Gitr binding proteins and uses thereof

Abstract

The present application relates to a GITR binding protein and its use, in particular to an isolated antigen binding protein comprising a HCDR3, which comprises an amino acid sequence as shown in SEQ ID NO: 3. The present application also provides the use of the antigen binding protein in preventing and treating diseases.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a GITR-binding protein and its uses. Background Technology

[0002] The immune checkpoint glucocorticoid-induced tumor necrosis factor receptor (GITR, also known as AITR, TNFRSF18) is a type I transmembrane protein belonging to the TNFRSF superfamily. Other members include OX40, CD27, CD40, and 4-1BB.

[0003] Currently, there are few GITR antibodies under research, and existing GITR antibodies have problems with poor safety and inadequate preventive and / or therapeutic effects. Therefore, there is an urgent need to develop more drugs that target GITR with high affinity and good activity to further improve the preventive and / or therapeutic effects of the disease and / or symptoms. Summary of the Invention

[0004] This application provides an isolated antigen-binding protein that may have one or more of the following advantages: (1) high affinity for GITR; (2) binding to human GITR expressed on cells; (3) agonist activity, such as activating the GITR-NFκB signaling pathway; and (4) good tumor-suppressive effect, such as reducing tumor volume in mice without affecting mouse weight.

[0005] On the one hand, this application provides an isolated antigen-binding protein comprising HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0006] In some embodiments, the isolated antigen-binding protein of this application comprises HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2.

[0007] In some embodiments, the isolated antigen-binding protein of this application comprises HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1.

[0008] In some embodiments, the isolated antigen-binding protein of this application comprises LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6.

[0009] In some embodiments, the isolated antigen-binding protein of this application comprises LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5.

[0010] In some embodiments, the isolated antigen-binding protein of this application comprises LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4.

[0011] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:36.

[0012] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0013] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR2, which is located between HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:37.

[0014] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR2, which is located between HCDR1 and HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0015] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR3, which is located between HCDR2 and HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:38.

[0016] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR3, which is located between HCDR2 and HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

[0017] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:39.

[0018] In some embodiments, the isolated antigen-binding protein of this application comprises H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:18.

[0019] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR1, wherein the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:40.

[0020] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR1, wherein the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and L-FR1 comprises the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0021] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR2, which is located between LCDR1 and LCDR2, and L-FR2 comprises the amino acid sequence shown in SEQ ID NO:41.

[0022] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR2, which is located between LCDR1 and LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.

[0023] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR3, which is located between LCDR2 and LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:42.

[0024] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR3, which is located between LCDR2 and LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

[0025] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:43.

[0026] In some embodiments, the isolated antigen-binding protein of this application comprises L-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28.

[0027] In some embodiments, the isolated antigen-binding protein of this application comprises HCDR1, HCDR2, and HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0028] In some embodiments, the isolated antigen-binding protein of this application comprises LCDR1, LCDR2, and LCDR3, wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0029] In some embodiments, the isolated antigen-binding protein of this application comprises a heavy chain variable region (VH) containing the amino acid sequence shown in SEQ ID NO:44.

[0030] In some embodiments, the isolated antigen-binding protein of this application comprises VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.

[0031] In some embodiments, the isolated antigen-binding protein of this application comprises a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:45.

[0032] In some embodiments, the isolated antigen-binding protein of this application comprises a VL, which contains the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.

[0033] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:44, and the VL comprises the amino acid sequence shown in SEQ ID NO:45.

[0034] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.

[0035] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:29, and the VL comprises the amino acid sequence shown in SEQ ID NO:33.

[0036] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:30, and the VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0037] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:30, and the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0038] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:31, and the VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0039] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:31, and the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0040] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0041] In some embodiments, the isolated antigen-binding protein of this application comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0042] In some embodiments, the isolated antigen-binding protein of this application comprises a constant region of the antibody heavy chain.

[0043] In some embodiments, the isolated antigen-binding protein of this application includes an antibody heavy chain constant region derived from a human antibody.

[0044] In some embodiments, the antibody heavy chain constant region in the isolated antigen-binding protein of this application includes a heavy chain constant region derived from IgG, IgA, IgD, IgE and / or IgM.

[0045] In some embodiments, the antibody heavy chain constant region in the isolated antigen-binding protein of this application includes a heavy chain constant region derived from IgG.

[0046] In some embodiments, the antibody heavy chain constant region in the isolated antigen-binding protein of this application includes heavy chain constant regions derived from IgG1, IgG2, IgG3 and / or IgG4.

[0047] In some embodiments, the antibody heavy chain constant region in the isolated antigen-binding protein of this application includes a heavy chain constant region derived from IgG1.

[0048] In some embodiments, the isolated antigen-binding protein of this application comprises a constant region of the antibody light chain.

[0049] In some embodiments, the isolated antigen-binding protein of this application includes a light chain constant region derived from a human antibody.

[0050] In some embodiments, the constant region of the antibody light chain in the isolated antigen-binding protein of this application comprises a constant region derived from human Igκ.

[0051] In some embodiments, the isolated antigen-binding protein of this application comprises an antibody or an antigen-binding fragment thereof.

[0052] In some embodiments, the isolated antigen-binding protein of this application includes a monoclonal antibody.

[0053] In some embodiments, the antibody in the isolated antigen-binding protein of this application is selected from one or more of the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0054] In some embodiments, the isolated antigen-binding protein of this application, wherein the antigen-binding fragment is selected from one or more of the group consisting of: Fab, Fab′, Fv fragment, F(ab′)2, F(ab)2, scFv, di-scFv, VHH, and dAb.

[0055] In some embodiments, the isolated antigen-binding protein of this application is capable of specifically binding to glucocorticoid-induced tumor necrosis factor receptor (GITR) or its functionally active fragments.

[0056] In some embodiments, the isolated antigen-binding protein of this application, the GITR, comprises human GITR.

[0057] In some embodiments, the isolated antigen-binding protein of this application has an EC50 of about 5.01 μg / ml or lower. 50 The ability to combine the value with the GITR or its functional segments.

[0058] In some embodiments, the isolated antigen-binding protein of this application, the GITR or a functional fragment thereof, comprises GITR or a functional fragment thereof expressed on cells.

[0059] In some embodiments, the isolated antigen-binding protein of this application has a K0 of about 3.01E-08M or lower. D The ability to combine the value with the GITR or its functional segments.

[0060] In some embodiments, the isolated antigen-binding protein of this application has the ability to activate the GITR or its functionally active fragment.

[0061] In some embodiments, the isolated antigen-binding protein of this application has an EC50 of about 3.20 μg / ml or lower. 50 The ability of the value to activate the GITR or its functional active fragment.

[0062] In some embodiments, the isolated antigen-binding protein of this application has the ability to activate the nuclear factor-κB (NF-κB) pathway.

[0063] In some embodiments, the isolated antigen-binding protein of this application has an EC50 of about 9.76 μg / ml or lower. 50 The ability to combine the value with the GITR or its functional segments.

[0064] In some embodiments, the isolated antigen-binding protein of this application has an EC50 of about 0.34 μg / ml or lower. 50 The ability to combine the value with the GITR or its functional segments.

[0065] In some implementations, the GITR includes human GITR and / or monkey GITR.

[0066] In some embodiments, the isolated antigen-binding protein of this application has tumor-suppressive capabilities.

[0067] In some embodiments, the isolated antigen-binding protein of this application has the ability to inhibit tumors with a tumor volume inhibition rate of about 38% or higher.

[0068] On the other hand, this application provides a polypeptide comprising the isolated antigen-binding protein of this application.

[0069] In some embodiments, the polypeptide of this application comprises a fusion protein.

[0070] On the other hand, this application provides nucleic acid molecules that encode the isolated antigen-binding protein and / or the polypeptide of this application.

[0071] On the other hand, this application provides a vector containing the nucleic acid molecules of this application.

[0072] On the other hand, this application provides an immunoconjugate comprising the isolated antigen-binding protein of this application and / or the polypeptide of this application.

[0073] On the other hand, this application provides cells that contain and / or express the isolated antigen-binding protein of this application, contain and / or express the polypeptide of this application, contain the nucleic acid molecule of this application, contain the vector of this application, and / or contain the immunoconjugate of this application.

[0074] On the other hand, this application provides a pharmaceutical composition comprising the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the carrier of this application, the immunoconjugate of this application, and / or the cell of this application, and optionally a pharmaceutically acceptable carrier.

[0075] In some embodiments, the pharmaceutical composition of this application further comprises one or more immune checkpoint inhibitors.

[0076] On the other hand, this application provides a pharmaceutical combination comprising the isolated antigen-binding protein of this application and / or the polypeptide of this application, as well as one or more immune checkpoint inhibitors.

[0077] In some embodiments, the drug combination of this application, wherein the immune checkpoint inhibitor is capable of inhibiting the interaction between PD-1 and PD-L1.

[0078] In some embodiments, the immune checkpoint inhibitor comprises a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0079] In some embodiments, the immune checkpoint inhibitor comprises a PD-1 antibody or an antigen-binding fragment thereof, and / or a PD-L1 antibody or an antigen-binding fragment thereof.

[0080] In some embodiments, the antibody comprises a selection from: murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0081] In some embodiments, the antigen-binding fragment is selected from: Fab, Fab′, Fv fragment, F(ab′)2, F(ab)2, scFv, di-scFv, VHH, and dAb.

[0082] In some embodiments, the immune checkpoint inhibitor comprises HCDR1-3 and LCDR1-3 of pembrolizumab.

[0083] In some embodiments, the immune checkpoint inhibitor comprises pembrolizumab VH and VL.

[0084] In some embodiments, the immune checkpoint inhibitor comprises pembrolizumab.

[0085] In some embodiments, the isolated antigen-binding protein and / or the polypeptide are not mixed with the immune checkpoint inhibitor in the drug combination.

[0086] In some embodiments, the isolated antigen-binding protein and / or the polypeptide are each present in separate containers from the immune checkpoint inhibitor.

[0087] In some embodiments, the pharmaceutical combination comprises a first formulation and a second formulation, the first formulation comprising the isolated antigen-binding protein and / or the polypeptide and a pharmaceutically acceptable first carrier, and the second formulation comprising the immune checkpoint inhibitor and a pharmaceutically acceptable second carrier.

[0088] On the other hand, this application provides a kit comprising the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, and / or the pharmaceutical combination of this application.

[0089] In some embodiments, the kit of this application is used to detect the presence and / or content of GITR in a sample.

[0090] On the other hand, this application provides a method for preparing the isolated antigen-binding protein and / or the polypeptide of this application, comprising culturing the cells of this application under conditions that cause the antigen-binding protein and / or the polypeptide to be expressed.

[0091] In some embodiments, the method of this application includes culturing the cells under conditions that cause the antigen-binding protein to be expressed.

[0092] In some embodiments, the method of this application includes culturing the cells under conditions that cause the polypeptide to be expressed.

[0093] On the other hand, this application provides a method for detecting GITR in a sample, comprising administering the isolated antigen-binding protein of this application, administering the polypeptide of this application, administering the nucleic acid molecule of this application, administering the carrier of this application, administering the immunoconjugate of this application, administering the cell of this application, administering the pharmaceutical composition of this application, administering the pharmaceutical combination of this application, and / or using the kit of this application.

[0094] On the other hand, this application provides the use of the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, and / or the pharmaceutical combination of this application in the preparation of a kit.

[0095] In some embodiments, the present application is used in which the kit is used to detect the presence and / or content of GITR in a sample.

[0096] On the other hand, this application provides the use of the isolated antigen-binding protein of this application, the polypeptide of this application, the nucleic acid molecule of this application, the vector of this application, the immunoconjugate of this application, the cell of this application, the pharmaceutical composition of this application, the pharmaceutical combination of this application and / or the kit of this application in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0097] In some embodiments, the use of this application includes the disease and / or condition comprising tumors and / or infections.

[0098] According to the purpose of this application, the disease and / or condition includes solid tumors and / or hematologic malignancies.

[0099] According to the purpose of this application, the diseases and / or conditions mentioned include breast cancer, stomach cancer, rectal cancer, and / or colon cancer.

[0100] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0101] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0102] Figure 1 The image shows the GITR antigen activity measured by ELISA.

[0103] Figure 2 The figure shows the serum titer curves of multiple immunized mice.

[0104] Figure 3 The figure shows the binding curves of GITR-binding protein and GITR expressed on the cell surface, as determined by flow cytometry.

[0105] Figure 4 The results show the biological activity of GITR-binding proteins detected using a luciferase reporter gene system.

[0106] Figure 5 and Figure 6 The figure shows the binding curves of GITR-binding protein and GITR expressed on the cell surface, as determined by flow cytometry.

[0107] Figure 7 , Figure 8 and Figure 9 The results show the biological activity of GITR-binding proteins detected using a luciferase reporter gene system.

[0108] Figure 10 , Figure 11 and Figure 12 The figure shows the binding curve between the GITR antigen-binding protein and the human GITR antigen.

[0109] Figure 13 , Figure 14 and Figure 15 The figure shows the binding curve of GITR antigen-binding protein to monkey GITR antigen.

[0110] Figure 16 This shows the antitumor efficacy of GITR-binding protein Ab0 in mice during the first trial.

[0111] Figure 17 The image shows the change in mouse body weight after administration of the GITR-binding protein Ab0 in the first experiment.

[0112] Figure 18 This shows the antitumor efficacy of GITR-binding protein Ab2 in mice during the second experiment.

[0113] Figure 19The image shows the changes in mouse body weight after administration of the GITR-binding protein Ab2 in the second experiment.

[0114] Figure 20 This shows the antitumor efficacy of the combination of GITR-binding protein Ab2 and PD-1 antibody in mice during the first trial.

[0115] Figure 21 The image shows the changes in mouse body weight after administration of the GITR-binding protein Ab2 and PD-1 antibody in combination during the first trial.

[0116] Figure 22 The image shows the antitumor efficacy of the combination of GITR-binding protein Ab5 and PD-1 antibody in mice during the second trial.

[0117] Figure 23 The image shows the changes in mouse body weight after administration of the GITR-binding protein Ab5 and PD-1 antibody in combination during the second experiment.

[0118] Figure 24 The image shows the antitumor efficacy of the combination of GITR-binding protein Ab5 and PD-L1 antibody in mice.

[0119] Figure 25 The image shows the changes in body weight in mice after administration of the GITR-binding protein Ab5 and the PD-L1 antibody in combination. Detailed Implementation

[0120] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0121] Terminology Definition

[0122] In this application, the term "GITR" generally refers to "glucocorticoid-induced tumor necrosis factor receptor," also known as TNF receptor superfamily 18 (TNFRSF18), and can be any native GITR from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats). For example, "GITR" in this application can include "full-length" GITR, unprocessed GITR, and / or any form of GITR resulting from cellular processing. "GITR" in this application can also include naturally occurring variants of GITR, such as splice variants or allelic variants. The amino acid sequence of human GITR can be found in GenBank accession number Q9Y5U5. The GITR protein in this application can also encompass its functionally active fragment, not limited to proteins containing functionally active fragments of GITR proteins resulting from cellular processing and / or modifications.

[0123] In this application, the term "NF-κB" generally refers to a transcription factor protein. For example, NF-κB can be a downstream pathway of GITR. Human NF-κB may include the protein listed in GenBank accession number Q04206. The NF-κB protein in this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of NF-κB proteins resulting from processing and / or modifications occurring in the cell.

[0124] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state can be called isolated. The term "isolated" may not exclude the presence of artificial or synthetic substances, nor may it exclude the presence of other impurities that do not affect the activity of the substance.

[0125] In this application, the term "isolated antigen-binding protein" generally refers to a protein that has antigen-binding ability after being removed from its native state. The "isolated antigen-binding protein" of this application may include a portion that binds to an antigen, and optionally, allow the antigen-binding portion to employ a framework or structural portion that promotes the conformation of the antigen-binding portion to bind to the antigen. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or CDR derivative. Such frameworks may include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, for example, biocompatible polymers. Examples of antigen-binding proteins may include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, VHH, IgD antibodies; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; and / or IgG4 antibodies and fragments thereof.

[0126] In this application, the term "CDR," also known as "complementarity-determining region," generally refers to a region within the variable structural domain of an antibody whose sequence can be highly variable and / or form a structurally defining loop. For example, an antibody may include six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In some embodiments, naturally occurring camel antibodies consisting only of the heavy chain can function normally and stably even in the absence of the light chain. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence number of the antigen-binding protein can be in accordance with the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system @imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000, Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203). For example, the CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242).

[0127] In this application, the term "FR" generally refers to a more conserved portion of the antibody variable domain, which is referred to as a frame region. For example, the variable domains of the natural heavy chain and light chain may each contain four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0128] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of the antibody heavy chain and / or light chain. Variable domains of the heavy and light chains may be referred to as "VH" and "VL" (or simply "VH" and "VL"). These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and may contain antigen-binding sites. In this application, the term "variable" generally refers to the possibility that certain segments of the variable domain may differ significantly in sequence between antibodies. Variable domains mediate antigen binding and determine the specificity of a particular antibody for its specific antigen. However, variability may not be uniformly distributed across the entire variable domain. It may typically be concentrated in three segments within the light and heavy chain variable domains, referred to as hypervariable regions (CDRs or HVRs). More highly conserved portions of the variable domain may be referred to as frame regions (FRs). The variable domains of the native heavy and light chains may each contain four FR regions, mostly in a β-sheet configuration, linked by three CDRs forming a ring link, and in some cases forming part of a β-sheet structure. CDRs in each chain can be held together closely by the FR region, and CDRs from the other chain together promote the formation of antigen-binding sites for antibodies.

