CD8α-binding polypeptides and uses thereof

Abstract

The present application relates to a CD8α binding polypeptide comprising at least one variable domain, the amino acid sequence of which has one or more amino acid substitutions, deletions or additions compared to the amino acid sequence represented by SEQ ID NO: 1. The present application also relates to compositions comprising said CD8α binding polypeptides and their use in the diagnosis and / or treatment of various diseases, including, for example, tumors.

Description

[Technical field]

[0001] The present application relates to the field of biopharmaceuticals, and in particular to CD8α binding polypeptides and uses thereof. [Background technology]

[0002] Emission Computed Tomography (ECT), which includes Single-Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), is used to diagnose tumors. ECT provides high-resolution imaging of tumors and allows quantitative analysis of the images.

[0003] In humans, CD8 is expressed primarily on cytotoxic T lymphocytes, but also on dendritic cells, natural killer cells, etc. The CD8 molecule can be either a homodimer composed of CD8α or a heterodimer composed of CD8α and CD8β, where the αβ heterodimer is more common.

[0004] The ability to monitor CD8+ tumor-infiltrating lymphocytes (TILs) in vivo has important implications for assessing immunotherapy responses and aiding in the development of more effective immune cell-targeted single agent and combination therapies. "Immuno-PET" imaging of tumor-infiltrating T cells can provide a specific and sensitive method to help select and determine whether a given immunotherapy regimen is effective.

[0005] There is a need in the art for detection agents for use in the detection of CD8 expressing cells, in particular CD8α antibody-based detection agents suitable for the detection of ECT. Summary of the Invention

[0006] [Disclosure of the Invention] On the other hand, the present application provides a CD8α binding polypeptide comprising at least one variable domain, wherein the amino acid sequence of the variable domain has an amino acid sequence in which one or more amino acids have been substituted, deleted or added compared to the amino acid sequence represented by SEQ ID NO: 1.

[0007] In some embodiments, the CD8α binding polypeptides described above have one or more of the following properties:

[0008] (i) capable of binding to the packing material of a PROTEIN A affinity chromatography column; (ii) is capable of binding to CD8α with the same or greater affinity as a reference antibody having the amino acid sequence set forth in SEQ ID NO: 1; (iii) having an increased expression level compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1; and (iv) has an increased isoelectric point compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1.

[0009] In some embodiments, the CD8α binding polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the antibodies include monoclonal antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies and / or fully human antibodies.

[0011] In some embodiments, the antigen-binding fragments include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0012] In some embodiments said VHHs are camelid, chimeric, human, partially humanized or fully humanized.

[0013] In some embodiments, the variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO:14.

[0014] In some embodiments, the variable domain comprises the CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:15 or SEQ ID NO:16.

[0015] In some embodiments, the variable domain comprises a CDR1 having the amino acid sequence represented by SEQ ID NO:19.

[0016] In some embodiments, the variable domain comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:17 or SEQ ID NO:18.

[0017] In some embodiments, the variable domain comprises a CDR2 having the amino acid sequence represented by SEQ ID NO:23.

[0018] In some embodiments, the variable domain comprises a CDR2 having an amino acid sequence represented by SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0019] In some embodiments, the variable domain comprises a CDR3 having an amino acid sequence represented by SEQ ID NO:27.

[0020] In some embodiments, the variable domain comprises a CDR3 having an amino acid sequence represented by SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

[0021] In some embodiments, the variable domain comprises a CDR1 having an amino acid sequence represented by SEQ ID NO: 19, a CDR2 having an amino acid sequence represented by SEQ ID NO: 23, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 27.

[0022] In some embodiments, the variable domain comprises a CDR1 having an amino acid sequence represented by SEQ ID NO: 17, a CDR2 having an amino acid sequence represented by SEQ ID NO: 20, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 24.

[0023] In some embodiments, the variable domain comprises a CDR1 having an amino acid sequence represented by SEQ ID NO: 17, a CDR2 having an amino acid sequence represented by SEQ ID NO: 20, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 28.

[0024] In some embodiments, the variable domain comprises a CDR1 having an amino acid sequence represented by SEQ ID NO: 18, a CDR2 having an amino acid sequence represented by SEQ ID NO: 23, and a CDR3 having an amino acid sequence represented by SEQ ID NO: 26.

[0025] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1, (a) E30; (b) S60; (c) E62; (d) S72; (e) Q78; (f) I83; (g) T85; (h) D109, and (i) M112.

[0026] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1, (a) E30S, E30G, E30T or E30Y; (b) S60Y; (c) E62D; (d) S72R, S72H or S72K; (e) Q78H or Q78T; (f) I83M; (g) T85S; (h) D109A, D109V, D109L, D109I, D109G, D109S or D109T, and (i) M112L, M112A, M112V, M112I, M112G, M112S or M112L.

[0027] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1, (i) S60Y / E62D / Q78H / I83M / T85S amino acid substitutions, (ii) an amino acid substitution of S72R, T85S, D109A, S72R / D109A or S72R / T85S / D109A, and (iii) has an amino acid substitution selected from one or more of the following amino acid substitutions: E30S / M112L.

[0028] In some embodiments, the variable domains are humanized.

[0029] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1, (a) L2; (b) A14; (c) G56; (d) N57; (e) A75; (f) K87; (g) P88, and (h) K117.

[0030] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1, (a) L2V; (b) A14P; (c) G56R; (d) N57R; (e) A75S; (f) K87R; (g) P88A, and (h) K117Q.

[0031] In some embodiments, the CD8 α binding polypeptide has the following amino acid substitutions compared to a reference antibody whose variable domain has the amino acid sequence set forth in SEQ ID NO:1: L2Y and A14P.

[0032] In some embodiments, the above-mentioned CD8 α binding polypeptide further comprises the following amino acid substitutions compared to a reference antibody whose variable domain has the amino acid sequence represented by SEQ ID NO:1: A75S, K87R, P88A and / or K117Q.

[0033] In some embodiments, the CD8 α binding polypeptides described above further comprise a G56R and / or N57R amino acid substitution compared to a reference antibody whose variable domain has the amino acid sequence represented by SEQ ID NO:1.

[0034] In some embodiments, the variable domain has an amino acid sequence represented by SEQ ID NO:14.

[0035] In some embodiments, the variable domain has an amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:15 or SEQ ID NO:16.

[0036] In some embodiments, the variable domain has an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[0037] In some embodiments, the CD8α binding polypeptide further comprises a cysteine ​​modification at the C-terminus.

[0038] In some embodiments, the cysteine ​​modifications are C, VDC, (GG) n C or (GS) m C, where n or m are each independently selected from 1, 2, 3, and 4.

[0039] In some embodiments, the CD8α binding polypeptide comprises one variable domain.

[0040] In some embodiments, the CD8α binding polypeptide has an amino acid sequence represented by SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:32.

[0041] On the other hand, the present application provided an isolated nucleic acid molecule encoding the above-mentioned CD8α-binding polypeptide.

[0042] On the other hand, the present application provided a vector comprising the above-mentioned nucleic acid molecule.

[0043] On the other hand, the present application provided a cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0044] On the other hand, the present application provided a method for preparing the claimed CD8α binding polypeptide, comprising culturing the above-mentioned cells under conditions permitting expression of the above-mentioned CD8α binding polypeptide.

[0045] In some embodiments, the method further comprises recovering the CD8α binding polypeptide expressed by the cell.

[0046] In some embodiments, the method further comprises purifying and / or modifying the CD8α binding polypeptide.

[0047] On the other hand, the present application provided an immunoconjugate comprising the above-mentioned CD8α-binding polypeptide.

[0048] In some embodiments, the immune complex further comprises at least one detectable label attached to the CD8α binding polypeptide.

