table 10. The binding activities of multiple nanobody-Fc fusion proteins to the antigen protein human PD-1 were equivalent to that of the parent MHPD1-A-7 H Fc.

[0212]

Table 10

[0213] A part of the fusion protein with high affinity for human PD-1 was selected, and its competitive binding activity with recombinant PD-L1 in binding to human PD-1 was detected. Recombinant human PD-L1-mFc (8 μg / mL) was added to a 96-well plate and coated overnight at 4°C. The plate was washed three times with PBST. 5% PBS-Milk was added and incubated at room temperature for 1 hour. All nanobody-Fc fusion proteins were serially diluted 3-fold starting from an initial concentration of 10 μg / mL, and 30 μL / well was added to each well and incubated at room temperature for 1 hour. Recombinant human PD-1-Fc-Biotin (0.5 μg / mL) was added to the antigen-coated 96-well plate at 30 μL / well and incubated at room temperature for 1 hour. The well plates were washed with PBST during different incubations. Detection was performed with horseradish peroxidase-labeled streptavidin (1:10000), and curves were fitted using Graphpad Prism software to calculate the IC50 values, which are shown in Table 11. From the results, it was revealed that after affinity maturation, the competitive inhibitory activity of MHPD1-A-15 H Fc against the antigen protein binding to the ligand h-PD-L1 was increased compared to that before affinity maturation, and the competitive inhibitory activities of multiple fusion proteins were superior to the positive control.

[0214]

Table 11

Claims

1. An isolated antigen-binding protein capable of binding to PD-1, comprising a heavy chain variable region VH, said VH comprising HCDR1, HCDR2, and HCDR3, and said HCDR1, HCDR2, and HCDR3 are 1) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 2) HCDR1: SEQ ID NO: 1, HCDR2: SEQ ID NO: 2, and HCDR3: SEQ ID NO: 3; 3) HCDR1: SEQ ID NO: 5, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 3; 4) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 3; 5) HCDR1: SEQ ID NO: 7, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 6) HCDR1: SEQ ID NO: 8, HCDR2: SEQ ID NO: 47, and HCDR3: SEQ ID NO: 3; 7) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 3; 8) HCDR1: SEQ ID NO: 11, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 3; 9) HCDR1: SEQ ID NO: 12, HCDR2: SEQ ID NO: 43, and HCDR3: SEQ ID NO: 3; 10) HCDR1: SEQ ID NO: 13, HCDR2: SEQ ID NO: 48, and HCDR3: SEQ ID NO: 3; 11) HCDR1: SEQ ID NO: 14, HCDR2: SEQ ID NO: 45, and HCDR3: SEQ ID NO: 3; 12) HCDR1: SEQ ID NO: 15, HCDR2: SEQ ID NO: 47, and HCDR3: SEQ ID NO: 3; 13) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 49, and HCDR3: SEQ ID NO: 3; 14) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3; 15) HCDR1: SEQ ID NO: 25, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 62; 16) HCDR1: SEQ ID NO: 34, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 64; 17) HCDR1: SEQ ID NO: 9, HCDR2: SEQ ID NO: 54, and HCDR3: SEQ ID NO: 3; and 18) HCDR1: SEQ ID NO: 39, HCDR2: SEQ ID NO: 46, and HCDR3: SEQ ID NO: 3 comprising an amino acid sequence of any one group selected from an isolated antigen-binding protein comprising VHH.

2. comprising H-FR1, the C-terminus of said H-FR1 being directly or indirectly linked to the N-terminus of said HCDR1, said H-FR1 comprising the amino acid sequence shown in SEQ ID NOs: 68, 144-148, 230 and / or 235, the isolated antigen-binding protein according to claim 1.

3. comprising H-FR2, said H-FR2 being located between said HCDR1 and said HCDR2, said H-FR2 comprising the amino acid sequence shown in SEQ ID NOs: 69, 149-151, and / or 231, the isolated antigen-binding protein according to claim 1 or 2.

4. comprising H-FR3, said H-FR3 being located between HCDR2 and HCDR3, said H-FR3 comprising the amino acid sequence shown in SEQ ID NOs: 70, 152-161, and / or 232, the isolated antigen-binding protein according to any one of claims 1 to 3.

5. comprising H-FR4, the N-terminus of said H-FR4 being directly or indirectly linked to the C-terminus of HCDR3, said H-FR4 comprising the amino acid sequence shown in SEQ ID NOs: 71, 162, and / or 233, the isolated antigen-binding protein according to any one of claims 1 to 4.

6. The isolated antigen-binding protein according to any one of claims 1 to 5, wherein the VH comprises an amino acid sequence shown in any one of SEQ ID NOs: 72, 181 to 191, 193, 201, 208, 215, 217, and 223.

7. The isolated antigen-binding protein according to any one of claims 1 to 6, wherein the antibody comprises a chimeric antibody and / or a humanized antibody.

8. The isolated antigen-binding protein according to any one of claims 1 to 7, further comprising an Fc region of an immunoglobulin.

9. The isolated antigen-binding protein according to claim 8, wherein the C-terminus of the VH of the antigen-binding protein is directly or indirectly linked to the N-terminus of the Fc region.

10. The isolated antigen-binding protein according to claim 8 or 9, wherein the Fc region comprises an amino acid sequence shown in any one of SEQ ID NOs: 121, 123 to 125 and / or 228 to 229.

11. An isolated nucleic acid molecule encoding the isolated antigen-binding protein according to any one of claims 1 to 10.

12. A pharmaceutical composition comprising the isolated antigen-binding protein according to any one of claims 1 to 10 and / or the isolated nucleic acid molecule according to claim 11, and optionally a pharmaceutically acceptable adjuvant and / or excipient.

13. The pharmaceutical composition according to claim 12, for preventing and / or treating a tumor.

14. The pharmaceutical composition according to claim 13, wherein the tumor is selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma, and breast cancer.

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