[0129] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide that includes an antigen-binding site, whether it is produced in vitro or in vivo. The term may include, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," such as in "complete antibody," for the purposes of this invention, the term "antibody" may also include antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that maintain antigen-binding function (e.g., specific binding to human GITR). Typically, such fragments may include an antigen-binding domain. The basic 4-chain antibody unit can be a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies can consist of five basic heterotetrameric units and another polypeptide called the J chain, containing 10 antigen-binding sites, while IgA antibodies can include 2-5 basic four-chain units that can bind to the J chain and polymerize to form a multivalent combination. For IgG, the four-chain unit is typically about 150,000 Daltons. Each L chain can be linked to an H chain via a covalent disulfide bond, and two H chains can be linked to each other via one or more disulfide bonds depending on the H chain isoform. Each H and L chain can also have regularly spaced intrachain disulfide bridges. Each H chain can have a heavy chain variable region (VH) at its N-terminus, followed by three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isoforms. Each L chain can have a light chain variable region (VL) at its N-terminus and a constant domain at its other end. VL and VH can correspond, and the light chain constant region (CL) can correspond to the first constant domain (CH1) of the heavy chain. Specific amino acid residues can be considered to form interfaces between the variable domains of the light and heavy chains. VH and VL can pair together to form a single antigen-binding site. L chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of the constant domain of the heavy chain (CH). Currently, there are five classes of immunoglobulins: IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, and / or IgG4), and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively.

[0130] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., GITR). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0131] In this application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, an intact antibody can be digested using papain. After digestion with papain, the antibody produces two identical antigen-binding fragments, namely the "Fab" fragment, and a residual "Fc" fragment (i.e., the Fc region). The Fab fragment may consist of a complete L chain with a variable region of a heavy chain and a first constant region (CH1) of the H chain (VH).

[0132] In this application, the term "F(ab)2" generally refers to the antigen-binding fragment of an antibody. For example, F(ab)2 can be composed of two Fab fragments linked together.

[0133] In this application, the term "Fab'" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which is slightly larger than the Fab fragment. For example, the Fab' fragment may include all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, the Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

[0134] In this application, the term "F(ab′)2" generally refers to an antibody fragment produced by digesting an intact antibody with pepsin. The F(ab′)2 fragment contains two Fab fragments held together by disulfide bonds and a partial hinge region. The F(ab′)2 fragment exhibits bivalent antigen-binding activity and is capable of cross-linking antigens.

[0135] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy chain variable region and light chain variable region, and lacking the heavy chain constant region and light chain constant region. The heavy chain variable region and light chain variable region include, for example, CDRs. For example, an Fv fragment comprises all or part of the amino-terminal variable region of about 110 amino acids of the heavy and light chains.

[0136] In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in this application, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0137] In this application, the term “dAb” generally refers to an antigen-binding fragment consisting of a VH domain or a VL domain, for example, Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11): 484-490.

[0138] In this application, the term "VHH" generally refers to an antibody containing a variable antigen-binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as a nanobody (Nb).

[0139] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually contain different antibodies targeting different determinants), each monoclonal antibody can target a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" can indicate the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in this application can be prepared in hybridoma cells or by recombinant DNA methods.

[0140] In this application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody from a laboratory animal such as a rodent ("parental antibody") and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to elicit an adverse immune response in human individuals compared to parental (e.g., mouse-derived) antibodies.

[0141] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids in the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. A humanized antibody may optionally contain at least a portion of the constant region of a human immunoglobulin. A "humanized antibody" may retain antigen specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., mouse) antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as a mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies can contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0142] In this application, the term "fully human antibody" generally refers to an antibody that contains only the protein sequence of human immunoglobulins. If it is produced in mice, in mouse cells, or in hybridomas derived from mouse cells, then a fully human antibody may contain mouse glycans. Similarly, "mouse antibody," "mouse antibody," or "rat antibody" respectively refers to an antibody that contains only mouse or rat immunoglobulin sequences. Fully human antibodies can be generated in humans or in transgenic animals with human immunoglobulin germline sequences via phage display or other molecular biology methods. Exemplary techniques that can be used to manufacture antibodies are known in the art.

[0143] In this application, the term "antigen-binding protein" generally refers to a protein containing a portion that binds to an antigen, and optionally, a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, provided they exhibit the desired antigen-binding activity. The "isolated antigen-binding protein" of this application may contain a portion that binds to an antigen and optionally, allow the antigen-binding portion to adopt a scaffold or backbone portion that promotes the binding of the antigen-binding portion to the antigen.

[0144] In this application, the terms "peptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two parts covalently linked together. Each part can be a polypeptide with different properties. These properties can be biological properties, such as in vitro or in vivo activity. They can also be simple chemical or physical properties, such as binding to target molecules, catalysis of reactions, etc. The two parts can be directly linked by a single peptide bond or through a peptide linker.

[0145] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0146] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted, thereby enabling the protein to be expressed. A vector can be transformed, transduced, or transfected into host cells, allowing the genetic material it carries to be expressed within the host cells. A vector may contain multiple elements controlling expression. Additionally, a vector may contain a replication origin site. A vector may also include components that facilitate its entry into the cell.

[0147] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this application. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in the morphology of total DNA complements or in the genome). Cells may include cells transfected in vitro using the vectors described in this application.

[0148] In this application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating the other reagent (e.g., chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents) with the antibody or its antigen-binding fragment (e.g., covalently linked by a linker molecule), which can deliver the other reagent to the target cell (e.g., tumor cells) by specifically binding the antibody or its antigen-binding fragment to the antigen on the target cell.

[0149] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise one or more suitable formulations such as a (pharmaceutically effective) carrier. The acceptable components of the composition are non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0150] In this application, the term "pharmaceutically acceptable carrier" generally refers to a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to cells or mammals exposed to it at the doses and concentrations used. Physiologically acceptable carriers may include suitable substances. It should be noted that a pharmaceutically acceptable carrier is generally not the same substance as a vector used in genetic engineering to insert nucleic acids.

[0151] In this application, the term "immune checkpoint inhibitor" generally refers to any agent capable of inhibiting the biological activity of immune checkpoints. In this application, the immune checkpoint can be a regulatory molecule that plays an inhibitory role in the immune system. The immune checkpoint can be expressed on immune cells, inhibiting their function. The immune checkpoint can be associated with tumor immune escape. In this application, the immune checkpoint can include PD-1 and / or PD-L1. In this application, the inhibitor can include any agent capable of inhibiting the interaction between ligands and receptors (e.g., PD-1 and PD-L1). In this application, the agent can be a chemical agent or a protein and / or peptide. For example, the agent can include an antibody or an antigen-binding fragment thereof.

[0152] In this application, the term "PD-1" generally refers to the CD28 family of immunosuppressive receptors. PD-1 can be expressed on pre-activated T cells and binds to two ligands, PD-L1 and / or PD-L2. In this application, the PD-1 may include human PD-1 (hPD-1) or its variants, isotypes, and species homologs, as well as analogs having at least one common epitope with hPD-1. The GenBank accession number for hPD-1 is U64863. The PD-1 protein in this application may also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of the PD-1 protein resulting from processing and / or modification occurring in cells.

[0153] In this application, the term "PD-L1" generally refers to one of the cell surface glycoprotein ligands of PD-1. PD-L1 can bind to PD-1. When PD-L1 binds to PD-1, it can downregulate T cell activation and / or cytokine secretion. In this application, PD-L1 can include human PD-L (hPD-L1) or its variants, isotypes, and species homologs, as well as analogs having at least one common epitope with hPD-L1. The GenBank accession number for hPD-L1 is Q9NZQ7. The PD-L1 protein in this application can also encompass its functionally active fragments, not limited to proteins containing functionally active fragments of PD-L1 protein resulting from processing and / or modification occurring in cells.

[0154] In this application, the term "solid tumor" generally refers to a tangible tumor that can be detected by clinical examination (e.g., X-ray, CT scan, ultrasound, or palpation). The tumor may include growths or solid lesions formed by abnormal cell growth.

[0155] In this application, the term "breast cancer" generally refers to a tumor that occurs in or originates from the glandular epithelial tissue of the breast, such as cancer that occurs in or originates from the glandular epithelial tissue of the breast.

[0156] In this application, the term "rectal cancer" generally refers to a tumor that occurs in or originates from the rectum. For example, a tumor that occurs in or originates from the area between the dentate line and the rectosigmoid junction may be a cancer that occurs in or originates from the area between the dentate line and the rectosigmoid junction.

[0157] In this application, the term "colon cancer" generally refers to a tumor of the digestive tract that occurs in or originates from the colon. For example, colon cancer may occur in or originate from the junction of the rectum and sigmoid colon. For example, colon cancer may be a cancer of the digestive tract that occurs in or originates from the colon.

[0158] In this application, the term "directly linked" is used in contrast to the term "indirectly linked," with "directly linked" generally referring to a direct connection. For example, direct linking can refer to a situation where substances are directly linked without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to a situation where substances are not directly linked. For example, indirect linking can refer to a connection via a spacer. For example, in the isolated antigen-binding protein described in this application, the C-terminus of L-FR1 and the N-terminus of LCDR1 can be directly or indirectly linked.

[0159] In this application, the term "infection" generally refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

[0160] In this application, the term "di-scFv" generally refers to a divalent scFv, such as a molecule in which two scFv molecules are joined together by a short connector.

[0161] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target is determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to the target with greater affinity, strength, ease, and / or duration than it binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0162] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0163] In this application, the term "tumor" generally refers to any new pathological growth of tissue. Tumor cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. In this application, the tumor can include benign tumors and malignant tumors. In this application, the tumor can include solid tumors and / or hematomas. In this application, the tumor can include cancer. In this application, examples of tumors include, but are not limited to, colon cancer.

[0164] In this application, the proteins, peptides and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0165] In this application, the variant can be, for example, a protein or polypeptide whose amino acid sequence has been substituted, deleted, or added with one or more amino acids, such as an antibody that specifically binds to GITR or a fragment thereof. For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conserved substitution. For example, the variant may also be a polypeptide encompassing its functionally active fragment, not limited to polypeptides containing the functionally active fragment of the protein resulting from processing and / or modification occurring in the cell.

[0166] In this application, the homolog can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to GITR or a fragment thereof).

[0167] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and DJ Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and DJ Lipman and W.R. Pearson, “A Fast and Sensitive Search for Protein Similarity,” Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, “A Basic Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0168] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0169] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 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% above or below a specified value. Invention Details

[0171] On the one hand, this application provides an isolated antigen-binding protein.

[0172] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses the CDR sequence partitioned according to any CDR partitioning method; it also encompasses variants thereof, including amino acid sequences of the CDR that have been substituted, deleted, and / or added to one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the CDR may be determined by the Kabat numbering scheme.

[0173] On one hand, this application provides an isolated antigen-binding protein that may contain HCDR3, wherein HCDR3 may contain the amino acid sequence shown in SEQ ID NO:3. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, the HCDR3 may contain HCDR3 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, the HCDR3 may contain HCDR3 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0174] In this application, the isolated antigen-binding protein may further comprise HCDR2, which may contain the amino acid sequence shown in SEQ ID NO:2. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, the HCDR2 may comprise HCDR2 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, the HCDR2 may comprise HCDR2 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0175] In this application, the isolated antigen-binding protein may further comprise HCDR1, which may contain the amino acid sequence shown in SEQ ID NO:1. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, the HCDR1 may comprise HCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, the HCDR1 may comprise HCDR1 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0176] In this application, the isolated antigen-binding protein may comprise HCDR3, HCDR2, and HCDR1. For example, HCDR3 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:3, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, and HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, HCDR3, HCDR2, and HCDR1 may comprise HCDR3, HCDR2, and HCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, HCDR3, HCDR2, and HCDR1 may comprise HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0177] In this application, the isolated antigen-binding protein may comprise LCDR3, which may contain the amino acid sequence shown in SEQ ID NO:6. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, the LCDR3 may comprise LCDR3 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, the LCDR3 may comprise LCDR3 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0178] In this application, the isolated antigen-binding protein may further comprise LCDR2, which may contain the amino acid sequence shown in SEQ ID NO:5. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, the LCDR2 may comprise LCDR2 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, the LCDR2 may comprise LCDR2 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0179] In this application, the isolated antigen-binding protein may further comprise LCDR1, which may contain the amino acid sequence shown in SEQ ID NO:4. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, the LCDR1 may comprise LCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, the LCDR1 may comprise LCDR1 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0180] In this application, the isolated antigen-binding protein may comprise LCDR3, LCDR2, and LCDR1. For example, LCDR3 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:6, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4. For example, the isolated antigen-binding protein of this application may have GITR binding capability. For example, the CDR may be determined by the Kabat numbering scheme. For example, LCDR3, LCDR2, and LCDR1 may comprise LCDR3, LCDR2, and LCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. For example, LCDR3, LCDR2, and LCDR1 may comprise LCDR3, LCDR2, and LCDR1 in the light chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9.

[0181] In this application, the isolated antigen-binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1. For example, the isolated antigen-binding protein HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3, HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:2, HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:1, LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:6, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:5, and LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:4. For example, the isolated antigen-binding protein of this application may have GITR binding ability. For example, HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9. In this application, the CDR can encompass CDR sequences partitioned according to any CDR partitioning method; it can also encompass its variants. For example, HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 can include HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab0, Ab2, Ab3, Ab5, Ab6, Ab8, or Ab9, and LCDR3, LCDR2, and LCDR1 in their light chain variable region. For example, HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 can include HCDR3, HCDR2, and HCDR1 in the heavy chain variable region of Ab0, and LCDR3, LCDR2, and LCDR1 in their light chain variable region.

[0182] In this application, the isolated antigen-binding protein may comprise H-FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of HCDR1. For example, H-FR1 may comprise an amino acid sequence as shown in X1VQLQESGPGLVKPSQX2LSLTCTVX3GX4SIX5, wherein X1, X2, X3, X4, and X5 may each be independently selected from any one amino acid, such as a natural amino acid. H-FR1 may comprise an amino acid sequence as shown in SEQ ID NO:36.

[0183] In this application, the isolated antigen-binding protein may include H-FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 may contain an amino acid sequence as shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0184] In this application, the isolated antigen-binding protein may comprise H-FR2, which may be located between HCDR1 and HCDR2. For example, H-FR2 may comprise an amino acid sequence as shown in WIRQX6PGX7X8LEWX9G, wherein X6, X7, X8, and X9 may each be independently selected from any one amino acid, such as a natural amino acid. H-FR2 may comprise an amino acid sequence as shown in SEQ ID NO:37.

[0185] In this application, the isolated antigen-binding protein may contain H-FR2, which may be located between HCDR1 and HCDR2, and H-FR2 may contain an amino acid sequence as shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0186] In this application, the isolated antigen-binding protein may include H-FR3, which may be located between HCDR2 and HCDR3. For example, H-FR3 may include RX. 10 X 11 IX 12 X 13 DTSKNQFX 14 LX 15 LX 16 SVTX 17 X 18 DTAX 19 YYCX 20 The amino acid sequence shown in R is X. 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 and X 20 Each amino acid can be independently selected from any amino acid, such as natural amino acids. The H-FR3 may contain an amino acid sequence as shown in SEQ ID NO:38.

[0187] In this application, the isolated antigen-binding protein may contain H-FR3, which may be located between HCDR2 and HCDR3, and the H-FR3 may contain an amino acid sequence as shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

[0188] In this application, the isolated antigen-binding protein may comprise H-FR4, the N-terminus of which may be linked to the C-terminus of HCDR3. For example, H-FR4 may comprise, for instance, WGQGTX. 21 X 22 The amino acid sequence shown in TVSS, where X 21 and X 22 Each amino acid can be independently selected from any amino acid, such as natural amino acids. The H-FR4 may contain an amino acid sequence as shown in SEQ ID NO:39.

[0189] In this application, the isolated antigen-binding protein may contain H-FR4, the N-terminus of which may be linked to the C-terminus of HCDR3, and the H-FR4 may contain an amino acid sequence as shown in SEQ ID NO:17 or SEQ ID NO:18.

[0190] In this application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4. For example, the isolated antigen-binding protein H-FR1, H-FR2, H-FR3, and H-FR4 may respectively comprise the amino acid sequences shown in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. For example, the isolated antigen-binding protein H-FR1, H-FR2, H-FR3, and H-FR4 may respectively comprise the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, and SEQ ID NO:17. For example, the isolated antigen-binding protein H-FR1, H-FR2, H-FR3, and H-FR4 may respectively comprise the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:18. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may, respectively, contain the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:18. For example, the isolated antigen-binding proteins H-FR1, H-FR2, H-FR3, and H-FR4 may, respectively, contain the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and SEQ ID NO:18.

[0191] In this application, the isolated antigen-binding protein may comprise L-FR1, and the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1. For example, L-FR1 may comprise DFX. 24 MTQTPLX 25 LSVTX 26 The amino acid sequence shown in GQPASISC, where X 24 X 25 and X 26 Each amino acid can be independently selected from any amino acid, such as natural amino acids. The L-FR1 may contain an amino acid sequence as shown in SEQ ID NO:40.

[0192] In this application, the isolated antigen-binding protein may include L-FR1, the C-terminus of which may be directly or indirectly linked to the N-terminus of LCDR1, and the L-FR1 may contain an amino acid sequence as shown in SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

[0193] In this application, the isolated antigen-binding protein may include L-FR2, which may be located between LCDR1 and LCDR2. For example, L-FR2 may include WLLQX. 28 PGQSPX 29 The amino acid sequence shown in RLIY, where X 28 and X 29 Each amino acid can be independently selected from any amino acid, such as natural amino acids. The L-FR2 may contain an amino acid sequence as shown in SEQ ID NO:41.

[0194] In this application, the isolated antigen-binding protein may include L-FR2, which may be located between LCDR1 and LCDR2, and L-FR2 may contain an amino acid sequence as shown in SEQ ID NO:22 or SEQ ID NO:23.

[0195] In this application, the isolated antigen-binding protein may include L-FR3, which may be located between LCDR2 and LCDR3. For example, L-FR3 may include GVPDRFX. 31 GSGSGTDFTLKISRVEAEDX 32 The amino acid sequence shown in GVYYC, where X 31 and X 32 Each amino acid can be independently selected from any amino acid, such as natural amino acids. The L-FR3 may contain an amino acid sequence as shown in SEQ ID NO:42.