[0049] In some embodiments, the detectable label is selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.

[0050] In some embodiments, the detectable label is 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C. 13 N, 15 O. 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 Contains Sr or other gamma-, beta- or positron emitters.

[0051] In some embodiments, the CD8α binding polypeptide is conjugated to the detectable label via a chelator.

[0052] In some embodiments, the chelating agent is selected from the group consisting of DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof.

[0053] In some embodiments, the detectable label is 68 Contains Ga.

[0054] In some embodiments, the chelator comprises NOTA or a derivative thereof, such as NODAGA or Maleimide-NODAGA.

[0055] On the other hand, the present application has provided a composition comprising the above-mentioned CD8α binding polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned cell and / or the above-mentioned immune complex, and optionally a pharma- ceutically acceptable carrier.

[0056] In some embodiments, the composition is a detection agent and comprises the CD8α binding polypeptide or the immune complex.

[0057] In some embodiments, the detection agent is an imaging agent.

[0058] In some embodiments, the imaging agent is an ECT imaging agent.

[0059] In some embodiments, the ECT contrast agent comprises a SPECT contrast agent or a PET contrast agent.

[0060] On the other hand, the present application provided the use of the above CD8α binding polypeptide, the above nucleic acid molecule, the above vector, the above cell, the above immunoconjugate and / or the above composition in the preparation of a pharmaceutical for monitoring, preventing, alleviating and / or treating a tumor.

[0061] On the other hand, the present application provides the use of the CD8α-binding polypeptide according to the present application or the immunoconjugate according to the present application in the preparation of a detection agent for detecting CD8-positive cells.

[0062] On the other hand, the present application provided a method for detecting the presence and / or amount of CD8 in a biological sample, the method comprising contacting the biological sample with the CD8α-binding polypeptide, the immune complex or the composition.

[0063] In some embodiments, the contacting is performed in vitro or ex vivo.

[0064] In some embodiments, the biological sample is a tissue.

[0065] In some embodiments, the tissue is selected from the group consisting of blood tissue, lymphatic tissue, and tumor tissue.

[0066] In some embodiments, the methods include detecting the presence and / or amount of CD8 positive cells in a biological sample.

[0067] In some embodiments, the presence and / or amount of CD8 positive cells in a biological sample is determined by imaging.

[0068] In some embodiments, the presence and / or amount of CD8 positive cells in a biological sample is determined by flow cytometry.

[0069] In some embodiments, the CD8 positive cells are CD8 positive T cells.

[0070] On the other hand, the present application provided a method for detecting and / or diagnosing a CD8-related disease or condition, comprising administering to a subject in need thereof the above-mentioned CD8α-binding polypeptide, the above-mentioned immune complex or the above-mentioned composition.

[0071] In some embodiments, the method further comprises imaging the subject. In some embodiments, the imaging comprises ECT imaging.

[0072] In some embodiments, the ECT imaging includes SPECT imaging or PET imaging.

[0073] In some embodiments, the CD8-related disease or condition comprises a tumor.

[0074] On the other hand, the present application relates to a method for treating a tumor, comprising: 1) administering to a subject in need thereof said CD8α binding polypeptide, said immunoconjugate or said composition, and 2) To determine whether the tumors of the subjects contain CD8-positive cells. Including, Here, a method is provided in which, if the presence of CD8 positive cells in said tumor is detected, said subject is administered an anti-tumor therapy.

[0075] On the other hand, the present application relates to a method for monitoring the efficacy of an antitumor therapy in a subject, comprising: 1) administering to a subject suffering from a tumor and being treated with an antitumor therapy, said CD8α-binding polypeptide, said immunoconjugate or said composition; and 2) A method was provided, which includes determining the amount of CD8-positive cells in the tumor of the subject.

[0076] In some embodiments, the presence and / or amount of CD8 positive cells in the subject's tumor is identified by imaging.

[0077] In some embodiments, the anti-tumor therapy is an immune checkpoint inhibitor therapy.

[0078] In some embodiments, the anti-tumor therapy is selected from administration of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, a LAG3 inhibitor, another T-cell co-inhibitor or ligand antagonist, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a vaccine against a tumor-specific antigen, an adjuvant that enhances antigen presentation, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, a cytokine, and an antibody-drug conjugate (ADC).

[0079] In some embodiments, the tumor comprises a solid tumor.

[0080] In some embodiments, the solid tumor is selected from the group consisting of colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, gastric cancer, esophageal cancer, head and neck cancer, salivary gland cancer, and myeloma.

[0081] On the other hand, the present application provided a method for isolating CD8-positive cells, the method comprising contacting a cell population containing CD8-positive cells with the above-mentioned CD8α-binding polypeptide or the above-mentioned immune complex, and recovering CD8-positive cells bound to the above-mentioned CD8α-binding polypeptide or the above-mentioned immune complex.

[0082] In some embodiments, the CD8 positive cells are CD8 positive T cells.

[0083] In some embodiments, the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMCs).

[0084] In some embodiments, said CD8α binding polypeptide or said immune complex is immobilized on a solid surface.

[0085] In some embodiments, the solid comprises a gel or a magnetic bead.

[0086] On the other hand, the present application provides a kit comprising the above-mentioned CD8α-binding polypeptide, the above-mentioned immunoconjugate or the above-mentioned composition.

[0087] Those skilled in the art can easily understand other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will recognize, the contents of the present application will enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which the present application pertains. Correspondingly, the drawings and descriptions in the specification of the present application are merely illustrative, not limiting. [Brief description of the drawings]

[0088] Particular features of the invention according to the present application are set forth in the appended claims. The features and advantages of the invention according to the present application can be better understood with reference to the exemplary embodiments described in detail below and the accompanying drawings, the brief description of which is as follows:

[0089] FIG. 1A shows the binding of antibody C37 to the PROTEIN A affinity packing described in this application.

[0090] FIG. 1B shows the binding of antibody C37-YDHMS to the PROTEIN A affinity packing described in this application.

[0091] FIG. 2 shows a graph of the SEC-HPLC purity analysis of antibody GSC-C37H described in this application before and after conjugation and after reduction.

[0092] FIG. 3 shows mass spectrometry data for the antibody GSC-C37H described in this application before and after conjugation to NODAGA.

[0093] FIG. 4 shows ELISA assay data of antibody GSC-C37H described in this application before and after conjugation to NODAGA.

[0094] FIG. 5A is a schematic diagram of the 68 1 shows the Radio-TLC results of the Ga-NODAGA-GSC-C37H single domain antibody.

[0095] FIG. 5B is a schematic diagram of the 68 1 shows the Radio-HPLC results of Ga-NODAGA-GSC-C37H single domain antibody.

[0096] FIG. 6 shows the results of the HSC-NPG animal model described in this application. 68 PET / CT imaging of Ga-NODAGA-GSC-C37H is shown.

[0097] FIG. 7 shows the results of the PBMC animal model described in this application. 68 Shown is micro-PET imaging of Ga-NODAGA-GSC-C37H.

[0098] FIG. 8A shows the biodistribution results of the ROI (1 hour post-administration) following micro-PET imaging of the PBMC-humanized animals described in this application.

[0099] FIG. 8B shows the biodistribution results of the ROIs (2 hours post-administration) 2 hours after micro-PET imaging of the PBMC-humanized animals described in this application.

[0100] FIG. 8C shows ex vivo biodistribution results (2 hours post-dosing) by dissection at 2 hours post-dosing of a PBMC-humanized animal described in this application.

[0101] FIG. 9A shows a trend plot of blood radioactivity ID% / g over time in the PBMC-humanized animals described in this application.

[0102] FIG. 9B shows the %ID / g TAC curves of blood uptake of different doses of radioactive material in the PBMC-humanized animals described in this application.

[0103] FIG. 10A is a schematic diagram of the 68 1 shows the Radio-TLC detection results for the in vitro serum stability of Ga-NODAGA-GSC-C37H single domain antibody.

[0104] FIG. 10B is a schematic diagram of the 68 1 shows the results of Radio-HPLC detection of the in vitro serum stability of Ga-NODAGA-GSC-C37H single domain antibody.

[0105] FIG. 11A shows the Radio-HPLC results for urine samples at different time points after administration of mice as described in this application.

[0106] FIG. 11B shows the Radio-HPLC results for blood samples at different time points after administration of the mice described in this application.

[0107] FIG. 12 is a schematic diagram of the 68 A synthetic route diagram of Ga-NODA-GA-GSC-C37H is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] Hereinafter, the embodiments of the present invention will be described with reference to specific examples. However, other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of this specification.

[0109] Definition of Terms The term "CD8" (cluster of differentiation 8) refers to a cell surface glycoprotein expressed primarily on cytotoxic T lymphocytes, but also on dendritic cells, natural killer cells, natural killer T cells, and a subset of γδ T cells. The glycoprotein is composed of two isoforms, α and β (encoded by different genes), expressed as αα homodimers or αβ heterodimers, with the latter predominating. For activation, the CD8 coreceptor stabilizes the T cell receptor-MHC-1 interaction and induces intracellular signaling via phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck) at CD3-associated immunoreceptor tyrosine-based activation motifs (ITAMs).

[0110] In the present application, the term "CD8α binding polypeptide" refers to any polypeptide capable of specifically binding to CD8α. In some embodiments, the binding polypeptide is an antibody. In other embodiments, the binding polypeptide is, for example, an antibody mimic, a cytokine, or a growth factor. For example, a single domain antibody that specifically binds to CD8α of the present application. "CD8α binding polypeptide" can also refer to monovalent polypeptides that bind to CD8α (i.e., a polypeptide that binds to one epitope of CD8α), as well as bivalent or multivalent binding polypeptides (i.e., a binding polypeptide that binds to more than one epitope). A "CD8α binding polypeptide" of the present application can include at least one variable domain that binds to CD8α. In some embodiments, a "CD8α binding polypeptide" of the present application can include two, three, four, or more variable domains that bind to CD8α. The CD8α-binding polypeptides of the present application may, in addition to a variable domain that binds to CD8α, comprise a half-life-prolonging domain (e.g., a variable domain that binds to serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or a linker such as an Fc region and / or a moiety with effector function. In some embodiments, the "CD8α-binding polypeptide" of the present application also encompasses bispecific antibodies comprising variable domains that bind to different antigens.

[0111] In this application, the term "antibody" is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity, such as binding to CD8α (human CD8α, mouse CD8α, cynomolgus monkey CD8α, or rhesus monkey CD8α) (Milleretal (2003) Jour. of Immunology 170:4854-4861). Antibodies can be murine antibodies, human antibodies, humanized antibodies, chimeric antibodies, or derived from other species.

[0112] A full-length antibody typically refers to an antibody composed of two "full-length antibody heavy chains" and two "full-length antibody light chains". A "full-length antibody heavy chain" is usually a polypeptide composed of, from the N-terminus to the C-terminus, an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), and is abbreviated as VH-CH1-HR-CH2-CH3, and optionally further comprises an antibody heavy chain constant domain 4 (CH4) in the case of an antibody of the IgE subclass. In some embodiments, a "full-length antibody heavy chain" is a polypeptide composed of, from the N-terminus to the C-terminus, VH, CH1, HR, CH2, and CH3. A "full-length antibody light chain" is usually a polypeptide composed of, from the N-terminus to the C-terminus, an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), and is abbreviated as VL-CL. The above antibody light chain constant domain (CL) can be kappa or lambda. Two full-length antibody chains are linked to each other via interpolypeptide disulfide bonds between the CL domain and the CH1 domain, and between the hinge region of the full-length antibody heavy chain. Typical full-length antibody examples are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE).

[0113] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains amino acids responsible for specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by an antibody is referred to as an "epitope" as described herein. An antigen-binding domain typically may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but need not include both. An Fd fragment, for example, has two VH regions and often retains some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include: (1) a Fab fragment, which is a monovalent fragment having a VL, VH, constant light chain (CL), and CH1 domains; (2) a F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) a dAb fragment having a VH domain (Ward, ES et al. "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature 341:544-546 (1989), which are incorporated herein in their entireties); (6) isolated complementarity determining regions (CDRs); and (7) single-chain Fvs (scFvs), e.g., derived from an scFV-library.The two domains of the Fv fragment, VL and VH, can be encoded by isolated genes and linked by a synthetic linker that allows recombinant methods to generate them as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)) (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)); and (8) VHH, which is a variable antigen-binding domain from a heavy chain antibody of the Camelidae family (camel, dromedary, llama, alpaca, etc.) (Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J. Immunol. 2002, 14:111-111). (See reviews in Biotechnol., 74, 277-302 and Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs are also sometimes called Nanobodies (Nbs) and / or single domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same way as the intact antibodies.

[0114] In this application, the term "variable domain" generally refers to the variable domain of an antibody capable of specifically binding to an antigen epitope. For example, the antibody variable domains VH and VL (VH domain and VL domain). Another example of a variable domain is the "VHH domain" (or simply referred to as "VHH"). The "VHH domain" is also called heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, and is the variable domain of an antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363,446-448 (1993)). The term "VHH domain" is used to distinguish the above variable domains from the heavy chain variable domains present in conventional four-chain antibodies (herein "VH domains") and the light chain variable domains present in conventional four-chain antibodies (herein "VL domains"). A VHH domain specifically binds an epitope without the need for another antigen-binding domain (this is in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains).

[0115] In the present application, "variable domains" usually have the same general structure, and each domain is composed of four framework (FR) regions with highly conserved sequences, where the FR region comprises four "framework regions": "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", and is linked by three "complementarity determining regions" or "CDRs", "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". The general structure or sequence of a variable domain is sometimes represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An antibody variable domain contains an antigen binding site and thus confers specificity to an antibody for an antigen.

[0116] In this application, the term "CDR" generally refers to the complementarity determining regions in the variable sequences of an antibody. There are three CDRs in each heavy and light chain variable region, called CDR1, CDR2 and CDR3 for each variable region. The exact boundaries of these CDRs have been defined differently in different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) provides not only an unambiguous residue numbering system that can be applied to any variable region of an antibody, but also the exact residue boundaries that define these three CDRs. These CDRs are called Kabat CDRs. Chothia and colleagues (Chothia & Lesk, J. MoI. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) have described the Kabat CDRs as follows: We found that several subportions within the CDRs adopt nearly identical peptide backbone conformations, with significant differences at the amino acid sequence level. These subportions are determined to be L1, L2, and L3, or H1, H2, and H3, where "L" and "H" refer to the light chain and heavy chain regions. These regions are sometimes called Chothia CDRs, and have boundaries that overlap with the Kabat CDRs. Other boundaries for defining CDRs that overlap with the Kabat CDRs are described in Padlan (FASEB J.9:133-139(1995)) and MacCallum (JMoI Biol 262(5):732-45(1996)). Other CDR boundaries may not strictly follow any of the above systems, but can be shortened or extended according to the following predictions or experimental findings, but still follow the Kabat CDRs. As used herein, unless otherwise specified, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia or IMGT).