[0196] In this application, the isolated antigen-binding protein may include L-FR3, which may be located between LCDR2 and LCDR3, and L-FR3 may include an amino acid sequence as shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

[0197] In this application, the isolated antigen-binding protein may comprise L-FR4, the N-terminus of which may be linked to the C-terminus of LCDR3. For example, L-FR4 may comprise FGX, etc. 33 The amino acid sequence shown in GTKLEIK, where X 33 It can be selected from any amino acid, such as a natural amino acid. The L-FR4 may contain an amino acid sequence as shown in SEQ ID NO:43.

[0198] In this application, the isolated antigen-binding protein may include L-FR4, the N-terminus of which may be linked to the C-terminus of LCDR3, and the L-FR4 may contain an amino acid sequence as shown in SEQ ID NO:27 or SEQ ID NO:28.

[0199] In this application, the isolated antigen-binding protein may comprise L-FR1, L-FR2, L-FR3, and L-FR4. For example, the isolated antigen-binding protein L-FR1, L-FR2, L-FR3, and L-FR4 may each sequentially comprise the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. For example, the isolated antigen-binding protein L-FR1, L-FR2, L-FR3, and L-FR4 may each sequentially comprise the amino acid sequences shown in SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:27. For example, the isolated antigen-binding protein L-FR1, L-FR2, L-FR3, and L-FR4 may each sequentially comprise the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:25, and SEQ ID NO:28. For example, the isolated antigen-binding proteins L-FR1, L-FR2, L-FR3 and L-FR4 may respectively contain the amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:26 and SEQ ID NO:28.

[0200] In this application, the isolated antigen-binding protein may contain a VH, which may contain the amino acid sequence shown in SEQ ID NO:44. For example, the isolated antigen-binding protein of this application may have GITR binding ability.

[0201] In this application, the isolated antigen-binding protein may contain a VH, which may contain the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. For example, the isolated antigen-binding protein of this application may have GITR binding ability.

[0202] In this application, the isolated antigen-binding protein may contain a VL, which may contain the amino acid sequence shown in SEQ ID NO:45. For example, the isolated antigen-binding protein of this application may have GITR binding ability.

[0203] In this application, the isolated antigen-binding protein may contain a VL, which may contain the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35. For example, the isolated antigen-binding protein of this application may have GITR binding ability.

[0204] In this application, the isolated antigen-binding protein may comprise the VH and VL. In some embodiments, the VH may comprise the amino acid sequence shown in SEQ ID NO:44, and the VL may comprise the amino acid sequence shown in SEQ ID NO:45.

[0205] For example, the VH may contain the amino acid sequence shown in SEQ ID NO:29, and the VL may contain the amino acid sequence shown in SEQ ID NO:33. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:30, and the VL may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:30, and the VL may contain the amino acid sequence shown in SEQ ID NO:35. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:31, and the VL may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:31, and the VL may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:31, and the VL may contain the amino acid sequence shown in SEQ ID NO:35. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:32, and the VL may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH may contain the amino acid sequence shown in SEQ ID NO:32, and the VL may contain the amino acid sequence shown in SEQ ID NO:35. For example, the isolated antigen-binding protein of this application may have GITR binding ability.

[0206] In this application, the isolated antigen-binding protein may contain at least one CDR from the VH described in this application. In this application, the isolated antigen-binding protein may contain at least one CDR from the VL described in this application. The CDR can be obtained according to any partitioning method. In this application, the CDR may cover CDR sequences partitioned according to any CDR partitioning method; it may also cover its variants.

[0207] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3 from the VH described in this application. The VH may contain the amino acid sequence shown in SEQ ID NO:44. For example, the isolated antigen-binding protein of this application may have GITR binding capability. In this application, the CDR may cover CDR sequences partitioned according to any CDR partitioning method; it may also cover its variants.

[0208] In this application, the isolated antigen-binding protein may include HCDR1, HCDR2, and HCDR3 as described in the VH of this application. The VH may contain the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. For example, the isolated antigen-binding protein of this application may have GITR binding capability. In this application, the CDR may cover CDR sequences partitioned according to any CDR partitioning method; it may also cover its variants.

[0209] In this application, the isolated antigen-binding protein may include LCDR1, LCDR2, and LCDR3 in the VL described in this application. The VL may contain the amino acid sequence shown in SEQ ID NO:45. For example, the isolated antigen-binding protein of this application may have GITR binding capability. In this application, the CDR may cover CDR sequences partitioned according to any CDR partitioning method; it may also cover its variants.

[0210] In this application, the isolated antigen-binding protein may include LCDR1, LCDR2, and LCDR3 as described in the VL of this application. The VL may contain the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35. For example, the isolated antigen-binding protein of this application may have GITR binding capability. In this application, the CDR may cover CDR sequences partitioned according to any CDR partitioning method; it may also cover its variants.

[0211] In this application, the isolated antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from the heavy chain constant regions of human IgG, IgA, IgD, IgE, and / or IgM. The antibody heavy chain constant region may be derived from the heavy chain constant region of human IgG. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the heavy chain constant regions of human IgG1, IgG2, IgG3, and / or IgG4. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the heavy chain constant region of human IgG1.

[0212] In this application, the isolated antigen-binding protein may include an antibody light chain constant region. The antibody light chain constant region may be derived from the human Igκ constant region.

[0213] In this application, the isolated antigen-binding protein may contain an antibody or an antigen-binding fragment thereof.

[0214] In some embodiments, the antigen-binding fragment may include Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, VHH and / or dAb.

[0215] In some embodiments, the antibody may include a monoclonal antibody. In some embodiments, the antibody may include a murine antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0216] For example, the VH of the chimeric antibody may contain the amino acid sequence shown in SEQ ID NO:29, and the VL of the chimeric antibody may contain the amino acid sequence shown in SEQ ID NO:33.

[0217] For example, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:30, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:30, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:35. For example, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:31, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:31, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:35. For example, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:32, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:34. For example, the VH of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:32, and the VL of the humanized antibody may contain the amino acid sequence shown in SEQ ID NO:35.

[0218] In some embodiments, the antibody may include a light chain constant region. For example, the light chain constant region may be a light chain constant region derived from a human antibody; for example, the light chain constant region may include the amino acid sequence shown in SEQ ID NO:46; for example, the light chain constant region may include the amino acid sequence shown in SEQ ID NO:47.

[0219] In some embodiments, the antibody may include a heavy chain constant region. For example, the heavy chain constant region may be a heavy chain constant region derived from a human antibody; for example, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:48; for example, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:49; for example, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:50; for example, the heavy chain constant region may include the amino acid sequence shown in SEQ ID NO:51.

[0220] Furthermore, it should be noted that the isolated antigen-binding protein described in this application may contain heavy chain and / or light chain sequences with one or more conserved sequence modifications. "Conserved sequence modification" refers to amino acid modifications that do not significantly affect or alter antibody binding properties. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding protein described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitution involves replacing an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), nonpolar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not cause the antigen to lose its binding ability. For example, see Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O′ Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0221] The GITR antigen-binding protein described in this application can be identified, screened, or characterized by various assays known in the art.

[0222] For example, the antigen-binding activity of the antigen-binding protein or fusion protein of this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0223] In this application, the isolated antigen-binding protein is capable of specifically binding to GITR or its functionally active fragment.

[0224] In some embodiments, GITR or its functionally active fragment can be a full-length GITR or a fragment that exerts GITR functional activity. For example, GITR functional activity can be the ability to activate downstream pathways of GITR, such as the nuclear factor-κB (NF-κB) pathway. In some embodiments, GITR or its functionally active fragment can be a separate GITR or a mixture of various forms of GITR or its functionally active fragments. For example, GITR or its functionally active fragment can be a human GITR or its functionally active fragment; for example, GITR or its functionally active fragment can be a GITR or its functionally active fragment expressed on the cell surface; for example, GITR or its functionally active fragment can be a GITR or its functionally active fragment bound to a solid medium.

[0225] In some embodiments, the isolated antigen-binding protein can be analyzed by measuring its binding activity to the antigen using a FACS method. For example, the antigen-binding protein described in this application can be analyzed at an EC50 concentration of less than or equal to about 5.00 μg / ml. 50The value binds to human GITR on the cell surface. For example, the antigen-binding protein described in this application may be present in concentrations less than or equal to about 5.10 μg / ml, less than or equal to about 5.00 μg / ml, less than or equal to about 4.50 μg / ml, less than or equal to about 4.00 μg / ml, less than or equal to about 3.70 μg / ml, less than or equal to about 3.60 μg / ml, less than or equal to about 3.50 μg / ml, less than or equal to about 3.00 μg / ml, less than or equal to about 2.50 μg / ml, less than or equal to about 2.00 μg / ml, less than or equal to about 1.50 μg / ml, less than or equal to about 1.00 μg / ml, less than or equal to about 0.90 μg / ml, less than or equal to about 0.89 μg / ml, less than or equal to about 0.88 μg / ml, less than or equal to about 0.87 μg / ml, less than or equal to about 0.86 μg / ml, less than or equal to about 0.85 μg / ml, and less than... Or equal to or less than or ... EC with concentrations of g / ml, less than or equal to about 0.50 μg / ml, less than or equal to about 0.45 μg / ml, less than or equal to about 0.40 μg / ml, less than or equal to about 0.37 μg / ml, less than or equal to about 0.36 μg / ml, less than or equal to about 0.365 μg / ml, less than or equal to about 0.358 μg / ml, less than or equal to about 0.35 μg / ml, or less than or equal to about 0.30 μg / ml 50 The value binds to human GITR on the cell surface.

[0226] In some embodiments, the affinity screening of the GITR-binding protein of this application is compared by detecting changes in luciferase expression in Jurkat-GITR-NFκB cells induced by the binding of the GITR-binding protein to cellularly expressed GITR. For example, the antigen-binding protein of this application can be expressed at an EC50 concentration of 3.20 μg / ml or lower. 50The value activates GITR signaling pathways, such as the NFκB signaling pathway. For example, the antigen-binding protein described in this application may be at a concentration of 3.20 μg / ml or lower, 3.10 μg / ml or lower, 3.00 μg / ml or lower, 2.50 μg / ml or lower, 2.00 μg / ml or lower, 1.50 μg / ml or lower, 1.30 μg / ml or lower, 1.25 μg / ml or lower, 1.23 μg / ml or lower, 1.00 μg / ml or lower, 0.90 μg / ml or lower, 0.80 μg / ml or lower, or 0.70 μg / ml. / ml or lower, at 0.68μg / ml or lower, at 0.67μg / ml or lower, at 0.66μg / ml or lower, at 0.65μg / ml or lower, at 0.64μg / ml or lower, at 0.60μg / ml or lower, at 0.50μg / ml or lower, at 0.40μg / ml or lower, at 0.39μg / ml or lower, at 0.35μg / ml or lower, at 0.33μg / ml or lower, at 0.32μg / ml or lower, or at 0.30μg / ml or lower EC 50 Signaling pathways that activate GITR, such as the NFκB signaling pathway.

[0227] In some embodiments, the isolated antigen-binding protein can be screened for affinity using Biacore. For example, the isolated antigen-binding protein can be screened for affinity at 3.10E-08M or lower. D The value binds to the GITR protein. For example, the isolated antigen-binding protein may have a value of 3.01E-08M or lower, 2.0E-08M or lower, 1.0E-08M or lower, 9.0E-09M or lower, 8.0E-09M or lower, 7.0E-09M or lower, 6.0E-09M or lower, 5.0E-09M or lower, 4.0E-09M or lower, 3.0E-09M or lower, 2.0E-09M or lower, 1.5E-09M or lower, 1.4E-09M or lower, 1.3E-09M or lower, or 1.0E... -0.9M or lower, with 9.0E-10M or lower, with 8.0E-10M or lower, with 7.0E-10M or lower, with 6.0E-10M or lower, with 5.0E-10M or lower, with 4.0E-10M or lower, with 3.0E-10M or lower, with 2.0E-10M or lower, with 1.6E-10M or lower, with 1.5E-10M or lower, with 1.4E-10M or lower, with 1.3E-10M or lower, with 1.2E-10M or lower, with 1.1E-10M or lower, or with 1.0E-10M or lower. D The value binds to the GITR protein.

[0228] In some embodiments, the isolated antigen-binding protein can be analyzed by enzyme-linked immunosorbent assay (ELISA) to determine its binding activity to the antigen. For example, the isolated antigen-binding protein may have an EC50 of about 9.76 μg / ml or lower. 50 The value refers to the ability to bind to the GITR or a functional fragment thereof. For example, the GITR or a functional fragment thereof may comprise a monkey GITR; the isolated antigen-binding protein may have an EC50 value of about 9.76 μg / ml or less, about 9.00 μg / ml or less, about 8.90 μg / ml or less, about 8.84 μg / ml or less, about 1.00 μg / ml or less, about 0.50 μg / ml or less, about 0.10 μg / ml or less, about 0.04 μg / ml or less, about 0.03 μg / ml or less, or about 0.02 μg / ml or less. 50 The value refers to the ability to bind to the GITR or a functional fragment thereof. For example, the GITR or a functional fragment thereof may comprise a human GITR; the isolated antigen-binding protein may have an EC50 of about 0.34 μg / ml or less, about 0.30 μg / ml or less, about 0.22 μg / ml or less, about 0.02 μg / ml or less, or about 0.01 μg / ml or less. 50 The ability to combine the value with the GITR or its functional segments.

[0229] In this application, the isolated antigen-binding protein is capable of inhibiting tumor growth. For example, the isolated antigen-binding protein is capable of inhibiting tumor growth with a tumor volume inhibition rate of about 40% or higher, such as in colon cancer. For example, the isolated antigen-binding protein is capable of inhibiting tumor growth with a tumor volume inhibition rate of about 38% or higher, about 39% or higher, about 40% or higher, about 41% or higher, about 42% or higher, about 43% or higher, about 45% or higher, about 50% or higher, about 55% or higher, about 56% or higher, about 57% or higher, about 58% or higher, or about 59% or higher.

[0230] On the one hand, this application provides a polypeptide molecule, nucleic acid molecule, carrier, immunoconjugate, cell and drug composition.

[0231] On the other hand, this application provides polypeptide molecules that may contain the isolated antigen-binding protein described in this application.

[0232] In some embodiments, the polypeptide molecule may comprise a fusion protein. In some embodiments, the polypeptide molecule may be a fusion protein. In some embodiments, the polypeptide molecule is a multispecific (e.g., bispecific, trispecific, or other multispecific) antibody. The multispecific antibody may comprise: 1) the isolated antigen-binding protein described in this application, and 2) one or more targeting moieties that bind other antigens and / or other epitopes of the same antigen. In some embodiments, the polypeptide molecule of this application may comprise structures other than amino acids; for example, the polypeptide molecule of this application may comprise nucleic acids, polysaccharides, lipids, small molecules, and any combination thereof.

[0233] On the other hand, this application provides isolated nucleic acid molecules that can encode the isolated antigen-binding protein described in this application. For example, it can be produced or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR); (ii) by clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0234] On the other hand, this application provides a vector that may contain the nucleic acid molecule described in this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. Furthermore, the vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0235] On the other hand, this application provides a cell that may contain the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain one or more of the nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more types (e.g., two or more) of the nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., *Escherichia coli*), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. The vectors described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0236] On the other hand, this application also provides immune conjugates that may contain the isolated antigen-binding proteins described in this application.

[0237] In some embodiments, the isolated antigen-binding protein or fragment thereof described in this application can be linked to another reagent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, etc. This linking can be via one or more covalent bonds, or non-covalent interactions, and may include chelation. A variety of adapters (which may be known in the art) can be used to form immunoconjugates. Furthermore, immunoconjugates can be provided in the form of fusion proteins, which may be expressed by polynucleotides encoding the immunoconjugate. The immunoconjugates may also comprise, for example, antibody-drug conjugates (ADCs). In an ADC, the antibody and the therapeutic agent can be cross-linked via an adapter, which can be a cleavable adapter, such as an in vivo and / or in vitro enzymatically cleavable adapter, for example, a peptide adapter, a disulfide adapter, or a hydrazone adapter.

[0238] On the other hand, this application also provides pharmaceutical compositions that may comprise the isolated antigen-binding protein described in this application, the polypeptide molecule described in this application, the immunoconjugate described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier.

[0239] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition may be non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0240] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0241] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0242] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient's body.

[0243] In some embodiments, the pharmaceutical composition further comprises one or more immune checkpoint inhibitors. For example, the immune checkpoint inhibitor may comprise a PD-1 inhibitor and / or a PD-L1 inhibitor. For example, the immune checkpoint inhibitor may comprise a PD-1 antibody or its antigen-binding fragment, and / or a PD-L1 antibody or its antigen-binding fragment. For example, the immune checkpoint inhibitor may comprise one CDR of pembrolizumab, such as LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, or HCDR3. For example, the immune checkpoint inhibitor may comprise three CDRs of pembrolizumab, such as any three of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, or HCDR3. For example, the immune checkpoint inhibitor may comprise six CDRs of pembrolizumab, such as LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. For example, an immune checkpoint inhibitor may contain one CDR of the heavy chain variable region (VH) of pembrolizumab, such as HCDR1, HCDR2, or HCDR3. For example, an immune checkpoint inhibitor may contain three CDRs of the heavy chain variable region (VH) of pembrolizumab. For example, an immune checkpoint inhibitor may contain one CDR of the light chain variable region (VL) of pembrolizumab, such as LCDR1, LCDR2, or LCDR3. For example, an immune checkpoint inhibitor may contain three CDRs of the light chain variable region (VL) of pembrolizumab. For example, an immune checkpoint inhibitor may contain three CDRs of both the heavy chain variable region (VH) and the light chain variable region (VL) of pembrolizumab. For example, the HCDR1 amino acid sequence of pembrolizumab can be as shown in SEQ ID NO:57, the HCDR2 amino acid sequence of pembrolizumab can be as shown in SEQ ID NO:58, the HCDR3 amino acid sequence of pembrolizumab can be as shown in SEQ ID NO:59, the LCDR1 amino acid sequence of pembrolizumab can be as shown in SEQ ID NO:60, the LCDR2 amino acid sequence of pembrolizumab can be as shown in SEQ ID NO:61, and the LCDR3 amino acid sequence of pembrolizumab can be as shown in SEQ ID NO:62. The CDR region can be determined using different coding systems based on the antibody's sequence and structure. Different coding systems may result in differences in the CDR region. In this application, the CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants thereof, including amino acid sequences of the CDR that have been substituted, deleted, and / or added with one or more amino acids.For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the CDR is determined by the Kabat numbering scheme. For example, an immune checkpoint inhibitor may comprise the heavy chain variable region VH of pembrolizumab. For example, an immune checkpoint inhibitor may comprise the light chain variable region VL of pembrolizumab. For example, an immune checkpoint inhibitor may comprise both the heavy chain variable region VH and the light chain variable region VL of pembrolizumab. For example, the VH amino acid sequence of the heavy chain variable region of pembrolizumab may be as shown in SEQ ID NO:63, and the VL amino acid sequence of the light chain variable region of pembrolizumab may be as shown in SEQ ID NO:64. For example, an immune checkpoint inhibitor may comprise the light chain of pembrolizumab. For example, an immune checkpoint inhibitor may comprise the heavy chain of pembrolizumab. For example, an immune checkpoint inhibitor may comprise both the light chain and the heavy chain of pembrolizumab; for example, the amino acid sequence of the heavy chain of pembrolizumab may be as shown in SEQ ID NO:65, and the amino acid sequence of the light chain of pembrolizumab may be as shown in SEQ ID NO:66.