[0117] In this application, the term "sequence identity" generally refers to a nucleic acid or amino acid sequence in which two or more compared sequences are identical when compared with a sequence comparison program. Here, the term "percentage of sequence identity" generally refers to the level of nucleic acid or amino acid sequence identity between two or more compared sequences when compared with a sequence comparison program. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is generally measured using sequence analysis software. For example, identity is determined using the BLAST program of the NCBI database. For determining sequence identity, see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, 20 von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0118] In this application, amino acid residues are indicated, for example, according to the standard three-letter or one-letter amino acid code generally known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" generally refers to the insertion, deletion or substitution of the indicated number of amino acid residues at a position of the reference sequence compared to another sequence. In some embodiments, the above-mentioned substitution is a conservative amino acid substitution, which means that the amino acid residue is replaced with another amino acid residue that has a similar chemical structure and has little or no effect on the function, activity or other biological properties of the polypeptide. The above conservative amino acid substitutions are well known in the art, for example, conservative amino acid substitutions are preferably those in which one amino acid residue within the following groups (i) to (v) is replaced by another amino acid residue within the same group: (i) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (iii) polar, positively charged residues: His, Arg, and Lys; (iv) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (v) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; Val to Ile or Leu. In some embodiments, the above substitutions are non-conservative amino acid substitutions, such as Ala to Asp, Asn, Glu or Gin.

[0119] In this application, the term "affinity" generally refers to the sum of the strengths of non-covalent interactions between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., a target or an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance, including those methods disclosed in this application. The higher the affinity of a molecule X for its binding partner Y, the lower the Kd value and / or EC50 value.

[0120] In this application, the term "isolated" typically refers to a molecule that is at least partially isolated from other molecules (e.g., antibodies, nucleic acids, etc.) with which it is normally associated in its natural state. An "isolated polypeptide" is essentially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, cellular debris, growth medium, etc. An "isolated nucleic acid" typically exists in a form or setting that is different from that in which it is found in nature.

[0121] In this application, the term "immunoconjugate" generally refers to a complex formed by binding an antibody or an antibody fragment thereof to another active agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, a radioactive element, an imaging probe, a spectroscopic probe, etc. The binding may be a covalent bond or a non-covalent interaction, such as by electrostatic forces. The immune complex may be formed by various linkers known in the art. The complex can deliver the other reagent to a target cell (e.g., a tumor cell) by specific binding of the antibody or its antigen-binding fragment to an antigen on the target cell. The immune complex may be provided in the form of a fusion protein expressible from a polynucleotide encoding the immune complex.

[0122] In this application, the term "chelating agent" generally refers to an organic molecule capable of forming a complex with a metal ion. Chelating agents are often used to label proteins and peptides. The final metal ion complexes are used in radioimmunodetection, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar techniques. Non-limiting examples of chelating or complexing agents are DTPA (diethylenetriaminepentaacetic anhydride) and its derivatives, NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid) and its derivatives, such as NODA-GA (NODAGA), Maleimide-NODAGA, DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) (coupled with radioactive metal ions) and its derivatives, TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N''''-tetraacetic acid) and its derivatives, and DTTA (N-(p-benzylisothiocyanate)-diethylenetriamine-N,N',N'',N''''-tetraacetic acid). These and other chelating agents are readily available commercially.

[0123] In this application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic substances that do not inhibit the effectiveness of the biological activity of the active ingredient. Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents, or encapsulating agents suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be used in the preparations described herein include, for example, flavorings, biocides, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (e.g., serum albumin, gelatin, casein), salt-forming counterions (such as sodium), and the like. These and other known pharmaceutical carriers, excipients and / or additives suitable for use in the preparations described herein are known in the art and may be found, for example, in "Remington: The Science & Practice of Pharmacy", 21st Edition, Lippincott Williams & Wilkins (2005) and "Physician's Desk Reference", 60th Edition, Medical Economics Publishing Company, Montvale, NJ (2005). Pharmaceutically acceptable carriers may be selected according to the mode of administration, solubility and / or stability desired or required, as is conventional.

[0124] In this application, the term "administer" and similar terms are generally not limited to administration to the body, and include in vitro, in vitro followed by in vivo, or in vivo methods as appropriate. For example, any administration method known to those skilled in the art that contacts a cell, organ, or tissue with a composition can be used. For example, the above compounds can be introduced into the body of a subject in need of treatment via any introduction or delivery route. In some embodiments, the compositions of the application can be administered orally, topically, intranasally, intramuscularly, subcutaneously, intradermally, intrathecally, intraperitoneally, or transdermally.

[0125] In this application, the terms "ex vivo" and "in vitro" are used interchangeably and generally refer to actions taken on cells, tissues and / or organs removed from within a subject's body in a controlled environment.

[0126] In this application, the term "diagnosis" generally refers to the detection of a disease or condition, or the measurement of the state or extent of a disease or condition. The term "diagnosis" may also include detecting the cause of a disease or condition, measuring the effectiveness of a pharmaceutical treatment, or predicting the pattern of response to a pharmaceutical treatment.

[0127] In this application, the term "treatment" generally refers to (i) preventing the occurrence of a disease, illness and / or condition in a patient who is susceptible to, but has not been diagnosed with, the disease, illness and / or condition; (ii) inhibiting the disease, illness or condition, i.e., arresting its progression; and (iii) alleviating the disease, illness or condition, i.e., causing regression of the disease, illness and / or condition and / or symptoms associated with the disease, illness and / or condition.

[0128] In this application, the terms "tumor" and "cancer" are used interchangeably and generally refer to neoplastic or malignant cell proliferation. Tumors in this application may be benign or malignant. Tumors in this application may be solid or non-solid tumors.

[0129] 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, monkeys, and the like.

[0130] In this application, the term "about" generally refers to a range of 0.5%-10% above and below a specified value, for example, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10% above or below a specified value.

[0131] In this application, the term "comprise" and variations thereof, including "containing," "having," etc., generally refer to the inclusion of other components, elements, values, steps, etc.

[0132] [Detailed Description of the Invention] CD8α-binding polypeptide On the other hand, the present application provides a CD8α binding polypeptide comprising at least one variable domain, wherein the amino acid sequence of the variable domain has an amino acid sequence in which one or more amino acids have been substituted, deleted or added compared to the amino acid sequence represented by SEQ ID NO: 1.

[0133] In some embodiments, the CD8α binding polypeptide may comprise one or two or three variable domains.

[0134] In some embodiments, the variable domain may have an amino acid sequence in which 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20 amino acids have been substituted, deleted, or added relative to SEQ ID NO:1.

[0135] In some embodiments, the amino acid sequence of the variable domain contains one or more amino acid substitutions compared to the amino acid sequence represented by SEQ ID NO:1, and may contain conservative and / or non-conservative substitutions.

[0136] In some embodiments, the amino acid mutations can be in the CDRs of the targeting moiety (e.g., the CDR1, CDR2, or CDR3 regions). In another embodiment, the amino acid changes can be in the framework regions (FRs) of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 regions).

[0137] The modification of amino acid sequence can be achieved by any technique known in the art, such as site-directed mutagenesis or PCR-based mutagenesis.Such techniques are described in detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0138] In some embodiments, the substitutions, deletions or additions do not substantially reduce the ability of a CD8α binding polypeptide of the present application to specifically bind to CD8α.

[0139] The CD8α binding polypeptides of the present application can have an equilibrium dissociation constant (KD) which can be used to describe the binding affinity to full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments of human CD8α and / or any other naturally occurring or synthetic analogs, variants or mutants (including monomers, dimers, heterodimers, multimers and / or related forms). The above CD8α binding polypeptides may comprise a targeting moiety that binds to full-length and / or mature and / or isoforms and / or splice variants and / or fragments of human CD8α and / or any other naturally occurring or synthetic analogs, variants or variants (including monomers, dimers, heterodimers, multimers and / or related forms), where the KD is less than about 1 uM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or about 1 nM.