[0244] On the one hand, this application provides a drug combination.

[0245] In some embodiments, the pharmaceutical combination of this application may comprise the isolated GITR-binding protein of this application and / or a polypeptide containing the GITR-binding protein, as well as one or more immune checkpoint inhibitors.

[0246] For example, the immune checkpoint inhibitor of this application can inhibit the interaction between PD-1 and PD-L1. For example, the immune checkpoint inhibitor of this application may comprise a PD-1 inhibitor and / or a PD-L1 inhibitor. For example, the immune checkpoint inhibitor of this application may comprise a PD-1 antibody or its antigen-binding fragment and / or a PD-L1 antibody or its antigen-binding fragment. In some embodiments, the antigen-binding fragment may include Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, VHH, and / or dAb. In some embodiments, the antibody may include a monoclonal antibody. In some embodiments, the antibody may include a murine antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0247] In some embodiments, the immune checkpoint inhibitor of this application may comprise pembrolizumab's HCDR1-3 and LCDR1-3. In some embodiments, the immune checkpoint inhibitor of this application may comprise pembrolizumab's VH and VL. For example, the immune checkpoint inhibitor of this application may comprise pembrolizumab or its antigen-binding fragment.

[0248] For example, the drug can inhibit tumor growth with a tumor volume inhibition rate of about 73% or higher or about 83% or higher.

[0249] In some embodiments, the isolated GITR-binding protein and / or polypeptide containing the GITR-binding protein of this application are not mixed with the immune checkpoint inhibitor in the drug combination. For example, the isolated GITR-binding protein and / or polypeptide containing the GITR-binding protein of this application and the immune checkpoint inhibitor are each present in separate containers.

[0250] In some embodiments, the pharmaceutical combination comprises a first formulation and a second formulation, the first formulation comprising the isolated GITR-binding protein of the present application and / or a polypeptide containing the GITR-binding protein and a pharmaceutically acceptable first carrier, and the second formulation comprising the immune checkpoint inhibitor and a pharmaceutically acceptable second carrier.

[0251] In some embodiments, it comprises a pharmaceutical composition, wherein the pharmaceutical composition contains the isolated GITR-binding protein of this application and / or a polypeptide containing the GITR-binding protein and the immune checkpoint inhibitor.

[0252] On the one hand, this application provides a reagent kit.

[0253] For example, the kit may contain the isolated GITR-binding protein of this application, a polypeptide containing the GITR-binding protein, a nucleic acid molecule encoding the isolated GITR-binding protein of this application, a carrier containing a nucleic acid molecule encoding the isolated GITR-binding protein of this application, an immunoconjugate containing the isolated GITR-binding protein of this application, cells containing the above substances, a pharmaceutical composition of this application, and / or a drug combination of this application.

[0254] In some implementations, the kit may be used to detect the presence and / or content of GITR in a sample.

[0255] On the one hand, this application provides a preparation method.

[0256] On the other hand, this application provides a method for preparing the isolated GITR-binding protein and / or polypeptides containing the GITR-binding protein of this application. The method may include culturing the host cells described in this application under conditions that cause the isolated GITR-binding protein and / or polypeptides containing the GITR-binding protein of this application to express. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0257] Any method suitable for producing monoclonal antibodies can be used to produce the antigen-binding protein of this application. For example, animals can be immunized using linked or naturally occurring GITR or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more routes. For example, hybridoma preparation methods can be used to obtain spleen cells from immunized mice, fuse them with SP2 / 0 myeloma cells, and select hybridoma cell lines through HAT screening.

[0258] Any suitable form of GITR can serve as an immunogen (antigen) to generate non-human antibodies specific to GITR and to screen for the biological activity of said antibodies. For example, the stimulating immunogen can be a full-length mature human GITR, including natural homodimers or peptides containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0259] Chimeric human antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. In this application, the antibody can be an IgG antibody, and IgG1, IgG2, IgG3, or IgG4 subtypes can be used. The necessary constant domain sequence can be optimized to produce the desired biological activity by screening antibodies using biological assays in the art. Similarly, any class of light chains can be used in the compounds and methods of this application. For example, the κ chain or a variant thereof can be used in the compounds and methods of this application.

[0260] On the one hand, this application provides a method and a use.

[0261] On the other hand, this application provides a method for detecting GITR in a sample, which may include administering the isolated GITR-binding protein of this application, administering a polypeptide containing the GITR-binding protein, administering a nucleic acid molecule encoding the isolated GITR-binding protein of this application, administering a carrier containing a nucleic acid molecule encoding the isolated GITR-binding protein of this application, administering an immunoconjugate containing the isolated GITR-binding protein of this application, administering cells containing the above substances, administering a pharmaceutical composition of this application, administering a pharmaceutical combination of this application, and / or using a kit of this application.

[0262] In some cases, the method for detecting GITR in a sample may be an in vitro method. In some cases, the method for detecting GITR in a sample may be a non-therapeutic method. In some cases, the method for detecting GITR in a sample may not be a diagnostic method.

[0263] On the other hand, this application provides the use of the isolated GITR-binding protein of this application, a polypeptide containing the GITR-binding protein, a nucleic acid molecule encoding the isolated GITR-binding protein of this application, a carrier containing a nucleic acid molecule encoding the isolated GITR-binding protein of this application, an immunoconjugate containing the isolated GITR-binding protein of this application, cells containing the above substances, the drug composition of this application, and / or the pharmaceutical combination of this application in the preparation of a kit. For example, the kit can be used to detect the presence and / or content of GITR in a sample.

[0264] On one hand, this application provides a preventive and / or therapeutic use. GITR can serve as a novel target, and antibodies targeting GITR can have the ability to activate the GITR-NFκB signaling pathway. Existing data suggest that antibodies targeting GITR can activate an immune response, and GITR antibodies can be developed into a drug for the prevention and / or treatment of diseases and / or conditions, or GITR antibodies can be used as a method for the prevention and / or treatment of diseases and / or conditions.

[0265] On the other hand, this application provides the use of the isolated GITR-binding protein of this application, a polypeptide containing the GITR-binding protein, a nucleic acid molecule encoding the isolated GITR-binding protein of this application, a carrier containing the nucleic acid molecule encoding the isolated GITR-binding protein of this application, an immunoconjugate containing the isolated GITR-binding protein of this application, a cell containing the above substances, a pharmaceutical composition of this application, a pharmaceutical combination of this application, and / or a kit of this application in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions. For example, the diseases and / or conditions of this application may be immune-related disorders and / or proliferative disorders. For example, the diseases and / or conditions of this application may be diseases related to abnormal GITR protein expression or dysfunction. For example, the diseases and / or conditions of this application may be GITR-positive tumors. For example, the diseases and / or conditions of this application may be GITR-overexpressing tumors.

[0266] In this application, the disease and / or condition may be a GITR-related disease and / or condition.

[0267] In this application, the disease and / or condition may include tumors and / or infections. In this application, the disease and / or condition may include conditions of excessive cell growth or proliferation. In this application, the disease and / or condition may include proliferative diseases, neoplastic diseases, and / or immune diseases. For example, the tumor and / or infection in this application may be a tumor and / or infection related to abnormal GITR protein expression or function. For example, the tumor in this application may be a GITR-positive tumor. For example, the disease and / or condition in this application may be a GITR-highly expressed tumor.

[0268] In this application, the tumor may include solid tumors and / or hematomas.

[0269] In this application, the disease and / or condition may include breast cancer, stomach cancer, rectal cancer, and / or colon cancer.

[0270] On the other hand, this application provides an isolated GITR-binding protein, a polypeptide containing the GITR-binding protein, a nucleic acid molecule encoding the isolated GITR-binding protein, a carrier containing the nucleic acid molecule encoding the isolated GITR-binding protein, an immunoconjugate containing the isolated GITR-binding protein, a cell containing the above substances, a pharmaceutical composition, a drug combination, and / or a kit, which can be used for the prevention and / or treatment of diseases and / or conditions. For example, the diseases and / or conditions of this application may be immune-related disorders and / or proliferative disorders. For example, the diseases and / or conditions of this application may be diseases related to abnormal GITR protein expression or functional abnormalities. For example, the diseases and / or conditions of this application may be GITR-positive tumors. For example, the diseases and / or conditions of this application may be GITR-overexpressing tumors.

[0271] In this application, the disease and / or condition may be a GITR-related disease and / or condition.

[0272] In this application, the disease and / or condition may include tumors and / or infections. In this application, the disease and / or condition may include conditions of excessive cell growth or proliferation. In this application, the disease and / or condition may include proliferative diseases, neoplastic diseases, and / or immune diseases. For example, the tumor and / or infection in this application may be a tumor and / or infection related to abnormal GITR protein expression or function. For example, the tumor in this application may be a GITR-positive tumor. For example, the disease and / or condition in this application may be a GITR-highly expressed tumor.

[0273] In this application, the tumor may include solid tumors and / or hematomas.

[0274] In this application, the disease and / or condition may include breast cancer, stomach cancer, rectal cancer, and / or colon cancer.

[0275] On the other hand, this application provides a method for preventing and / or treating diseases and / or symptoms, which may include administering to a subject in need the isolated GITR-binding protein of this application, a polypeptide containing the GITR-binding protein, a nucleic acid molecule encoding the isolated GITR-binding protein of this application, a carrier containing a nucleic acid molecule encoding the isolated GITR-binding protein of this application, an immunoconjugate containing the isolated GITR-binding protein of this application, a cell containing the above substances, a drug composition of this application, a pharmaceutical combination of this application, and / or a kit of this application.

[0276] In this application, the application can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal application.

[0277] In this application, the disease and / or condition may be a GITR-related disease and / or condition.

[0278] In this application, the disease and / or condition may include tumors and / or infections. In this application, the disease and / or condition may include conditions of excessive cell growth or proliferation. In this application, the disease and / or condition may include proliferative diseases, neoplastic diseases, and / or immune diseases. For example, the tumor and / or infection in this application may be a tumor and / or infection related to abnormal GITR protein expression or function. For example, the tumor in this application may be a GITR-positive tumor. For example, the disease and / or condition in this application may be a GITR-highly expressed tumor.

[0279] In this application, the tumor may include solid tumors and / or hematomas.

[0280] In this application, the disease and / or condition may include breast cancer, stomach cancer, rectal cancer, and / or colon cancer.

[0281] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the proteins, preparation methods and uses of this application, and are not intended to limit the scope of the invention.

[0282] Implementation Plan

[0283] 1. An isolated antigen-binding protein comprising HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0284] 2. The isolated antigen-binding protein according to embodiment 1, comprising HCDR2, wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2.

[0285] 3. The isolated antigen-binding protein according to any one of embodiments 1-2, comprising HCDR1, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0286] 4. The isolated antigen-binding protein according to any one of embodiments 1-3, comprising LCDR3, wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0287] 5. The isolated antigen-binding protein according to any one of embodiments 1-4, comprising LCDR2, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5.

[0288] 6. The isolated antigen-binding protein according to any one of embodiments 1-5, comprising LCDR1, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0289] 7. The isolated antigen-binding protein according to any one of embodiments 1-6, comprising H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:36.

[0290] 8. The isolated antigen-binding protein according to any one of embodiments 1-7, comprising H-FR1, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0291] 9. The isolated antigen-binding protein according to any one of embodiments 1-8, comprising H-FR2 located between HCDR1 and HCDR2, and wherein H-FR2 comprises the amino acid sequence shown in SEQ ID NO:37.

[0292] 10. The isolated antigen-binding protein according to any one of embodiments 1-9, comprising H-FR2 located between HCDR1 and HCDR2, and wherein the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0293] 11. The isolated antigen-binding protein according to any one of embodiments 1-10, comprising H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 comprising the amino acid sequence shown in SEQ ID NO:38.

[0294] 12. The isolated antigen-binding protein according to any one of embodiments 1-11, comprising H-FR3, said H-FR3 being located between said HCDR2 and said HCDR3, and said H-FR3 comprising the amino acid sequence shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

[0295] 13. The isolated antigen-binding protein according to any one of embodiments 1-12, comprising H-FR4, wherein the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and wherein H-FR4 comprises the amino acid sequence shown in SEQ ID NO:39.

[0296] 14. The isolated antigen-binding protein according to any one of embodiments 1-13, comprising H-FR4, wherein the N-terminus of H-FR4 is directly or indirectly linked to the C-terminus of HCDR3, and wherein H-FR4 comprises the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:18.

[0297] 15. The isolated antigen-binding protein according to any one of embodiments 1-14, comprising L-FR1, wherein the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:40.

[0298] 16. The isolated antigen-binding protein according to any one of embodiments 1-15, comprising L-FR1, wherein the C-terminus of L-FR1 is directly or indirectly linked to the N-terminus of LCDR1, and wherein L-FR1 comprises the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0299] 17. The isolated antigen-binding protein according to any one of embodiments 1-16, comprising L-FR2 located between LCDR1 and LCDR2, and wherein L-FR2 comprises the amino acid sequence shown in SEQ ID NO:41.

[0300] 18. The isolated antigen-binding protein according to any one of embodiments 1-17, comprising L-FR2 located between LCDR1 and LCDR2, and wherein L-FR2 comprises the amino acid sequence shown in SEQ ID NO:22 or SEQ ID NO:23.

[0301] 19. The isolated antigen-binding protein according to any one of embodiments 1-18, comprising L-FR3 located between LCDR2 and LCDR3, and wherein the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:42.

[0302] 20. The isolated antigen-binding protein according to any one of embodiments 1-19, comprising L-FR3 located between LCDR2 and LCDR3, and wherein the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

[0303] 21. The isolated antigen-binding protein according to any one of embodiments 1-20, comprising L-FR4, wherein the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and wherein L-FR4 comprises the amino acid sequence shown in SEQ ID NO:43.

[0304] 22. The isolated antigen-binding protein according to any one of embodiments 1-21, comprising L-FR4, wherein the N-terminus of L-FR4 is directly or indirectly linked to the C-terminus of LCDR3, and wherein L-FR4 comprises the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28.

[0305] 23. The isolated antigen-binding protein according to any one of embodiments 1-22, comprising HCDR1, HCDR2 and HCDR3, wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.

[0306] 24. The isolated antigen-binding protein according to any one of embodiments 1-23, comprising LCDR1, LCDR2 and LCDR3, wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.

[0307] 25. The isolated antigen-binding protein according to any one of embodiments 1-24, comprising a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO:44.

[0308] 26. The isolated antigen-binding protein according to any one of embodiments 1-25, comprising VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.

[0309] 27. The isolated antigen-binding protein according to any one of embodiments 1-26, comprising a light chain variable region (VL) comprising the amino acid sequence shown in SEQ ID NO:45.

[0310] 28. The isolated antigen-binding protein according to any one of embodiments 1-27, comprising a VL, said VL comprising the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.

[0311] 29. The isolated antigen-binding protein according to any one of embodiments 1-28, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:44, and the VL comprises the amino acid sequence shown in SEQ ID NO:45.

[0312] 30. The isolated antigen-binding protein according to any one of embodiments 1-29, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36.

[0313] 31. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:29 and the VL comprises the amino acid sequence shown in SEQ ID NO:33.

[0314] 32. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:30, and the VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0315] 33. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:30, and the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0316] 34. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:31, and the VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0317] 35. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:31, and the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0318] 36. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:34.

[0319] 37. The isolated antigen-binding protein according to any one of embodiments 1-30, comprising VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:32, and the VL comprises the amino acid sequence shown in SEQ ID NO:35.

[0320] 38. The isolated antigen-binding protein according to any one of embodiments 1-37, comprising an antibody heavy chain constant region.

[0321] 39. The isolated antigen-binding protein according to embodiment 38, wherein the antibody heavy chain constant region comprises a heavy chain constant region derived from a human antibody.

[0322] 40. The isolated antigen-binding protein according to any one of embodiments 38-39, wherein the antibody heavy chain constant region comprises a heavy chain constant region derived from IgG, IgA, IgD, IgE and / or IgM.

[0323] 41. The isolated antigen-binding protein according to any one of embodiments 38-40, wherein the antibody heavy chain constant region comprises a heavy chain constant region derived from IgG.

[0324] 42. The isolated antigen-binding protein according to any one of embodiments 38-41, wherein the antibody heavy chain constant region comprises a heavy chain constant region derived from IgG1, IgG2, IgG3 and / or IgG4.

[0325] 43. The isolated antigen-binding protein according to any one of embodiments 38-42, wherein the antibody heavy chain constant region comprises a heavy chain constant region derived from IgG1.

[0326] 44. The isolated antigen-binding protein according to any one of embodiments 1-43, comprising the constant region of the antibody light chain.