[0140] In some embodiments, the CD8α binding polypeptides described above may have one or more of the following properties: (i) capable of binding to the packing material of a PROTEIN A affinity chromatography column; (ii) is capable of binding to CD8α with the same or greater affinity as a reference antibody having the amino acid sequence set forth in SEQ ID NO: 1; (iii) having an increased expression level compared to a reference antibody having an amino acid sequence represented by SEQ ID NO: 1; and (iv) an increased isoelectric point compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1.

[0141] In some embodiments, the CD8α binding polypeptides of the present application may have expression of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1.

[0142] In some embodiments, the isoelectric point (PI) value of the reference antibody (designated C37) having the amino acid sequence represented by SEQ ID NO: 1 is 4.56, and the PI value of the CD8α binding polypeptide is 4.56 or more. For example, the PI value of the CD8α binding polypeptide can be about 5, about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.

[0143] In some embodiments, the CD8α binding polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0144] In some embodiments, the antibodies may include monoclonal antibodies, multispecific antibodies (eg, bispecific antibodies), chimeric antibodies, humanized antibodies and / or fully human antibodies.

[0145] In some embodiments, the antigen-binding fragment may include a Fab, a Fab', a F(ab)2, an Fv fragment, a F(ab')2, a scFv, a di-scFv, a VHH, and / or a dAb. For example, the antibody or antigen-binding fragment thereof may be a VHH.

[0146] In some embodiments, the VHHs are not limited to a particular biological source or a particular preparation method. For example, the VHHs generally include (1) the VHHs of naturally occurring heavy chain antibodies. H (2) isolating the naturally occurring V H (3) expressing a nucleotide sequence encoding a naturally occurring VH domain; H The H domain may be "humanized" or such a humanized V H(4) "camelizing" a naturally occurring VH domain from any animal species, particularly a mammal such as human, or expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" e.g. a "domain antibody" or "Dab" as described in the art, or expressing a nucleic acid encoding such a camelized VH domain; (6) preparing a protein, polypeptide or other amino acid sequence by synthetic or semi-synthetic techniques known in the art; (7) preparing a nucleic acid encoding a VHH by nucleic acid synthesis techniques known in the art, followed by expressing the nucleic acid so obtained; and / or (8) any combination of one or more of the above.

[0147] In some embodiments, said CD8α binding polypeptides are "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain of a conventional four-chain antibody with one or more amino acid residues present at the corresponding positions in the VHH domain of a camelid heavy chain antibody VHH. In some embodiments, such "camelization" substitutions are inserted at amino acid positions formed and / or present at the VH-VL interface and / or at so-called camelid hallmark residues (see, for example, WO9404678, which is incorporated by reference in its entirety into the present application). In some embodiments, the VH sequence used as starting material or starting point for producing or designing a camelized VHH is a VH sequence of mammalian origin, such as a human VH sequence, such as a VH3 sequence. Said camelized VHH can be obtained by any suitable method known in the art (i.e., for example according to points (1) to (8) above) and is therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VH domain as starting material.

[0148] In some embodiments, the VHHs may be camelid, chimeric, human, partially humanized, or fully humanized.

[0149] In some embodiments, the CD8α binding polypeptides are “humanized”, i.e., have a naturally occurring V H The VHH is "humanized" by replacing one or more amino acid residues in the amino acid sequence of the H sequence (specifically in the framework sequence) with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional four-chain antibody of human origin. This can be done by humanization techniques known in the art. In some embodiments, humanizable substitutions or combinations of humanizing substitutions can be identified by methods known in the art, such as, for example, performing a comparison between the sequence of the VHH and the sequence of a naturally occurring human VH domain. In some embodiments, the humanizing substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. Generally, as a result of humanization, the VHH of the present application is more "human-like" than the corresponding naturally occurring VHH domain, while retaining advantageous properties such as reduced immunogenicity. The humanized VHH of the present application can be obtained by any suitable method known in the art, and is therefore not strictly limited to polypeptides obtained using a polypeptide comprising a naturally occurring VHH domain as a starting material.

[0150] In some embodiments, the variable domain may comprise CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO: 14. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs or Contact CDRs. In some embodiments, the CDRs are Chothia CDRs.

[0151] In some embodiments, the variable domain may comprise the CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:15 or SEQ ID NO:16.

[0152] In some embodiments, the variable domain may comprise a CDR1 having the amino acid sequence represented by SEQ ID NO:19.

[0153] In some embodiments, the variable domain may comprise a CDR1 having the amino acid sequence represented by SEQ ID NO:17 or SEQ ID NO:18.

[0154] In some embodiments, the variable domain may comprise a CDR2 having the amino acid sequence represented by SEQ ID NO:23.

[0155] In some embodiments, the variable domain may comprise a CDR2 having an amino acid sequence represented by SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22.

[0156] In some embodiments, the variable domain comprises a CDR3 having an amino acid sequence represented by SEQ ID NO:27.

[0157] In some embodiments, the variable domain comprises a CDR3 having an amino acid sequence represented by SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.

[0158] For example, the above variable domain may comprise a CDR1 having the amino acid sequence represented by SEQ ID NO: 19, a CDR2 having the amino acid sequence represented by SEQ ID NO: 23, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 27.

[0159] For example, the above variable domain may comprise a CDR1 having the amino acid sequence represented by SEQ ID NO: 17, a CDR2 having the amino acid sequence represented by SEQ ID NO: 20, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 24.

[0160] For example, the above variable domain may comprise a CDR1 having the amino acid sequence represented by SEQ ID NO: 17, a CDR2 having the amino acid sequence represented by SEQ ID NO: 20, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 28.

[0161] For example, the above variable domain may comprise a CDR1 having the amino acid sequence represented by SEQ ID NO: 18, a CDR2 having the amino acid sequence represented by SEQ ID NO: 23, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 26.

[0162] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, is It may contain amino acid substitutions at one or more positions selected from the group consisting of: (a) E30; (b) S60; (c) E62; (d) S72; (e) Q78; (f) I83; (g) T85; (h) D109, and (i) M112.

[0163] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at positions (b) S60; (c) E62; (e) Q78; (f) I83, and (g) T85, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1.

[0164] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at positions (d) S72; (f) I83; and / or (h) D109, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1.

[0165] For example, the above CD8α binding polypeptide may comprise, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, an amino acid substitution in the variable domain at positions (a) E30; and / or (i) M112.

[0166] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at the following positions compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1: (a) E30; (b) S60; (c) E62; (d) S72; (e) Q78; (f) I83; (g) T85; (h) D109, and (i) M112.

[0167] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, is (a) E30S, E30G, E30T or E30Y; (b) S60Y; (c) E62D; (d) S72R, S72H or S72K; (e) Q78H or Q78T; (f) I83M; (g) T85S; (h) D109A, D109V, D109L, D109I, D109G, D109S or D109T, and (i) M112L, M112A, M112V, M112I, M112G, M112S or M112L.

[0168] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, is It may contain one or more amino acid substitutions selected from the group consisting of: (a) E30S; (b) S60Y; (c) E62D; (d) S72R; (e) Q78H; (f) I83M; (g) T85S; (h) D109A, and (i) M112L.

[0169] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, is (i) S60Y / E62D / Q78H / I83M / T85S amino acid substitutions; (ii) an amino acid substitution of S72R, T85S, D109A, S72R / D109A or S72R / T85S / D109A, and (iii) may contain an amino acid substitution selected from one or more of the groups consisting of the E30S / M112L amino acid substitution.

[0170] For example, the CD8α binding polypeptide described above may comprise the following amino acid substitutions in the variable domain compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1: S60Y, E62D, Q78H, I83M and T85S.

[0171] For example, the CD8α binding polypeptide described above may comprise the following amino acid substitutions in the variable domain as compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1: S72R, T85S, D109A.

[0172] For example, the CD8α binding polypeptide described above may comprise the following amino acid substitutions in the variable domain as compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1: E30S, M112L.

[0173] Also, for example, the above CD8α binding polypeptide may contain the following amino acid substitutions in the variable domain compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1: E30S, S60Y, E62D, S72R, Q78H, I83M, T85S, D109A, M112L.

[0174] In some embodiments, the variable domains may be humanized.

[0175] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, is It may contain an amino acid substitution at one or more positions selected from the group consisting of: (a) L2; (b) A14; (c) G56; (d) N57; (e) A75; (f) K87; (g) P88, and (h) K117.

[0176] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at positions (a) L2; (b) A14; (e) A75; (f) K87 and (g) P88, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1.

[0177] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at positions (a) L2; (b) A14; (e) A75; (f) K87; (g) P88, and (h) K117, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1.

[0178] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at the following positions, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1: (a) L2; (b) A14; (c) G56; (e) A75; (f) K87; (g) P88, and (h) K117.

[0179] For example, the above CD8α binding polypeptide may include amino acid substitutions in the variable domain at the following positions, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1: (a) L2; (b) A14; (d) N57; (e) A75; (f) K87; (g) P88, and (h) K117.

[0180] In some embodiments, the CD8 α binding polypeptide has a variable domain that, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, is It may contain one or more amino acid substitutions selected from the group consisting of: (a) L2V; (b) A14P; (c) G56R; (d) N57R; (e) A75S; (f) K87R; (g) P88A, and (h) K117Q.

[0181] For example, the CD8α binding polypeptide described above may comprise the following amino acid substitutions in the variable domain: L2Y and A14P, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1.

[0182] For example, the above CD8α binding polypeptides may further comprise the following amino acid substitutions in the variable domain compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1: A75S, K87R, P88A and / or K117Q.

[0183] For example, the above CD8α binding polypeptides may further comprise the following amino acid substitutions in the variable domain as compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1: G56R and / or N57R.

[0184] Also, for example, the above CD8α binding polypeptide may contain the following amino acid substitutions in the variable domain compared to a reference antibody having the amino acid sequence represented by SEQ ID NO:1: L2Y, A14P, A75S, K87R and P88A.

[0185] Also, for example, the above CD8α binding polypeptides may contain the following amino acid substitutions in the variable domain compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1: L2Y, A14P, E30S, S60Y, E62D, S72R, A75S, Q78H, I83M, T85S, K87R, P88A, D109A and M112L.

[0186] In some embodiments, the variable domain has an amino acid sequence represented by SEQ ID NO:14.

[0187] For example, the variable domain has the amino acid sequence represented by SEQ ID NO:2, SEQ ID NO:15 or SEQ ID NO:16.

[0188] In some embodiments, the variable domain may comprise an amino acid sequence having at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[0189] Also, for example, the above variable domain has an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[0190] In some embodiments, the CD8 alpha binding polypeptide may comprise one variable domain. For example, the CD8 alpha binding polypeptide may comprise an amino acid sequence having at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

[0191] Also, for example, the above CD8α binding polypeptide has an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[0192] In some embodiments, the C-terminus of the above CD8α binding polypeptide may further comprise a cysteine ​​modification.

[0193] In some embodiments, the cysteine ​​modifications are C, VDC, (GG) n C or (GS) m C, where n or m is each independently selected from 1, 2, 3, and 4.

[0194] In some embodiments, the CD8α binding polypeptide may comprise one variable domain.

[0195] In some embodiments, the CD8α binding polypeptide has an amino acid sequence represented by SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:32.

[0196] Nucleic acids, vectors and cells On the other hand, the present application provided an isolated nucleic acid molecule capable of encoding the above-mentioned CD8α binding polypeptide.

[0197] In some embodiments, the nucleic acid can encode an amino acid sequence comprising a VL and / or a VH (e.g., a light chain and / or a heavy chain of an antibody) comprising an antibody. In some embodiments, the nucleic acid can encode an amino acid sequence comprising a VHH comprising an antibody. In some embodiments, an isolated nucleic acid encoding an anti-CD8 alpha heavy chain variable region is provided, the isolated nucleic acid having a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleic acid sequence encoding SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.

[0198] On the other hand, the present application provided a vector comprising the above-mentioned nucleic acid molecule.

[0199] On the other hand, the present application provided a cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0200] The nucleic acid encoding the CD8α-binding polypeptide of the present application can be incorporated (linked) into an expression vector that can be introduced into a host cell by transfection, transformation or transduction techniques. For example, the nucleic acid encoding the CD8α-binding polypeptide of the present application can be introduced into a host cell by reverse transcription viral transduction. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, the host cell is a prokaryotic cell, such as an E. coli cell.

[0201] On the other hand, the present application provided a method for producing a CD8α binding polypeptide, comprising culturing a cell containing, for example, a nucleic acid encoding an antibody provided herein, under conditions suitable for expression of the CD8α binding polypeptide, and optionally recovering said CD8α binding polypeptide from the cells (or cell culture medium).

[0202] In some embodiments, the above methods may further comprise purifying and / or modifying said CD8α binding polypeptide.

[0203] Specific expression and purification conditions will vary depending on the expression system used. For example, if the gene is to be expressed in E. coli, the engineered gene is first cloned into an expression vector by placing it downstream of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in eukaryotic cells, such as CHO cells, it is first inserted into an expression vector that includes, for example, a suitable eukaryotic promoter, secretion signal, enhancer, and various introns. The gene construct can be introduced into the host cell using transfection, transformation, or transduction techniques.

[0204] The CD8α binding polypeptide of the present application can also be expressed in vivo, for example in a patient. For example, in some embodiments, the CD8α binding polypeptide of the present application can be administered in the form of a nucleic acid encoding the CD8α binding polypeptide of the present application. The nucleic acid can be DNA or RNA. In some embodiments, the CD8α binding polypeptide of the present application is encoded by a modified mRNA, i.e., an mRNA comprising one or more modified nucleotides. In some embodiments, the present application relates to a gene therapy vector comprising the modified mRNA. In some embodiments, the present application relates to a gene therapy method comprising the gene therapy vector. In some embodiments, the nucleic acid is expressed in the form of an oncolytic virus, such as an adenovirus, a reovirus, a measles virus, a herpes simplex virus, a Newcastle disease virus, or a vaccinia virus.

[0205] immune complex On the other hand, the present application provided an immunoconjugate comprising the above-mentioned CD8α-binding polypeptide.

[0206] In some embodiments, the immune complex further comprises at least one detectable label attached to the CD8α binding polypeptide.

[0207] In some embodiments, the detectable label may be selected from the group consisting of a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.

[0208] Fluorescent agents that can be conjugated include, but are not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescent amines; chemiluminescent agents that can be conjugated include, but are not limited to, luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters; bioluminescent agents that can be conjugated include, but are not limited to, luciferin, luciferase, and aequorin. Paramagnetic ions available for complexation are chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III) and erbium(III), or barium, diatrizoate, ethiodized oil, gallium citrate, yttrium(III) and yttrium(III). Radiopaque materials include, but are not limited to, occarmic acid, iosetamic acid, iodamide, iodipamide, iodoxamic acid, ioglamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosephamic acid, ioceric acid, iosuramide meglumine, iotazur, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoic acid, propriodone, and thallium oxide. Enzymes that can be used for conjugation include, but are not limited to, horseradish peroxidase, and the like.