[0327] 45. The isolated antigen-binding protein according to embodiment 44, wherein the antibody light chain constant region comprises a light chain constant region derived from a human antibody.

[0328] 46. ​​The isolated antigen-binding protein according to any one of embodiments 44-45, wherein the constant region of the antibody light chain comprises a constant region derived from human Igκ.

[0329] 47. An isolated antigen-binding protein according to any one of embodiments 1-46, comprising an antibody or an antigen-binding fragment thereof.

[0330] 48. The isolated antigen-binding protein according to embodiment 47, wherein the antibody comprises a monoclonal antibody.

[0331] 49. The isolated antigen-binding protein according to any one of embodiments 47-48, wherein the antibody is selected from one or more of the group consisting of murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0332] 50. The isolated antigen-binding protein according to embodiment 47, wherein the antigen-binding fragment is selected from one or more of the group consisting of: Fab, Fab′, Fv fragment, F(ab′)2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0333] 51. The isolated antigen-binding protein according to any one of embodiments 1-50, which is capable of specifically binding to glucocorticoid-induced tumor necrosis factor receptor (GITR) or its functionally active fragment.

[0334] 52. The isolated antigen-binding protein according to embodiment 51, wherein the GITR comprises human GITR.

[0335] 53. The isolated antigen-binding protein according to any one of embodiments 51-52, having an EC50 of about 5.01 μg / ml or less. 50The ability to combine the value with the GITR or its functional segments.

[0336] 54. The isolated antigen-binding protein according to embodiment 53, wherein the GITR or a functional fragment thereof comprises a GITR or a functional fragment thereof expressed on cells.

[0337] 55. The isolated antigen-binding protein according to any one of embodiments 51-54, having a K0 of about 3.01E-08M or lower. D The ability to combine the value with the GITR or its functional segments.

[0338] 56. The isolated antigen-binding protein according to any one of embodiments 51-55, which has the ability to activate the GITR or a functionally active fragment thereof.

[0339] 57. The isolated antigen-binding protein according to any one of embodiments 51-56, having an EC50 of about 3.20 μg / ml or less. 50 The ability of the value to activate the GITR or its functional active fragment.

[0340] 58. The isolated antigen-binding protein according to any one of embodiments 1-57, which has the ability to activate the nuclear factor-κB (NF-κB) pathway.

[0341] 59. The isolated antigen-binding protein according to any one of embodiments 51-58, having an EC50 of about 9.76 μg / ml or less. 50 The ability to combine the value with the GITR or its functional segments.

[0342] 60. The isolated antigen-binding protein according to any one of embodiments 51-59, having an EC50 of about 0.34 μg / ml or less. 50 The ability to combine the value with the GITR or its functional segments.

[0343] 61. The isolated antigen-binding protein according to embodiments 59-60, wherein the GITR comprises human GITR and / or monkey GITR.

[0344] 62. The isolated antigen-binding protein according to any one of embodiments 1-61 has tumor-suppressive ability.

[0345] 63. The isolated antigen-binding protein according to any one of embodiments 1-62, which has the ability to inhibit tumors with a tumor volume inhibition rate of about 38% or higher.

[0346] 64. A polypeptide comprising the isolated antigen-binding protein of any one of embodiments 1-63.

[0347] 65. The polypeptide according to embodiment 64, comprising a fusion protein.

[0348] 66. A nucleic acid molecule encoding an isolated antigen-binding protein as described in any one of embodiments 1-63 and / or a polypeptide as described in any one of embodiments 64-65.

[0349] 67. A vector comprising the nucleic acid molecule described in embodiment 66.

[0350] 68. An immunoconjugate comprising the isolated antigen-binding protein of any one of embodiments 1-63 and / or the polypeptide of any one of embodiments 64-65.

[0351] 69. Cells comprising and / or expressing the isolated antigen-binding protein of any one of embodiments 1-63, comprising and / or expressing the polypeptide of any one of embodiments 64-65, comprising the nucleic acid molecule of embodiment 66, comprising the vector of embodiment 67, and / or comprising the immunoconjugate of embodiment 68.

[0352] 70. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of embodiments 1-63, the polypeptide of any one of embodiments 64-65, the nucleic acid molecule of embodiment 66, the carrier of embodiment 67, the immunoconjugate of embodiment 68, and / or the cell of embodiment 69, and optionally a pharmaceutically acceptable carrier.

[0353] 71. The pharmaceutical composition according to embodiment 70 further comprises one or more immune checkpoint inhibitors.

[0354] 72. A pharmaceutical combination comprising the isolated antigen-binding protein of any one of embodiments 1-63 and / or the polypeptide of any one of embodiments 64-65, and one or more immune checkpoint inhibitors.

[0355] 73. The drug combination according to embodiment 72, wherein the immune checkpoint inhibitor is capable of inhibiting the interaction between PD-1 and PD-L1.

[0356] 74. The drug combination according to any one of embodiments 72-73, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0357] 75. The drug combination according to any one of embodiments 72-74, wherein the immune checkpoint inhibitor comprises a PD-1 antibody or an antigen-binding fragment thereof, and / or a PD-L1 antibody or an antigen-binding fragment thereof.

[0358] 76. The drug combination according to embodiment 75, wherein the antibody comprises a subset selected from: murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0359] 77. The drug combination according to embodiment 75, wherein the antigen-binding fragment is selected from: Fab, Fab′, Fv fragment, F(ab′)2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0360] 78. The drug combination according to any one of embodiments 72-77, wherein the immune checkpoint inhibitor comprises HCDR1-3 and LCDR1-3 of pembrolizumab.

[0361] 79. The drug combination according to any one of embodiments 72-78, wherein the immune checkpoint inhibitor comprises pembrolizumab VH and VL.

[0362] 80. The drug combination according to any one of embodiments 72-79, wherein the immune checkpoint inhibitor comprises pembrolizumab.

[0363] 81. The pharmaceutical combination according to any one of embodiments 72-80, wherein the isolated antigen-binding protein and / or the polypeptide and the immune checkpoint inhibitor are not mixed with each other in the pharmaceutical combination.

[0364] 82. The pharmaceutical combination according to any one of embodiments 72-81, wherein the isolated antigen-binding protein and / or the polypeptide and the immune checkpoint inhibitor are each contained in separate containers.

[0365] 83. The pharmaceutical combination according to any one of embodiments 72-82, wherein the pharmaceutical combination comprises a first formulation and a second formulation, the first formulation comprising the isolated antigen-binding protein and / or the polypeptide and a pharmaceutically acceptable first carrier, and the second formulation comprising the immune checkpoint inhibitor and a pharmaceutically acceptable second carrier.

[0366] 84. A kit comprising the isolated antigen-binding protein of any one of embodiments 1-63, the polypeptide of any one of embodiments 64-65, the nucleic acid molecule of embodiment 66, the vector of embodiment 67, the immunoconjugate of embodiment 68, the cell of embodiment 69, the pharmaceutical composition of any one of embodiments 70-71, and / or the pharmaceutical combination of any one of embodiments 72-83.

[0367] 85. The kit according to embodiment 84 is used to detect the presence and / or content of GITR in a sample.

[0368] 86. A method for preparing the isolated antigen-binding protein of any one of embodiments 1-63 and / or the polypeptide of any one of embodiments 64-65, comprising culturing the cells of embodiment 69 under conditions that cause the antigen-binding protein and / or the polypeptide to be expressed.

[0369] 87. The method according to embodiment 86, comprising culturing the cells under conditions that cause the antigen-binding protein to be expressed.

[0370] 88. The method according to embodiment 86, comprising culturing the cells under conditions that cause the polypeptide to be expressed.

[0371] 89. A method for detecting GITR in a sample, comprising administering the isolated antigen-binding protein as described in any one of embodiments 1-63, administering the polypeptide as described in any one of embodiments 64-65, administering the nucleic acid molecule as described in embodiment 66, administering the carrier as described in embodiment 67, administering the immunoconjugate as described in embodiment 68, administering the cell as described in embodiment 69, administering the pharmaceutical composition as described in any one of embodiments 70-71, administering the pharmaceutical combination as described in any one of embodiments 72-83, and / or using the kit as described in any one of embodiments 84-85.

[0372] 90. Use of the isolated antigen-binding protein of any one of embodiments 1-63, the polypeptide of any one of embodiments 64-65, the nucleic acid molecule of embodiment 66, the carrier of embodiment 67, the immunoconjugate of embodiment 68, the cell of embodiment 69, the pharmaceutical composition of any one of embodiments 70-71, and / or the pharmaceutical combination of any one of embodiments 72-83 in the preparation of a kit.

[0373] 91. The use according to embodiment 90, wherein the kit is used to detect the presence and / or content of GITR in a sample.

[0374] 92. Use of the isolated antigen-binding protein of any one of embodiments 1-63, the polypeptide of any one of embodiments 64-65, the nucleic acid molecule of embodiment 66, the carrier of embodiment 67, the immunoconjugate of embodiment 68, the cell of embodiment 69, the pharmaceutical composition of any one of embodiments 70-71, the pharmaceutical composition of any one of embodiments 72-83, and / or the kit of any one of embodiments 84-85 in the preparation of a medicament for the prevention and / or treatment of diseases and / or conditions.

[0375] 93. The use according to embodiment 92, wherein the disease and / or condition includes tumors and / or infections.

[0376] 94. The use according to any one of embodiments 92-93, wherein the disease and / or condition comprises solid tumors and / or hematologic malignancies.

[0377] 95. The use according to any one of embodiments 92-94, wherein the disease and / or condition includes breast cancer, gastric cancer, rectal cancer, and / or colon cancer.

[0378] Example

[0379] Example 1: Preparation of GITR antigen

[0380] 1. Antigen coating: Prepare serially diluted GITR antigen (Human GITR Fc, Genscript, catalog number: Z03440) solutions using DPBS solution. Add 100 μl to each well of a 96-well plate and coat at 37°C for 1 hour.

[0381] 2. Washing: Wash three times with PBST (containing 0.05% Tween 20) solution.

[0382] 3. Blocking: Block the antigen with DPBS (containing 3% BSA), add 200 μl to each well, and incubate at room temperature for 1 hour.

[0383] 4. Ligand binding: Dilute the GITR ligand (Bio-GITR Ligand, Acrobiosystem, catalog number: GIL-H82F8) to 1 μg / ml with DPBS (containing 1% BSA), add 100 μl to each well, and coat at 37°C for 1 hour.

[0384] 5. Secondary antibody conjugation: Discard the reaction solution, wash three times with PBST solution, add 100 μl of secondary antibody (Goat anti-Ms IgG F(ab′)2 Secondary Antibody, HRP conjugate, source: invitrogen, catalog number: 31436) to each well, and incubate at 37°C for 1 hour.

[0385] 6. ELISA assay: Discard the reaction solution and wash three times with PBST solution; add 100 μl of TMB solution to each well and gently shake until a blue color appears; add 50 μl of H2SO4 to each well and gently shake until a yellow color appears; detect the OD value at 450 nm.

[0386] like Figure 1 As shown, the GITR antigen activity was measured by ELISA.

[0387] Example 2 Preparation of GITR-binding protein

[0388] GITR binding protein was obtained by immunizing 6-week-old female BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., weighing 18 grams).

[0389] Hybridoma technology involves fusing antigen-immunized mouse spleen cells and mouse myeloma cells SP2 / 0, thereby simultaneously preserving the main characteristics of both cell types. Mouse spleen cells (B lymphocytes) immunized with specific antigens are characterized by their antibody-secreting function but cannot be continuously cultured in vitro. Mouse myeloma cells, on the other hand, possess so-called immortality, capable of unlimited division and proliferation under culture conditions. Under selective culture conditions, only hybrid cells resulting from the fusion of B cells and myeloma cells can form cell clones possessing both antibody-secreting function and immortality. In this experiment, mice were immunized with human GITR protein (Genscript, catalog number: Z03440) and an adjuvant (Sigma Adjuvant System, Sigma, catalog number: S6322). The resulting mouse spleen cells were then fused with SP2 / 0 cells to obtain hybridoma cells capable of expressing positive antibodies.

[0390] Table 1 shows the experimental animals and their immunization information.

[0391] Table 1. Experimental animals and immunization information

[0392] Immune antigens GITR antigen Immunization methods 25μg / animal, intramuscular injection Number of immunizations 3 Finally, strengthen immunity. 50 μg GITR protein, intraperitoneal injection (ip), three days before fusion.

[0393] Serum titer of immunized mice after immunization

[0394] 1. Antigen coating: Prepare a 0.5 μg / ml solution of GITR antigen (GITR his, Sinobiological, catalog number: H13643-H08H) using DPBS solution. Add 100 μl to each well of a 96-well plate and coat at 37°C for 1 hour.

[0395] 2. Washing: Wash three times with PBST (containing 0.05% Tween 20) solution.

[0396] 3. Blocking: Block the antigen with DPBS (containing 3% BSA), add 200 μl to each well, and incubate at room temperature for 1 hour.

[0397] 4. Antibody binding: Dilute the serum solution with DPBS (containing 1% BSA). The first concentration was diluted 300-fold, and then serially diluted 3-fold to obtain 8 concentrations. Add 100 μl to each well. The negative control was serum from unimmunized mice. The concentration of the positive control well was 1 μg / ml. Incubate at room temperature for 1 hour.

[0398] 5. Secondary antibody conjugation: Discard the reaction solution and wash three times with PBST solution; add 100 μl of secondary antibody (Goat anti-Ms IgG F(ab′)2 Secondary Antibody, HRP conjugate, source: invitrogen, catalog number: 31436) to each well and incubate at room temperature for 1 hour.

[0399] 6. ELISA assay: Discard the reaction solution and wash three times with PBST solution; add 100 μl of TMB solution to each well and gently shake until a blue color appears; add 50 μl of H2SO4 to each well and gently shake until a yellow color appears; detect the OD value at 450 nm.

[0400] like Figure 2 As shown, the serum titers of multiple immunized mice.

[0401] Table 2 shows the serum efficacy values ​​of multiple immunized mice.

[0402] Table 2 Serum titers of immunized mice

[0403] 512K 512K 1024K 1024K 512K

[0404] Hybridoma fusion

[0405] Preparation of electrofusion tank: After thoroughly soaking the electrofusion tank with 1% sodium hydroxide and 75% ethanol, clean it with PBS and then dry it in a clean bench for later use.

[0406] Isolation of spleen cells

[0407] 1. Dissection: The spleen of the immunized mouse was obtained by routine dissection.

[0408] 2. Collect spleen cells: Place the spleen in a sterile petri dish, lift it up while adding 3-4 ml of serum-free DMEM for washing, then transfer it to another sterile petri dish containing 20 ml of serum-free DMEM, place a filter screen and grind it with a syringe core, aspirate all the supernatant, centrifuge at 1200 rpm for 5 minutes, and collect the spleen cells.

[0409] 3. Lysis of red blood cells: Remove the supernatant and resuspend the cells in 10 ml of ACK lysis buffer (Gibco). Then add 5 ml of ACK lysis buffer. After the suspension stands for 5 minutes, centrifuge at 1200 rpm for 5 minutes. Remove the supernatant and resuspend the cells in 10 ml of basal culture medium, then add 20 ml of PBS and centrifuge at 1200 rpm for 5 minutes. Remove the supernatant, resuspend the cells in 20 ml of PBS and count them.

[0410] 4. Collect SP2 / 0 cells: Collect SP2 / 0 cells, centrifuge at 1200 rpm for 5 minutes and discard the supernatant. Dilute the collected SP2 / 0 cells 10-fold with PBS, mix well, and then take 10 μl of the solution and mix it with 10 μl of AOPI staining solution for cell counting.

[0411] 5. Cell Fusion: Mix SP2 / 0 cells and spleen cells, centrifuge at 1000 rpm for 6 minutes. After removing the supernatant, resuspend the cells in an appropriate amount of fusion buffer. Add 9 ml of cell suspension to each electrofusion dish for electrofusion. After fusion, allow the cells to stand in the electrode holder for 5 minutes. The parameters of the electrofusion instrument can be set according to common practices in the field.

[0412] 6. Cell Plating: Remove the cell suspension and transfer it to selection medium. Gently rinse the chamber twice with electrofusion buffer, then transfer the rinsed cells to the medium and plate them. Incubate the cell plates in a cell culture incubator. Replace the selection medium on day 7 after fusion. Perform selection after day 10 (or longer, depending on cell growth status). Screen for hybridoma cells expressing the specific GITR-binding protein using FACS and ELISA. The selection medium can be prepared using methods commonly used in the art.

[0413] Positive hybridoma cell subclones

[0414] Subcloning procedure: Prepare a 96-well plate. Add 100 μl of the basal culture medium as described above to columns 2 through 12 of the 96-well cell plate. Prepare a cell suspension from the positive wells selected through fusion screening and add it to column 1. Take 100 μl of the cell suspension from column 1 and add it to column 2. Mix thoroughly, then add 100 μl to the next column. Repeat the above steps until the last column. Let the serially diluted cell suspension stand for 5-10 minutes until all cells have settled to the bottom. Observe and count under a microscope. Take the volume corresponding to 100 cells and add it to 20 ml of the basal culture medium as described above, mix well, and plate the plate, 200 μl per well. Observe under a microscope after one week, identify and mark the single-clone wells, and pick out the positive wells for testing.

[0415] Positive hybridoma single-cell screening

[0416] Hybridoma monoclonal cells expressing specific GITR-binding proteins were screened using FACS and ELISA.

[0417] Cell cryopreservation: Observe the cell status. When the cells grow well and the viability is >90%, centrifuge at 1000 rpm for 5 minutes and collect the supernatant. Resuspend the cells in cryopreservation buffer (90% FBS, 10% DMSO) and aliquot them into cryovials. Place them in a programmed cooling box and freeze at -80°C.

[0418] Trizol sample collection: Observe cell status. When cells grow well and viability is >90%, centrifuge at 1000 rpm for 5 minutes and collect the supernatant. Resuspend cells in Trizol solution and aliquot into centrifuge tubes, then freeze at -80°C.