[0209] In some embodiments, the detectable label may be a radionuclide. Radionuclides available for conjugation include: 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y,89 Zr, 94m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C. 13 N, 15 O. 186 Re, 188 Re, 51 Mn, 52m Mn, 72 As, 75 Br, 76 Br, 82m Rb, 83 It can be a radionuclide with an energy between 20-4000 KeV, including but not limited to Sr or other γ-, β- or positron emitters.

[0210] In some embodiments, the detectable label is 68 Ga or 125 It can be I.

[0211] Methods for conjugating detectable labels to polypeptides are well known to those of skill in the art. For example, in some embodiments, the CD8α binding polypeptides can be conjugated to the detectable labels via chelators.

[0212] In some embodiments, the CD8α binding polypeptide of the present application 68To label with a radionuclide such as Ga, the CD8α binding polypeptides of the present application must be reacted with a reagent that has a long tail incorporating multiple integrating groups for binding ions, such as polylysine, polysaccharides or other polymers that have derivatized or derivatizable chains with side groups that can incorporate chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid), NOTA, TETA, NETA, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes and similar groups known to be useful for such purposes.

[0213] In some embodiments, the detectable label is conjugated to the CD8α binding polypeptide of the present application via a chelator, which includes, but is not limited to, DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof.

[0214] In some embodiments, the detectable label is 68 It can be Ga.

[0215] In some embodiments, the chelator can be NOTA or a derivative thereof. For example, the detectable label can be 68 Ga, the chelator can be NOTA. For example, the detectable label can be 68 Ga, the chelating agent can be NODAGA.

[0216] On the other hand, the present application 68A method for preparing a radionuclide, such as a Ga-labeled immunoconjugate, comprising the steps of: 1) conjugating a CD8α binding polypeptide of the present application to a chelating agent to produce a conjugate of said CD8α binding polypeptide and a chelating agent; and 2) reacting the product of step 1). 68 By contacting it with a radioactive nuclide such as Ga, 68 and labeling the CD8α binding polypeptide of the present application with a radionuclide such as Ga by chelating with a chelator. In some embodiments, the chelator is NOTA, and in step 1), the CD8α binding polypeptide is reacted with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA to form a conjugate between the CD8 binding polypeptide and NOTA. In other embodiments, the chelator is NODAGA, and in step 1), the CD8α binding polypeptide is reacted with Maleimide-NODAGA to form a conjugate between the CD8 binding polypeptide and NODAGA.

[0217] On the other hand, the present application 125 A method for preparing a I-labeled conjugated molecule, comprising: 1) reacting a CD8α binding polypeptide of the present application in the presence of chloramine T. 125 I; and 2) quenching the reaction with sodium metabisulfite.

[0218] composition On the other hand, the present application provided a composition comprising the above-mentioned CD8α-binding polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned cell and / or the above-mentioned immune complex, and optionally a pharma- ceutically acceptable carrier.

[0219] The CD8α binding polypeptides described in the present application may have a functional group that is sufficiently basic to react with inorganic or organic acids, or a carboxyl group that can react with inorganic or organic bases to form pharma-ceutically acceptable salts. For example, as is well known in the art, pharma-ceutically acceptable acid addition salts are formed from pharma-ceutically acceptable acids. Such salts include, for example, pharma-ceutically acceptable salts described in Journal of Pharmaceutical Science, 66, 2-19 (1977), and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. PH Stahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety. In some embodiments, the compositions described in the present application are in the form of pharma-ceutically acceptable salts.

[0220] Any of the compositions described in this application can be administered to a subject as a component of a composition that includes a pharma- ceutically acceptable carrier. Such compositions can optionally include a suitable amount of a pharma- ceutically acceptable excipient to provide the form for proper administration.

[0221] The pharmaceutical excipient may be a liquid, such as water or an oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical excipient may be, for example, saline, gum arabic, gelatin, gelatinized starch, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, the pharma- ceutical acceptable excipient is sterile when administered to a subject. When any of the formulations described in this application are administered intravenously, water is a useful excipient. Saline, aqueous glucose and aqueous glycerol solutions may also be employed as liquid excipients, particularly for injections. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, nonfat dry milk, glycerin, propylene, ethylene glycol, water, ethanol, and the like. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (ed. Alfonso R. Gennaro, 19th ed., 1995), which is incorporated herein by reference in its entirety.

[0222] The present application includes various preparation forms of the compositions (and / or other therapeutic agents) described herein. Any of the compositions (and / or other therapeutic agents) of the present invention described herein may be in the form of a solution, suspension, emulsion, drops, tablets, pills, elixirs, capsules, liquid-containing capsules, gelatin capsules, powders, sustained release preparations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized agents, frozen suspensions, dry powders, or any other suitable form. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in the form of a lozenge. In another embodiment, the composition is formulated in the form of a softgel capsule. In another embodiment, the composition is formulated in the form of a gelatin capsule. In another embodiment, the composition is formulated as a liquid.

[0223] Any of the compositions described in this application are formulated in accordance with conventional procedures into compositions suitable for the modes of administration described in this application.

[0224] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intrathecal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In some embodiments, the administration is oral. In other embodiments, the administration is by parenteral injection. The mode of administration is left to the discretion of the physician and will depend in part on the site of the medical condition.

[0225] In some embodiments, the composition includes the immunoconjugate of the present application and can be a detection agent. In some embodiments, the detection agent can be an imaging agent. For example, the imaging agent can be an ECT imaging agent. Also, for example, the ECT imaging agent can include a SPECT imaging agent or a PET imaging agent.

[0226] Detection / Diagnostic Use On the other hand, the present application provided the use of the above CD8α binding polypeptide, the above nucleic acid molecule, the above vector, the above cell, the above immunoconjugate and / or the above composition in the preparation of a pharmaceutical for monitoring, preventing, alleviating and / or treating a tumor.

[0227] On the other hand, the present application provides the use of the CD8α binding polypeptide of the present application or the immunoconjugate of the present application in the preparation of a detection agent for detecting CD8 positive cells. In some embodiments, the detection agent can be used to detect the presence and / or amount of CD8 positive cells in tissues in a subject. In some embodiments, the tissue is a tumor tissue. In some embodiments, the CD8 positive cells can be CD8 positive T cells. In some embodiments, the detection agent can be an imaging agent. In some embodiments, the imaging agent can be an ECT imaging agent, such as a SPECT imaging agent or a PET imaging agent.

[0228] On the other hand, the present application has provided a method for detecting the presence and / or amount of CD8 in a biological sample, the method comprising contacting the biological sample with the CD8α-binding polypeptide, the immune complex, the composition or the detection agent.

[0229] In some embodiments, the contacting may occur in vitro or ex vivo.

[0230] In some embodiments, the biological sample may be a tissue.

[0231] In some embodiments, the tissue may be selected from the group consisting of blood tissue, lymphatic tissue, and tumor tissue.

[0232] In some embodiments, the above methods may include detecting the presence and / or amount of CD8 positive cells in a biological sample.

[0233] In some embodiments, the presence and / or amount of CD8 positive cells in a biological sample may be identified by imaging.

[0234] In some embodiments, the presence and / or amount of CD8 positive cells in a biological sample may be determined by flow cytometry.

[0235] In some embodiments, the CD8 positive cells may be CD8 positive T cells.

[0236] On the other hand, the present application has provided a method for detecting and / or diagnosing a CD8-related disease or illness, which may comprise administering to a subject in need thereof the above-mentioned CD8α-binding polypeptide, the above-mentioned immune complex, the above-mentioned composition or the above-mentioned detection agent.

[0237] In some embodiments, the method may further include imaging the subject.

[0238] In some embodiments, the imaging may include ECT imaging.

[0239] In some embodiments, the ECT imaging may include SPECT imaging or PET imaging.