[0419] antigen-binding protein sequencing

[0420] This application utilizes molecular biology techniques to obtain the antigen-binding protein sequence in GITR-positive hybridoma cells, and then performs chimerism and humanization modification.

[0421] Humanized design of GITR-binding proteins

[0422] To reduce the immunogenicity of murine antibodies, the selected biologically active antibodies can optionally be humanized. Humanization of murine monoclonal antibodies is performed according to methods published in numerous publications in the field. In short, the constant domain of the parental (murine antibody) can be replaced with a human antibody constant domain, and a human germline antibody sequence is selected based on the homology between the murine and human antibodies for CDR transplantation. Then, based on the three-dimensional structure of the murine antibody, reverse mutations can be performed on the amino acid residues of VL and VH to replace the constant region of the murine antibody with the human constant region, yielding the final humanized binding protein.

[0423] As shown in Tables 3, 4 and 5, the amino acid sequences of the CDR, heavy chain variable region VH, light chain variable region VL, FR, light chain constant region CL and heavy chain constant region CH of the antigen-binding protein of this application are presented.

[0424] Table 3. Sequence information of the CDR, heavy chain variable region VH, and light chain variable region VL of the antigen-binding protein in this application.

[0425]

[0426]

[0427] Table 4. Sequence information of the antigen-binding protein FR in this application.

[0428]

[0429] Table 5. Sequence information of the light chain constant region CL and heavy chain constant region CH of the antigen-binding protein in this application.

[0430] Lambda, the constant region of light chains SEQ ID NO:47 Heavy chain constant region IgG1 SEQ ID NO:48 Heavy chain constant region IgG2 SEQ ID NO:49 Heavy chain constant region IgG3 SEQ ID NO:50 Heavy chain constant region IgG4 SEQ ID NO:51

[0431] Expression and purification of antibody proteins

[0432] A plasmid was constructed based on the antigen-binding protein sequence of this application. The antibody expression plasmid was extracted using a high-volume extraction kit and transiently transfected into HEK293 cells. Solution 1: Dilute 15 μg of plasmid with 1 ml of culture medium and mix well. Solution 2: Dilute 60 μl of transfection reagent with 1 ml of culture medium and mix well. Add Solution 2 to Solution 1, mix well, incubate at 37°C for 15 minutes, then add the mixed transfection solution dropwise to the cell culture medium while shaking. Incubate on a shaker for one week, collect the supernatant, and centrifuge at 8000 rpm for 5 minutes.

[0433] Protein A affinity chromatography column was used to purify antibody protein. (1) Equilibrate the column: 1×PBS, flow rate 1ml / min, 20ml (2) Load sample: flow rate 1ml / min (3) Wash: 1×PBS, flow rate 1ml / min, 20ml (4) Elute: citrate buffer (pH 3.4), 1ml / min, collected in separate tubes, approximately 500μl per tube. A total of 10 tubes were collected, and the absorbance value at 280nm was read using a NanoDrop instrument. (5) Dialysis: The high concentration of protein was aspirated into a dialysis bag and placed in a beaker containing 1×PBS for dialysis. The purity was tested using a high performance liquid chromatograph LC-20AT and a gel chromatography column, and the endotoxin test was passed.

[0434] Example 3: Binding of GITR-binding protein to GITR expressed on cells

[0435] The binding affinity of GITR-binding proteins to human GITR expressed on the surface of HT1080 cells was determined by flow cytometry. The binding affinity was assessed by comparing the binding curves of different GITR-binding proteins to human GITR expressed on the surface of HT1080 cells.

[0436] (1) HT1080 / hGITR cells (Kangyuan Bochuang) were seeded in a 96-well plate.

[0437] (2) Prepare positive control antibody 1 (B-011B-PC-1, for example, the full-length heavy chain sequence can be as shown in SEQ ID NO:52, and the full-length light chain sequence can be as shown in SEQ ID NO:53), negative control antibody (human IgG) and antigen-binding protein antibody of this application with PBS containing 2% FBS. The maximum concentration is 15 μg / ml. Dilute 5 times, 8 spots, add 100 μl to each tube, and incubate at 4 degrees Celsius for 1 hour.

[0438] (3) Wash the plate three times with PBS containing 2% FBS.

[0439] (4) Dilute PE-labeled human IgG (Biolegend, 410708) with PBS containing 2% FBS at a ratio of 1:2000, add 100 μl to each well, and incubate at 4 degrees Celsius for 0.5 hours.

[0440] (5) Wash the plate twice with PBS containing 2% FBS. Resuspend the cells in 100 μl of PBS containing 2% FBS.

[0441] (6) The median fluorescence value (MFI) of the PE channel was determined using flow cytometry.

[0442] The above experiments demonstrate that the chimeric GITR-binding protein of this application has the ability to bind to human GITR overexpressed on HT1080 cells.

[0443] like Figure 3 As shown, the binding curves of GITR-binding protein and GITR expressed on the cell surface were determined by flow cytometry.

[0444] As shown in Table 6, the binding of GITR-binding protein to GITR expressed on the cell surface in ECs was determined by flow cytometry. 50 Value and highest MFI value.

[0445] Table 6 ECs of GITR-binding proteins and GITR binding 50 Value and highest MFI value

[0446] Positive control antibody 1 0.5337 51807.92 negative control antibody - 436.06 Ab0 0.5306 48846.22

[0447] Example 4 Biological activity of GITR-binding proteins

[0448] GITR is a cellular co-stimulatory target; binding proteins to it can activate the GITR pathway. This study compared the functions of different GITR-binding proteins by detecting changes in luciferase expression in Jurkat-GITR-NFκB cells after GITR-binding proteins bound to cellularly expressed GITR, using a luciferase reporter gene system.

[0449] (1) To screen for biologically active GITR binding proteins, cDNA encoding human GITR (amino acid sequence as shown in SEQ ID NO:55, nucleotide sequence as shown in SEQ ID NO:56) was cloned into the pLVX-IRES-Puro (takara, 632186) vector. The obtained plasmid and the NF-kappaB luciferace reporter gene plasmid (Promega) were co-transfected into Jurkat cells (Chinese Academy of Sciences Cell Bank, TCU123) to generate Jurkat-GITR-NFκB cells that overexpress human GITR and carry the NF-kappaB luciferace reporter gene system.

[0450] Jurkat-GITR-NFκB cells were seeded in 96-well transparent white plates (Corning, 3903) and placed in a cell culture incubator.

[0451] (2) Antibody crosslinking: The anti-human secondary antibody (AffiniPure F(ab′)2Fragment, Goat Anti-Human IgG, Fcγ fragment specific, Jackson Immunoresearch, 109-006-008), positive control antibody 1 (B-011B-PC-1), negative control antibody (human IgG), or the antigen-binding protein of this application were added to the detection medium, i.e., 1640 medium containing 10% FBS, so that the final concentration of the secondary antibody was 80 μg / ml and the final concentration of the antigen-binding protein of this application was 20 μg / ml. After gently mixing, the mixture was incubated in a cell culture incubator for 30 minutes to carry out the crosslinking reaction.

[0452] (3) After the antibody cross-linking reaction is completed, perform a 5-fold serial dilution, resulting in a total of 8 concentration gradients (including the 0 point). Take 40 μl of diluted antibody, add it to the cell wells, and then incubate it in a cell culture incubator for 5 hours.

[0453] (4) After incubation, add Luciferase reagent (Promega, E6120) to each well of the cell plate at a volume of 80 μl / well, and shake the plate at room temperature in the dark for 5 minutes. After shaking, detect the biofluorescence value of the 96-well plate using a microplate reader.

[0454] The above experiments demonstrate that the antigen-binding protein of this application has the ability to activate the NFκB signaling pathway by binding to human GITR overexpressed on Jurkat-hGITR-NFκB cells.

[0455] like Figure 4 As shown, the biological activity of GITR-binding proteins was detected using a luciferase reporter gene system.

[0456] As shown in Table 7, the ability of GITR-binding proteins to activate the NFκB signaling pathway was detected using a luciferase reporter gene system.

[0457] Table 7. Ability of GITR-binding proteins to activate the NFκB signaling pathway

[0458] Positive control antibody 1 0.3523 7915 negative control antibody - 4330 Ab0 0.2532 7102

[0459] Example 5: Binding of GITR-binding protein to GITR expressed on cells

[0460] The binding ability of GITR-binding proteins to human GITR expressed on the surface of HT1080 cells was determined by flow cytometry. The binding ability was determined by comparing the binding curves of different GITR-binding proteins of this application with human GITR expressed on the surface of HT1080 cells.

[0461] (1) HT1080 / hGITR cells (Kangyuan Bochuang) were seeded in a 96-well plate.

[0462] (2) Prepare positive control antibody 1 (B-011B-PC-1), positive control antibody 2 (B-011B-PC-2, for example, the full-length heavy chain sequence can be as shown in SEQ ID NO:54, and the full-length light chain sequence can be as shown in SEQ ID NO:53), negative control antibody (human IgG) and antigen-binding protein of this application with PBS containing 2% FBS. The maximum concentration is 15 μg / ml. Dilute 5 times, 8 spots, add 100 μl to each tube, and incubate at 4 degrees Celsius for 1 hour.

[0463] (3) Wash the plate three times with PBS containing 2% FBS.

[0464] (4) Dilute PE-labeled human IgG (Biolegend, 410708) with PBS containing 2% FBS at a ratio of 1:2000, add 100 μl to each well, and incubate at 4 degrees Celsius for 0.5 hours.

[0465] (5) Wash the plate twice with PBS containing 2% FBS. Resuspend the cells in 100 μl of PBS containing 2% FBS.

[0466] (6) The median fluorescence value (MFI) of the PE channel was determined using flow cytometry.

[0467] The above experiments demonstrate that the humanized GITR-binding protein of this application has the ability to bind to human GITR overexpressed on HT1080 cells.

[0468] like Figure 5 and Figure 6 As shown, the binding curves of GITR-binding protein and GITR expressed on the cell surface were determined by flow cytometry.

[0469] As shown in Table 8, the binding of GITR-binding protein to GITR expressed on the cell surface in ECs was determined by flow cytometry. 50 Value and highest MFI value.

[0470] Table 8 ECs of GITR-binding proteins and GITR binding 50 Value and highest MFI value

[0471]

[0472]

[0473] Example 6 Biological activity of GITR-binding proteins

[0474] GITR is a cellular co-stimulatory target; binding proteins to it can activate the GITR pathway. This study compared the functions of different GITR-binding proteins by detecting changes in luciferase expression in Jurkat-GITR-NFκB cells after GITR-binding proteins bound to cellularly expressed GITR, using a luciferase reporter gene system.

[0475] (1) To screen for biologically active GITR binding proteins, the cDNA encoding human GITR was cloned into the pLVX-IRES-Puro (takara, 632186) vector. The obtained plasmid and the NF-kappaB luciferace reporter gene plasmid (Promega) were co-transfected into Jurkat cells (Chinese Academy of Sciences Cell Bank, TCU123) to generate Jurkat-GITR-NFκB cells that overexpress human GITR and carry the NF-kappaB luciferace reporter gene system.

[0476] Jurkat-GITR-NFκB cells were seeded in 96-well transparent white plates (Corning, 3903) and placed in a cell culture incubator.

[0477] (2) Antibody crosslinking: The anti-human secondary antibody (AffiniPure F(ab′)2Fragment, Goat Anti-Human IgG, Fcγ fragment specific, Jackson Immunoresearch, 109-006-008) and positive control antibody 1 (B-011B-PC-1), positive control antibody 2 (B-011B-PC-2), negative control antibody (human IgG) or the antigen-binding protein of this application were added to the detection medium, i.e., 1640 medium containing 10% FBS, so that the final concentration of the secondary antibody was 80 μg / ml and the final concentration of the antigen-binding protein of this application was 20 μg / ml. After gently mixing, the mixture was incubated in a cell culture incubator for 30 minutes to carry out the crosslinking reaction.

[0478] (3) After the antibody cross-linking reaction is completed, perform a 5-fold serial dilution, resulting in a total of 8 concentration gradients (including the 0 point). Take 40 μl of diluted antibody, add it to the cell wells, and then incubate it in a cell culture incubator for 5 hours.

[0479] (4) After incubation, add Luciferase reagent (Promega, E6120) to each well of the cell plate at a volume of 80 μl / well, and shake the plate at room temperature in the dark for 5 minutes. After shaking, detect the biofluorescence value of the 96-well plate using a microplate reader.

[0480] like Figure 7 , Figure 8 and Figure 9 As shown, the biological activity of GITR-binding proteins was detected using a luciferase reporter gene system.

[0481] The above experiments demonstrate that the applicant's derived antigen-binding protein has the ability to activate the NFκB signaling pathway by binding to human GITR overexpressed on Jurkat-hGITR-NFκB cells.

[0482] As shown in Table 9, the ability of GITR-binding proteins to activate the NFκB signaling pathway was detected using the luciferase reporter gene system.

[0483] Table 9 ECGs of GITR-binding proteins activating the NFκB signaling pathway 50 Value and relative light unit

[0484] Positive control antibody 1 0.3658 5549.5 Positive control antibody 2 0.5740 5610 negative control antibody - 2145.5 Ab0 0.3202 5468 Ab2 0.6584 5143 Ab3 0.6699 5591.5 Ab5 0.6717 5524 Ab6 0.3992 5405 Ab8 1.2330 4869 Ab9 3.1830 4573.5

[0485] Example 7 Binding affinity of GITR-binding proteins

[0486] The binding affinity of different GITR-binding proteins to the antigen (GITR recombinant human protein (ECD, His Tag, SinoBiological, 13643-H08H)) was determined using Biacore.

[0487] Operating reagents: containing 10 mM N-(2-hydroxyethyl)piperazine-N-2-sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20, pH adjusted to 7.4; Human IgG (Fc) Capture Kit (catalog number: BR-1008-39, GE), including: mouse anti-human IgG (Fc) antibody (0.5 mg / ml), fixation reagent (10 mM sodium acetate, pH 5.0), regeneration reagent (3 M magnesium chloride); Amino-conjugated Kit (catalog number: BR100050, GE), including: 115 mg N-hydroxysuccinimide (NHS), 750 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10.5 ml 1 M ethanolamine (pH 8.5). Add 10 ml of deionized water to each tube of EDC and NHS, aliquot and store at -18°C or lower. Shelf life is two months. (Refer to GE Amino Coupling Instruction Manual 22-0510-62 AG).

[0488] Chip preparation. Mouse anti-human IgG (Fc) antibody was diluted to 25 μg / ml with a fixation reagent (10 mM sodium acetate, pH 5.0). Approximately 100 μl of mouse anti-human IgG (Fc) antibody was used per channel of the chip, and approximately 10 μl of mouse anti-human IgG (Fc) antibody was added to 190 μl of fixation reagent to fix both channels. First, the surface of the CM5 chip was activated for 420 seconds with 400 mM EDC and 100 mM NHS at a flow rate of 10 μl / min. Next, 25 μg / ml of mouse anti-human IgG (Fc) antibody was injected into the experimental channel (FC2) at a flow rate of 10 μl / min for approximately 420 seconds, with a fixation volume of approximately 9000 to 14000 RU. Finally, the chip was blocked with 1M ethanolamine at a flow rate of 10 μl / min for 420 seconds. The reference channel (FC1) was prepared using the same procedure as the experimental channel (FC2). (Refer to the GE Human IgG (Fc) Capture Kit Instruction Manual 22-0648-88 AD).

[0489] Ligand capture. Dilute the positive control antibody 1 (B-011B-PC-1) or the antigen-binding protein of this application to 5 μg / ml with the running reagent and inject it into the experimental channel (FC2) at a flow rate of 10 μl / min for 40 seconds. Ligand capture is not required in the reference channel (FC1).

[0490] Single-concentration analysis of analytes. The antigen was diluted to 200 nM with the running reagent. After capturing the antibody, the diluted antigen was sequentially injected into the experimental and reference channels at a flow rate of 30 μl / min for 90 seconds followed by dissociation for 210 seconds. A zero concentration was set for each antigen-binding protein. After each antigen-binding protein analysis, the chip was regenerated with 3M magnesium chloride at a flow rate of 20 μl / min for 30 seconds to wash away ligands and undissociated analytes.

[0491] The equilibrium dissociation constant (K0) of each antigen-binding protein in this application was calculated using Biacore 8K analysis software. D (Value). The reference channel (FC1) is used for background subtraction.

[0492] The above experiments demonstrate that the antigen-binding protein of this application has binding affinity to antigens.

[0493] As shown in Table 10, the binding affinity of different GITR-binding proteins to antigen proteins was detected using Biacore.

[0494] Table 10 Binding affinity of GITR-binding proteins to antigen proteins

[0495] Positive control antibody 1 8.09E+05 7.68E-04 9.49E-10 Ab0 3.86E+10 4.40E+00 1.14E-10 Ab2 8.60E+05 2.59E-02 3.01E-08 Ab3 4.70E+05 6.16E-04 1.31E-09 Ab5 5.57E+07 6.46E-03 1.16E-10 Ab6 2.47E+08 3.36E-02 1.36E-10 Ab8 2.26E+08 3.02E-02 1.34E-10 Ab9 3.50E+10 5.33E+00 1.52E-10

[0496] Example 8: Species Cross-Reactivity Assay of GITR-Binding Proteins

[0497] This experiment detected the binding of GITR-binding proteins to human GITR (Human GITR-Fc, Acro, catalog number GIR-H5254) and monkey GITR (Rhesus macaque GITR Fc, Acro, catalog number GIR-C5255) proteins, respectively.

[0498] Prepare 1 μg / ml human GITR antigen and monkey GITR antigen separately with PBS, add to 96-well plates (Corning, catalog number 9018), and incubate overnight at 4°C. The next day, discard the coating solution and wash the plate three times with 0.05% Tween 20 PBS. Incubate with PBS containing 3% BSA at 37°C for 2 hours, and wash three times with 0.05% Tween 20 DPBS. Prepare 12 concentration points of the target antibody with PBS containing 1% BSA, with a maximum concentration of 15 μg / ml, 5-fold dilution. Add to 96-well plates and incubate at room temperature for 1 hour. Wash three times with 0.05% Tween 20 PBS. Dilute the secondary antibody (HRP-Goat anti-Human IgG F(ab′)2, Jackson, catalog number 109-006-008) with PBS containing 1% BSA according to the product instructions. Add the diluted sample to 96-well plates and incubate at room temperature for 0.5 hours. Wash the plate three times with 0.05% Tween 20 PBS. Add 100 μl of TMB substrate chromogenic solution to each well. Add 50 μl of 2N H2SO4 stop solution to each well, and read and record the values ​​at 450 nm using a microplate reader.

[0499] The above experiments demonstrate that the GITR antigen-binding protein of this application has the ability to bind to human GITR antigen and monkey GITR antigen.

[0500] like Figure 10 , Figure 11 and Figure 12 The image shows the binding curves of the GITR antigen-binding protein to the human GITR antigen. Figure 13 , Figure 14 and Figure 15 As shown, the binding curves of GITR antigen-binding protein and monkey GITR antigen are displayed.

[0501] Table 11 shows the binding ability of GITR antigen-binding proteins to human GITR antigen and monkey GITR antigen.

[0502] Table 11. Binding ability of GITR antigen-binding proteins to human and monkey GITR antigens.

[0503]

[0504] Example 9: Antitumor efficacy test of GITR-binding protein

[0505] This study used MC38 cells inoculated into hGITR transgenic mice to detect the antitumor effect of the GITR binding protein in this application.

[0506] Human GITR transgenic mice: Female C57BL / 6 background human GITR transgenic mice (approximately 6 weeks old) were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. The mice were acclimatized for 7 days after arrival before the study began.

[0507] Cells: Mouse MC38 cells were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd., and were routinely passaged and cultured according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in serum-free medium. On day 0, 0.1 ml of the cell suspension was subcutaneously inoculated into the right axilla of human GITR transgenic mice to establish an MC38-hGITR tumor-bearing mouse model.

[0508] Administration:

[0509] On day 7 after tumor cell inoculation, the tumor volume of each mouse can be measured, and mice with tumor volumes of 20 mm or less can be selected. 3 ~60mm 3 Mice within the target range were divided into groups of 6 mice each based on average tumor volume. The mice were administered the drug on days 7, 10, 13, 16, 19, and 22 post-vaccination, along with a negative control antibody (human IgG), a positive control antibody 1 (B-011B-PC-1, provided by Taizhou Baiying Biotechnology Co., Ltd., catalog number: B166705), and the antigen-binding protein described in this application. Tumor volume and body weight changes in each group were monitored twice per week for 3 consecutive weeks during the administration period. Body weight and tumor volume were measured before each administration. The tumor volume inhibition rate (TGI%) was calculated on day 25 post-vaccination using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100; where Ti: median tumor volume in the administered group, T0: median tumor volume in the administered group on day D0, Vi: median tumor volume in the negative control antibody group, and V0: median tumor volume in the negative control antibody group on day D0. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: TV = 1 / 2 × a × b 2 Weight was measured using an electronic balance.

[0510] Table 12 shows the dosage and method of administration.

[0511] Table 12 Drug administration study design

[0512] negative control antibody 20mg / kg Q3DX6* Intraperitoneal injection Positive control antibody 1 20mg / kg Q3DX6* Intraperitoneal injection Ab0 20mg / kg Q3DX6* Intraperitoneal injection Ab2 20mg / kg Q3DX6* Intraperitoneal injection

[0513] *Administer the medication every 3 days, for a total of 6 times.

[0514] like Figure 16 As shown, the antitumor efficacy of GITR-binding protein Ab0 in mice was detected in the first experiment.

[0515] like Figure 17 As shown, the change in mouse body weight after administration of GITR-binding protein Ab0 in the first experiment.

[0516] like Figure 18 As shown, the antitumor efficacy of GITR-binding protein Ab2 in mice was detected in the second experiment.

[0517] like Figure 19 As shown, the change in mouse body weight after administration of GITR-binding protein Ab2 in the second experiment.

[0518] The above experiments show that the GITR-binding protein of this application significantly inhibits tumor growth and has no significant effect on animal body weight.

[0519] As shown in Tables 13 and 14, in the first trial, on day 25 after inoculation, the GITR-binding protein Ab0 of this application significantly inhibited tumor growth at a dose of 20 mg / kg, with a tumor volume inhibition rate of 38%; in the second trial, on day 27 after inoculation, the GITR-binding protein Ab2 of this application significantly inhibited tumor growth at a dose of 20 mg / kg, with a tumor volume inhibition rate of 40%.

[0520] Table 13. In vivo tumor growth inhibition ability of the GITR-binding protein of this application on day 25 of the first trial.

[0521] negative control antibody 2167.76±236.44 - Positive control antibody 1 1236.04±80.82** 44％ Ab0 1362.47±232.91* 38％

[0522] Where * indicates that P<0.05 compared with the tumor volume of the negative control antibody; ** indicates that P<0.01 compared with the tumor volume of the negative control antibody.

[0523] Table 14. In vivo tumor growth inhibition ability of the GITR-binding protein of this application on day 27 of the second trial.

[0524] negative control antibody 1929.96±260.70 - Positive control antibody 1 1572.94±315.60 19％ Ab2 1171.34±218.20 40％

[0525] Example 10: Combination of GITR-binding protein with PD-1 antibody

[0526] This study used MC38 / hPDL1 cells to inoculate human hPD-1 / / hGITR transgenic mice to detect the antitumor effect of the GITR binding protein combined with PD-1 antibody as described in this application.

[0527] Human hPD-1 / / hGITR transgenic mice: Female B-hPD-1 / / hGITR transgenic mice (approximately 6 weeks old) with a C57BL / 6 background were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. The mice were acclimatized for 7 days after arrival before the study began.

[0528] Cells: Mouse MC38 / hPDL1 (mPDL1 KO) cells were purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., and were routinely passaged and cultured according to the manufacturer's instructions for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in serum-free medium. On day 0, 0.1 ml of the cell suspension was subcutaneously inoculated into the right axilla of human B-hPD-1 / / hGITR transgenic mice to establish an MC38 / hPDL1 tumor-bearing mouse model.

[0529] Administration:

[0530] On day 7 after tumor cell inoculation, the tumor volume of each mouse was measured, and mice with tumor volumes of 100 mm were selected. 3 ~200mm 3 Mice within the target range were divided into groups of 6 mice each based on average tumor volume. A single dose of PD-1 antibody (Pembrolizumab, source: Taizhou Baiying Biotechnology Co., Ltd., catalog number: B2014-CHO) was administered on day 7 post-inoculation. Subsequent doses were administered on days 7, 10, 14, 17, 21, and 24 post-inoculation, along with a negative control antibody (human IgG) and the antigen-binding protein described in this application. Tumor volume and body weight changes in each group were monitored twice per week for 3 consecutive weeks during the administration period. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on days 31 and 28 post-inoculation using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100; where Ti: mean tumor volume in the administered group, T0: mean tumor volume in the administered group on day D0, Vi: mean tumor volume in the negative control antibody group, and V0: mean tumor volume in the negative control antibody group on day D0. Tumor volume measurement: The long diameter (a) and wide diameter (b) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: TV = 1 / 2 × a × b 2 Weight was measured using an electronic balance.

[0531] Table 15 shows the dosage and method of administration.

[0532] Table 15 Drug administration study design

[0533] negative control antibody 10mg / kg BIWX6* Intraperitoneal injection PD-1 antibody 10mg / kg single Intraperitoneal injection Ab2 10mg / kg BIWX6* Intraperitoneal injection Ab5 10mg / kg BIWX6* Intraperitoneal injection PD-1 antibody + Ab2 10mg / kg + 10mg / kg Single + BIWX6* Intraperitoneal injection PD-1 antibody + Ab5 10mg / kg + 10mg / kg Single + BIWX6* Intraperitoneal injection

[0534] *Administer twice a week for a total of 6 weeks.

[0535] like Figure 20 As shown, the antitumor efficacy of the combination of GITR-binding protein Ab2 and PD-1 antibody in mice was tested in the first experiment.

[0536] like Figure 21 As shown, the changes in mouse body weight after administration of GITR-binding protein Ab2 and PD-1 antibody in combination in the first experiment.

[0537] like Figure 22 As shown, the second experiment tested the antitumor efficacy of the combination of GITR-binding protein Ab5 and PD-1 antibody in mice.

[0538] like Figure 23 As shown, the changes in mouse body weight after administration of GITR-binding protein Ab5 and PD-1 antibody in combination in the second experiment.

[0539] The above experiments show that the combination of the GITR-binding protein and PD-1 antibody in this application significantly inhibits tumor growth and has no significant effect on animal body weight.

[0540] As shown in Table 16, in the first trial, on day 31 after inoculation, the tumor volume inhibition rate of the GITR-binding protein Ab2 of this application was 33% at a dose of 10 mg / kg, the tumor volume inhibition rate of the PD-1 antibody was 59% at a dose of 10 mg / kg, and the tumor volume inhibition rate of the combined dose of GITR-binding protein Ab2 and PD-1 antibody was 73%. The combined use of GITR-binding protein and PD-1 antibody of this application showed a significant synergistic ability to inhibit tumor growth.

[0541] Table 16. In vivo tumor growth inhibition capacity of the combination of GITR-binding protein Ab2 and PD-1 in this application on day 31 of the first trial.

[0542] negative control antibody 2629.71±442.02 - PD-1 antibody 1155.44±205.42* 59％ Ab2 1802.89±162.09 33％ PD-1 antibody + Ab2 817.66±406.86* 73％

[0543] * indicates that the tumor volume compared with the negative control antibody tumor volume is P<0.05.

[0544] As shown in Table 17, in the second trial, on day 28 after inoculation, the tumor volume inhibition rate of the GITR-binding protein Ab5 of this application was 62% at a dose of 10 mg / kg, the tumor volume inhibition rate of the PD-1 antibody was 51% at a dose of 10 mg / kg, and the tumor volume inhibition rate of the combined dose of GITR-binding protein Ab5 and PD-1 antibody was 83%. The combined use of GITR-binding protein and PD-1 antibody of this application showed a significant synergistic ability to inhibit tumor growth.

[0545] Table 17. In vivo tumor growth inhibition capacity of the combination of GITR-binding protein Ab5 and PD-1 in this application on day 28 of the second trial.

[0546] negative control antibody 1715.51±272.45 - PD-1 antibody 914.40±142.19* 51％ Ab5 750.18±138.27* 62％ PD-1 antibody + Ab5 409.20±122.41** 83％

[0547] Where * indicates that P<0.05 compared with the tumor volume of the negative control antibody, and ** indicates that P<0.01 compared with the tumor volume of the negative control antibody.

[0548] Example 11: Combination of GITR-binding protein with PD-L1 antibody

[0549] This study used MC38 / hPDL1 cells to inoculate human B-hPD-1 / hPD-L1 / hGITR transgenic mice to detect the antitumor effect of the GITR binding protein combined with the PD-L1 antibody in this application.

[0550] Human B-hPD-1 / hPD-L1 / hGITR transgenic mice:

[0551] Female B-hPD-1 / hPD-L1 / hGITR transgenic mice (approximately 6 weeks old) with a C57BL / 6 background were purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd. The mice were acclimatized for 7 days after arrival before the study began.

[0552] Cells: Mouse MC38 / hPDL1 cells were constructed by Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., and routinely passaged for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in serum-free medium. On day 0, 0.1 ml of the cell suspension was subcutaneously seeded into the right axilla of human B-hPD-1 / hPD-L1 / hGITR transgenic mice to establish an MC38 / hPDL1 tumor-bearing mouse model.

[0553] Administration:

[0554] On day 6 after tumor cell inoculation, the tumor volume of each mouse was measured. Mice with tumor volumes within an appropriate range were selected and grouped into groups of 6 mice each. The mice were then administered the drug on days 6, 9, 13, 16, and 20 after inoculation. The drugs included negative control antibody (human IgG), PD-L1 antibody (atezolizumab, source: Hangzhou Haoyang Biotechnology Co., Ltd., catalog number: HSP099-20), positive control antibody 1 (B-011B-PC-1), and the antigen-binding protein Ab5 of this application. During the drug administration period, the tumor volume and body weight of each group of mice were monitored twice a week for 3 consecutive weeks. Body weight and tumor volume were measured before each administration. Tumor volume inhibition rate (TGI%) was calculated on day 23 after cell seeding using the following formula: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100; where Ti: tumor volume statistics (e.g., mean) in the administered group, T0: tumor volume statistics (e.g., mean) in the administered group on day D0, Vi: tumor volume statistics (e.g., mean) in the negative control antibody group, and V0: tumor volume statistics (e.g., mean) in the negative control antibody group on day D0. Tumor volume was measured using calipers (length (a) and width (b)). The tumor volume was calculated using the following formula: TV = 1 / 2 × a × b 2 Weight was measured using an electronic balance.

[0555] Table 18 shows the dosage and method of administration.

[0556] Table 18 Drug administration test design

[0557] negative control antibody 10mg / kg BIWX5* Intraperitoneal injection PD-L1 antibody 3mg / kg BIWX5* Intraperitoneal injection Positive control antibody 1 10mg / kg BIWX5* Intraperitoneal injection Ab5 10mg / kg BIWX5* Intraperitoneal injection PD-L1 antibody + positive control antibody 1 3mg / kg + 10mg / kg BIWX5*+BIWX5* Intraperitoneal injection PD-L1 antibody + Ab5 3mg / kg + 10mg / kg BIWX5*+BIWX5* Intraperitoneal injection

[0558] *Administer twice a week for a total of 5 weeks.

[0559] like Figure 24 As shown, the antitumor efficacy of the combination of GITR-binding protein Ab5 and PD-L1 antibody in mice was detected.

[0560] like Figure 25 As shown, the changes in mouse body weight after combined administration of GITR-binding protein Ab5 and PD-L1 antibody.

[0561] The above experiments show that the combination of the GITR-binding protein and the PD-L1 antibody in this application significantly inhibits tumor growth and has no significant effect on animal body weight.

[0562] As shown in Table 19, on day 23 post-vaccination, the tumor volume inhibition rate of GITR-binding protein Ab5 at a dose of 10 mg / kg was 36%, the tumor volume inhibition rate of PD-L1 antibody at a dose of 3 mg / kg was 29%, and the tumor volume inhibition rate of the combined dose of GITR-binding protein Ab5 and PD-L1 antibody was 63%. The combined use of GITR-binding protein Ab5 and PD-1 antibody showed significant inhibition of tumor growth.

[0563] Table 19. In vivo tumor growth inhibition capacity of the combination of GITR-binding protein and PD-L1 anti-tumor agents in this application on day 23.

[0564]

[0565] Note: *: P<0.05 compared with negative control antibody tumor volume; **: P<0.01 compared with negative control antibody tumor volume.

[0566] The above experiments show that the combination of the GITR-binding protein and the PD-L1 antibody in this application significantly inhibits tumor growth and has no significant effect on animal body weight.

[0567] The combination of the GITR-binding protein and the PD-L1 antibody in this application demonstrates a significant synergistic ability to inhibit tumor growth.

[0568] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents. sequence list <110> Shanghai Pharmaceuticals Holding Co., Ltd. <120> GITR-binding proteins and their uses <130> 0124-PA-016 <160> 66 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Ser Asp Tyr Ala Trp Asn 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Ser Gly Val Tyr Asp Leu Phe Asp Tyr 1 5 <210> 4 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Trp Gln Gly Thr His Phe Pro Tyr Thr 1 5 <210> 7 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> HFR1 Ab0 <400> 7 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 20 25 30 <210> 8 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> HFR1 Ab1-3 <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser 20 25 30 <210> 9 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> HFR1 Ab4-9 <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr 20 25 30 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HFR2 Ab0 <400> 10 Trp Ile Arg Gln Phe Pro Gly Asp Lys Leu Glu Trp Met Gly 1 5 10 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HFR2 Ab1-3 <400> 11 Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly 1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HFR2 Ab4-9 <400> 12 Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Met Gly 1 5 10 <210> 13 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HFR3 Ab0 <400> 13 Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln 1 5 10 15 Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Arg 20 25 30 <210> 14 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HFR3 Ab1-3 <400> 14 Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 15 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HFR3 Ab4-6 <400> 15 Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 <210> 16 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HFR3 Ab7-9 <400> 16 Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 1 5 10 15 Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr Arg 20 25 30 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HFR4 Ab0 <400> 17 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 1 5 10 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HFR4 Ab1-9 <400> 18 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 19 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> LFR1 Ab0 <400> 19 Asp Phe Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 <210> 20 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> LFR1 Ab2+5+8 <400> 20 Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 <210> twenty one <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> LFR1 Ab3+6+9 <400> twenty one Asp Phe Leu Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 <210> twenty two <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LFR2 Ab0 <400> twenty two Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr 1 5 10 15 <210> twenty three <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LFR2 Ab2-3+5-6+8-9 <400> 23 Trp Leu Leu Gln Lys Pro Gly Gln Ser Pro Gln Arg Leu Ile Tyr 1 5 10 15 <210> 24 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LFR3 Ab0 <400> 24 Gly Val Pro Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 20 25 30 <210> 25 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LFR3 Ab1-2+4-5+7-8 <400> 25 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> 26 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LFR3 Ab3+6+9 <400> 26 Gly Val Pro Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 20 25 30 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LFR4 Ab0 <400> 27 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LFR4 AB1-9 <400> 28 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 29 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH Ab0 <400> 29 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asp Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly Val Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 30 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH Ab1-3 <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Val Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 31 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH Ab4-6 <400> 31 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly Val Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH Ab7-9 <400> 32 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ser Gly Val Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 33 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL Ab0 <400> 33 Asp Phe Leu Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 34 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL Ab2+5+8 <400> 34 Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL Ab3+6+9 <400> 35 Asp Phe Leu Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 36 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> HFR1 general formula <220> <221> misc_feature <222> (1)..(1) <223> Xaa = Asp or Gln <220> <221> misc_feature <222> (17)..(17) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (25)..(25) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (27)..(27) <223> Xaa = Gly or Tyr <220> <221> misc_feature <222> (30)..(30) <223> Xaa = Ser or Thr <400> 36 Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Xaa Leu Ser Leu Thr Cys Thr Val Xaa Gly Xaa Ser Ile Xaa 20 25 30 <210> 37 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HFR2 general formula <220> <221> misc_feature <222> (5)..(5) <223> Xaa = Phe or His <220> <221> misc_feature <222> (8)..(8) <223> Xaa = Asp or Lys <220> <221> misc_feature <222> (9)..(9) <223> Xaa = Gly or Lys <220> <221> misc_feature <222> (13)..(13) <223> Xaa = Ile or Met <400> 37 Trp Ile Arg Gln Xaa Pro Gly Xaa Xaa Leu Glu Trp Xaa Gly 1 5 10 <210> 38 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> HFR3 general formula <220> <221> misc_feature <222> (2)..(2) <223> Xaa = Ile or Val <220> <221> misc_feature <222> (3)..(3) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (5)..(5) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Arg or Val <220> <221> misc_feature <222> (14)..(14) <223> Xaa = Phe or Ser <220> <221> misc_feature <222> (16)..(16) <223> Xaa = Lys or Gln <220> <221> misc_feature <222> (18)..(18) <223> Xaa = Asn or Ser <220> <221> misc_feature <222> (22)..(22) <223> Xaa = Ala or Thr <220> <221> misc_feature <222> (23)..(23) <223> Xaa = Ala or Glu <220> <221> misc_feature <222> (27)..(27) <223> Xaa = Thr or Val <220> <221> misc_feature <222> (31)..(31) <223> Xaa = Ala or Thr <400> 38 Arg Xaa Xaa Ile Xaa Xaa Asp Thr Ser Lys Asn Gln Phe Xaa Leu Xaa 1 5 10 15 Leu Xaa Ser Val Thr Xaa Xaa Asp Thr Ala Xaa Tyr Tyr Cys Xaa Arg 20 25 30 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HFR4 general formula <220> <221> misc_feature <222> (6)..(6) <223> Xaa = Leu or Thr <220> <221> misc_feature <222> (7)..(7) <223> Xaa = Leu or Val <400> 39 Trp Gly Gln Gly Thr Xaa Xaa Thr Val Ser Ser 1 5 10 <210> 40 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> LFR1 general formula <220> <221> misc_feature <222> (3)..(3) <223> Xaa = Leu or Val <220> <221> misc_feature <222> (10)..(10) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (15)..(15) <223> Xaa = Ile or Pro <400> 40 Asp Phe Xaa Met Thr Gln Thr Pro Leu Xaa Leu Ser Val Thr Xaa Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys 20 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> LFR2 general formula <220> <221> misc_feature <222> (5)..(5) <223> Xaa = Lys or Arg <220> <221> misc_feature <222> (11)..(11) <223> Xaa = Lys or Gln <400> 41 Trp Leu Leu Gln Xaa Pro Gly Gln Ser Pro Xaa Arg Leu Ile Tyr 1 5 10 15 <210> 42 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> LFR3 general formula <220> <221> misc_feature <222> (7)..(7) <223> Xaa = Ala or Ser <220> <221> misc_feature <222> (27)..(27) <223> Xaa = Leu or Val <400> 42 Gly Val Pro Asp Arg Phe Xaa Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Tyr Cys 20 25 30 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LFR4 general formula <220> <221> misc_feature <222> (3)..(3) <223> Xaa = Gly or Gln <400> 43 Phe Gly Xaa Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 44 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH general formula <220> <221> misc_feature <222> (1)..(1) <223> Xaa = Asp or Gln <220> <221> misc_feature <222> (17)..(17) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (25)..(25) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (27)..(27) <223> Xaa = Gly or Tyr <220> <221> misc_feature <222> (30)..(30) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (41)..(41) <223> Xaa = Phe or His <220> <221> misc_feature <222> (44)..(44) <223> Xaa = Asp or Lys <220> <221> misc_feature <222> (45)..(45) <223> Xaa = Gly or Lys <220> <221> misc_feature <222> (49)..(49) <223> Xaa = Ile or Met <220> <221> misc_feature <222> (68)..(68) <223> Xaa = Ile or Val <220> <221> misc_feature <222> (69)..(69) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (71)..(71) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (72)..(72) <223> Xaa = Arg or Val <220> <221> misc_feature <222> (80)..(80) <223> Xaa = Phe or Ser <220> <221> misc_feature <222> (82)..(82) <223> Xaa = Lys or Gln <220> <221> misc_feature <222> (84)..(84) <223> Xaa = Asn or Ser <220> <221> misc_feature <222> (88)..(88) <223> Xaa = Ala or Thr <220> <221> misc_feature <222> (89)..(89) <223> Xaa = Ala or Glu <220> <221> misc_feature <222> (93)..(93) <223> Xaa = Thr or Val <220> <221> misc_feature <222> (97)..(97) <223> Xaa = Ala or Thr <220> <221> misc_feature <222> (113)..(113) <223> Xaa = Leu or Thr <220> <221> misc_feature <222> (114)..(114) <223> Xaa = Leu or Val <400> 44 Xaa Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Xaa Leu Ser Leu Thr Cys Thr Val Xaa Gly Xaa Ser Ile Xaa Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Xaa Pro Gly Xaa Xaa Leu Glu Trp 35 40 45 Xaa Gly Tyr Ile Ser Asn Ser Gly Tyr Thr Ile Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Xaa Xaa Ile Xaa Xaa Asp Thr Ser Lys Asn Gln Phe Xaa 65 70 75 80 Leu Xaa Leu Xaa Ser Val Thr Xaa Xaa Asp Thr Ala Xaa Tyr Tyr Cys 85 90 95 Xaa Arg Ser Gly Val Tyr Asp Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Xaa Xaa Thr Val Ser Ser 115 <210> 45 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL general formula <220> <221> misc_feature <222> (3)..(3) <223> Xaa = Leu or Val <220> <221> misc_feature <222> (10)..(10) <223> Xaa = Ser or Thr <220> <221> misc_feature <222> (15)..(15) <223> Xaa = Ile or Pro <220> <221> misc_feature <222> (44)..(44) <223> Xaa = Lys or Arg <220> <221> misc_feature <222> (50) <223> Xaa = Lys or Gln <220> <221> misc_feature <222> (68)..(68) <223> Xaa = Ala or Ser <220> <221> misc_feature <222> (88)..(88) <223> Xaa = Leu or Val <220> <221> misc_feature <222> (105)..(105) <223> Xaa = Gly or Gln <400> 45 Asp Phe Xaa Met Thr Gln Thr Pro Leu Xaa Leu Ser Val Thr Xaa Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Xaa Pro Gly Gln Ser 35 40 45 Pro Xaa Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Xaa Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Xaa Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Xaa Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 46 <211> 107 <212> PRT <213> Homo sapiens <400> 46 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 47 <211> 106 <212> PRT <213> Homo sapiens <400> 47 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 48 <211> 330 <212> PRT <213> Homo sapiens <400> 48 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 49 <211> 326 <212> PRT <213> Homo sapiens <400> 49 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 50 <211> 377 <212> PRT <213> Homo sapiens <400> 50 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 51 <211> 327 <212> PRT <213> Homo sapiens <400> 51 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 52 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> B011-B-PC-1 heavy chain <400> 52 Gln Val Gln Leu Val Glu Ser Gly Gly Asp Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Trp Tyr Ala Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Met Val Arg Gly Leu Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly Lys 450 <210> 53 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> B011-B-PC-1+2 light chain <400> 53 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 54 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> B011-B-PC-2 heavy chain <400> 54 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly 450 <210> 55 <211> 241 <212> PRT <213> Homo sapiens <400> 55 Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu 1 5 10 15 Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro 20 25 30 Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg 35 40 45 Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu 50 55 60 Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His 65 70 75 80 Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro 85 90 95 Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys 100 105 110 Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys 115 120 125 Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro 130 135 140 Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala 145 150 155 160 Glu Pro Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys 165 170 175 Val Leu Leu Leu Thr Ser Ala Gln Leu Gly Leu His Ile Trp Gln Leu 180 185 190 Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln Leu Leu Leu Glu Val 195 200 205 Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln Phe Pro Glu Glu Glu 210 215 220 Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg Leu Gly Asp Leu Trp 225 230 235 240 Val <210> 56 <211> 726 <212> DNA <213> Homo sapiens <400> 56 atggcacagc acggggcgat gggcgcgttt cgggccctgt gcggcctggc gctgctgtgc 60 gcgctcagcc tgggtcagcg ccccaccggg ggtcccgggt gcggccctgg gcgcctcctg 120 cttgggacgg gaacggacgc gcgctgctgc cgggttcaca cgacgcgctg ctgccgcgat 180 tacccgggcg aggagtgctg ttccgagtgg gactgcatgt gtgtccagcc tgaattccac 240 tgcggagacc cttgctgcac gacctgccgg caccaccctt gtcccccagg ccagggggta 300 cagtcccagg ggaaattcag ttttggcttc cagtgtatcg actgtgcctc ggggaccttc 360 tccgggggcc acgaaggcca ctgcaaacct tggacagact gcacccagtt cgggtttctc 420 actgtgttcc ctgggaacaa gacccacaac gctgtgtgcg tcccagggtc cccgccggca 480 gagccgcttg ggtggctgac cgtcgtcctc ctggccgtgg ccgcctgcgt cctcctcctg 540 acctcggccc agcttggact gcacatctgg cagctgagga gtcagtgcat gtggccccga 600 gagacccagc tgctgctgga ggtgccgccg tcgaccgag acgccagag ctgccagttc 660 cccgaggaag agcggggcga gcgatcggca gaggagagg ggcggctggg agacctgtgg 720 gtgtga 726 <210> 57 <211> 5 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> pembrolizumab HCDR1 <400> 57 Asn Tire Tire Met Tire 1 5 <210> 58 <211> 17 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> pembrolizumab HCDR2 <400> 58 Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Asn <210> 59 <211> 11 <212> PRT <213> Private Sequence(Artificial Sequence) <220> <223> pembrolizumab HCDR3 <400> 59 Arg Asp Tyr Arg Phe Asp Meth Gly Phe Asp Tyr 1 5 10 <210> 60 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab LCDR1 <400> 60 Arg Ala Ser Lys Gly Val Ser Thr Ser Gly Tyr Ser Tyr Leu His 1 5 10 15 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab LCDR2 <400> 61 Leu Wing Ser Tyr Leu Glu Ser 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab LCDR3 <400> 62 Gln His Ser Arg Asp Leu Pro Leu Thr 1 5 <210> 63 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab VH <400> 63 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 64 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab VL <400> 64 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 65 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab heavy chain <400> 65 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 66 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> pembrolizumab light chain <400> 66 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. The isolated anti-GITR antibody or its antigen-binding fragment, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

2. The anti-GITR antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain constant region of the antibody includes a heavy chain constant region derived from human antibodies.

3. The anti-GITR antibody or its antigen-binding fragment as described in claim 2, characterized in that, The heavy chain constant region of the antibody includes heavy chain constant regions derived from IgG, IgA, IgD, IgE and / or IgM.

4. The anti-GITR antibody or its antigen-binding fragment as described in claim 3, characterized in that, The heavy chain constant region of the antibody includes a heavy chain constant region derived from IgG.

5. The anti-GITR antibody or its antigen-binding fragment as described in claim 4, characterized in that, The heavy chain constant region of the antibody includes heavy chain constant regions derived from IgG1, IgG2, IgG3 and / or IgG4.

6. The anti-GITR antibody or its antigen-binding fragment as described in claim 5, characterized in that, The heavy chain constant region of the antibody includes a heavy chain constant region derived from IgG1.

7. The anti-GITR antibody or its antigen-binding fragment as described in claim 1, characterized in that, The light chain constant region of the anti-GITR antibody includes a light chain constant region derived from a human antibody.

8. The anti-GITR antibody or its antigen-binding fragment as described in claim 7, characterized in that, The light chain constant region of the antibody includes a constant region derived from human Igκ.

9. The anti-GITR antibody or its antigen-binding fragment as described in claim 1, characterized in that, The anti-GITR antibody is selected from: The antibody has an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:29 and an amino acid sequence of the light chain variable region as shown in SEQ ID NO:

33. The antibody has the heavy chain variable region amino acid sequence as shown in SEQ ID NO:30 and the light chain variable region amino acid sequence as shown in SEQ ID NO:

34. The antibody has the heavy chain variable region amino acid sequence as shown in SEQ ID NO:30 and the light chain variable region amino acid sequence as shown in SEQ ID NO:

35. Antibodies with heavy chain variable region amino acid sequences as shown in SEQ ID NO:32 and light chain variable region amino acid sequences as shown in SEQ ID NO:34; or The antibody has an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:32 and an amino acid sequence of the light chain variable region as shown in SEQ ID NO:

35.

10. The anti-GITR antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antigen-binding fragment is selected from one or more of the following groups: Fab, Fab′, Fv fragment, F(ab\')2, F(ab)2, scFv, di-scFv, VHH and dAb.

11. The anti-GITR antibody or its antigen-binding fragment as described in claim 1, characterized in that, The GITR mentioned is a human GITR.

12. The anti-GITR antibody or its antigen-binding fragment as described in claim 11, characterized in that, The antibody has the ability to bind the GITR or its functional fragment at an EC50 value of 5.01 μg / ml or lower.

13. The anti-GITR antibody or its antigen-binding fragment as described in claim 12, characterized in that, The GITR or its functional fragments comprise GITR or its functional fragments expressed on cells.

14. The anti-GITR antibody or its antigen-binding fragment as described in claim 11, characterized in that, The antibody's ability to bind the GITR or its functional fragment with a KD value of 3.01E-08M or lower.

15. The anti-GITR antibody or its antigen-binding fragment as described in claim 14, characterized in that, The antibody has the ability to activate the GITR or its functional active fragment.

16. The anti-GITR antibody or its antigen-binding fragment as described in claim 15, characterized in that, The antibody has the ability to activate the GITR or its functional active fragment at an EC50 value of 3.2 μg / ml or lower.

17. The anti-GITR antibody or its antigen-binding fragment as described in claim 11, characterized in that, The antibody has the ability to activate the NF-κB pathway.

18. The anti-GITR antibody or its antigen-binding fragment as described in claim 1, characterized in that, The antibody has tumor-suppressing ability, and the tumor is colon cancer.

19. The anti-GITR antibody or its antigen-binding fragment as described in claim 18, characterized in that, The antibody has the ability to inhibit tumors with a tumor volume inhibition rate of 38% or higher.

20. A nucleic acid molecule encoding the anti-GITR antibody or antigen-binding fragment thereof as described in any one of claims 1-19.

21. A vector comprising the nucleic acid molecule of claim 20.

22. A cell comprising and / or expressing the anti-GITR antibody or antigen-binding fragment thereof as described in any one of claims 1-19, comprising the nucleic acid molecule as described in claim 20, or comprising the vector as described in claim 21.

23. A pharmaceutical composition comprising the anti-GITR antibody or its antigen-binding fragment as described in any one of claims 1-19, the nucleic acid molecule as described in claim 20, the vector as described in claim 21, and / or the cell as described in claim 22, and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition according to claim 23, characterized in that, It also contains immune checkpoint inhibitors.

25. A pharmaceutical combination comprising an anti-GITR antibody or an antigen-binding fragment thereof as described in any one of claims 1-19, and one or more immune checkpoint inhibitors; said immune checkpoint inhibitor inhibits the interaction between PD-1 and PD-L1, comprising a PD-1 inhibitor and / or a PD-L1 inhibitor; said immune checkpoint inhibitor comprises a PD-1 antibody or an antigen-binding fragment thereof, and / or a PD-L1 antibody or an antigen-binding fragment thereof.

26. The pharmaceutical combination as described in claim 25, characterized in that, The immune checkpoint inhibitors include pembrolizumab HCDR1-3 and LCDR1-3.

27. The pharmaceutical combination as described in claim 26, characterized in that, The immune checkpoint inhibitors include pembrolizumab VH and VL.

28. The pharmaceutical combination as described in claim 27, characterized in that, The immune checkpoint inhibitor is pembrolizumab.

29. The pharmaceutical combination as described in claim 25, characterized in that, The anti-GITR antibody or its antigen-binding fragment is not mixed with the immune checkpoint inhibitor in the drug combination.

30. The pharmaceutical combination as described in claim 29, characterized in that, The anti-GITR antibody or its antigen-binding fragment and the immune checkpoint inhibitor are each contained in separate containers.

31. The pharmaceutical combination as described in claim 25, characterized in that, The drug combination comprises a first formulation and a second formulation, the first formulation comprising the anti-GITR antibody or its antigen-binding fragment and a pharmaceutically acceptable first carrier, and the second formulation comprising the immune checkpoint inhibitor and a pharmaceutically acceptable second carrier.

32. A kit comprising the anti-GITR antibody or its antigen-binding fragment as described in any one of claims 1-19, the nucleic acid molecule as described in claim 20, the vector as described in claim 21, the cell as described in claim 22, the pharmaceutical composition as described in any one of claims 23-24, and / or the pharmaceutical combination as described in any one of claims 25-31.

33. The kit according to claim 32, characterized in that, It is used to detect the presence and / or content of GITR in a sample.

34. Use of the anti-GITR antibody or antigen-binding fragment thereof of any one of claims 1-19, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22, the pharmaceutical composition of any one of claims 23-24, or the pharmaceutical combination of any one of claims 25-31 in the preparation of a kit for detecting the presence and / or content of GITR in a sample.

35. The use as described in claim 34, wherein the kit is used to detect the presence and / or content of GITR in a sample.

36. Use in the preparation of a medicament of any one of claims 1-19, the anti-GITR antibody or its antigen-binding fragment thereof, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22, the pharmaceutical composition of any one of claims 23-24, or the pharmaceutical combination of any one of claims 25-31, for the treatment of a tumor; the tumor being colon cancer.

Images