[0240] In some embodiments, the CD8-related disease or condition may include a tumor.

[0241] On the other hand, the present application relates to a method for treating a tumor, comprising: 1) administering to a subject in need thereof the CD8α-binding polypeptide, the immunoconjugate, the composition, or the detection agent; and 2) To determine whether the tumors of the subjects contain CD8-positive cells. Includes Here, a method is provided in which, if the presence of CD8 positive cells in said tumor is detected, said subject is administered an anti-tumor therapy.

[0242] On the other hand, the present application relates to a method for monitoring the efficacy of an antitumor therapy in a subject, comprising: 1) administering to a subject suffering from a tumor and being treated with an antitumor therapy, the CD8α-binding polypeptide, the immunoconjugate, the composition, or the detection agent; and 2) A method was provided, which includes determining the amount of CD8-positive cells in the tumor of the subject.

[0243] In some embodiments, the presence and / or amount of CD8 positive cells in the subject's tumor is identified by imaging.

[0244] In some embodiments, the anti-tumor therapy is an immune checkpoint inhibitor therapy.

[0245] In some embodiments, the anti-tumor therapy may be selected from the group consisting of administration of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, a LAG3 inhibitor, another T-cell co-inhibitor or ligand antagonist, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a vaccine against a tumor-specific antigen, an adjuvant that enhances antigen presentation, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, a cytokine, and an antibody-drug conjugate (ADC).

[0246] In some embodiments, the tumor may comprise a solid tumor.

[0247] In some embodiments, the solid tumor may be selected from the group consisting of colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer, and myeloma.

[0248] On the other hand, the present application provided a method for isolating CD8-positive cells, which may include contacting a cell population containing CD8-positive cells with the above-mentioned CD8α-binding polypeptide or the above-mentioned immune complex, and recovering CD8-positive cells bound to the above-mentioned CD8α-binding polypeptide or the above-mentioned immune complex.

[0249] In some embodiments, the CD8 positive cells may be CD8 positive T cells.

[0250] In some embodiments, the cell population containing CD8-positive cells may be human peripheral blood mononuclear cells (PBMCs).

[0251] In some embodiments, the CD8α binding polypeptide or the immune complex may be immobilized on a solid surface.

[0252] In some embodiments, the solid may include a gel or magnetic beads.

[0253] On the other hand, the present application has provided a kit which may comprise the above-mentioned CD8α-binding polypeptide, the above-mentioned immunoconjugate or the above-mentioned composition.

[0254] The kit may include instructions for use. The instructions typically include tangible expressions describing techniques to be employed to achieve a desired therapeutic outcome, such as the treatment of cancer, using the components of the kit. The kit may also optionally include other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipettes or measuring devices, packaging materials or other useful accessories, as would be readily understood by one of ordinary skill in the art.

[0255] The materials and components incorporated in the kit can be provided to one skilled in the art for storage in any convenient and appropriate manner that maintains operability and usefulness. By way of example, the components can be provided at room temperature, refrigerated temperature, or frozen temperature. The components are typically contained in suitable packaging. In some embodiments, the packaging is constructed by well-known methods, preferably to provide a sterile, contamination-free environment. The packaging may have an exterior label indicating the contents and / or purpose of the kit and / or its components.

[0256] The following embodiments are intended only to interpret the antibodies, preparation methods and uses of the present application without being limited by any theory, and are not intended to limit the inventive scope of the present application. In the present application, the CD8 single domain antibody or CD8α single domain antibody is as disclosed in CN202010703962.9, and the above documents are incorporated by reference in their entirety into the present application. EXAMPLES

[0257] Example 1 1.1 Optimization of the sequence of the C37 antibody for binding to the affinity packing After early screening and identification, C37-cHis was selected from the candidate antibodies as the final antibody sequence (including histidine tag), and at the same time, a tag-free recombinant plasmid C37 was constructed. The single domain antibody C37 fusion protein plasmid constructed by the above-mentioned recombination was transfected into transfection HEK293 cells to express the antibody. Then, after purification and identification by PROTEIN A affinity chromatography, it was found that the C37 antibody did not bind to PROTEIN A (Protein A) affinity chromatography packing, making it more difficult to obtain the protein by subsequent purification, so it is necessary to optimize and modify the C37 antibody sequence so that it can be purified via affinity chromatography.

[0258] The framework region of the C37 antibody sequence was analyzed, and important amino acids related to binding to the PROTEIN A affinity packing were selected and mutated. After multiple rounds of mutation, the final mutant C37-YDHMS was able to bind to the PROTEIN A affinity chromatography packing.

[0259] TIFF2024523560000001.tif205170

[0260] The recombinant plasmid C37 and the mutated recombinant plasmid C37-YDHMS were respectively used to express antibodies in HEK293 cells, and purified with PROTEIN A affinity chromatography packing. The binding status to the affinity packing after mutation was verified, and the analysis results after antibody purification by SDS-PAGE before and after mutation are shown in Figure 1. Figure 1A shows the C37 purification SDS-page diagram, as shown in the figure, C37 did not bind to the packing PROTEIN A, and the target protein partially passed through the flow-through solution directly, and partially eluted with the equilibration buffer buffer A. As shown in Figure 1B, the target protein was not found in the flow-through solution and equilibration buffers A and B, and the elution buffers C and D contained pure target protein, suggesting that the modified C37-YDHMS can successfully bind to the packing PROTEIN A.

[0261] 1.2 Sequence optimization of C37 antibody for binding activity, expression level, and PI value Molecules such as antibodies generally require an isoelectric point (PI) higher than 7 so that the molecules can be applied to ion exchange chromatography in downstream purification process platforms. In addition, the isoelectric point of host DNA is mainly distributed between 4 and 4.5, and if the antibody PI value is close to that, it is difficult to achieve a removal effect. The PI value of the C37 antibody is 4.56, which is close to the isoelectric point of DNA, and a poor removal effect is expected, and in order to make it easier to select an ion exchange packing in downstream purification, it is necessary to modify the C37 antibody sequence by mutation to improve the PI value.

[0262] The FR (Framework region) and CDR (Complementarity-determining region) regions of the C37 antibody sequence were analyzed, and amino acids related to affinity, expression level, and PI value were selected and mutated. First, one amino acid was selected and mutated, and then multiple amino acids were mutated, for a total of 15 sets of mutations in the parts related to expression level and binding activity.

[0263] TIFF2024523560000002.tif245170

[0264] TIFF2024523560000003.tif186170

[0265] For each mutant, the expression level and binding activity are detected respectively.For the convenience of detection, this is carried out by using a recombinant plasmid with histidine tag to carry out transient transfection, and after transient transfection, the recombinant protein is purified by affinity chromatography using the corresponding Ni+ resin gel, and the obtained target protein can be used to detect the expression level and binding activity.

[0266] Detection of affinity activity: CD8α-Fc fusion protein was coated on the plate at 0.5μg / well overnight at 4℃, washed with PBST, blocked with 3% BSA, and then gradient dilution series of each mutant C37 single domain antibody protein was added and reacted at room temperature for 1 hour. After washing, anti-his horseradish peroxidase-labeled antibody was added and reacted at room temperature for 1 hour. After washing, color development solution was added and absorbance was read at 450nm. Software SotfMaxPro v5.4 was applied for data processing and graph analysis to obtain binding curves of mutant antibodies to CD8α by 4-parameter fitting, and C37 antibody was used as a control to compare the relative activity of different mutants, reflecting the affinity of the antibodies to CD8α.

[0267] Calculation of expression levels: Expression levels were calculated based on the total amount of target protein obtained after purification by one-step affinity chromatography.

[0268] TIFF2024523560000004.tif152170

[0269] A comprehensive analysis of the affinity and expression level of each mutant for the CD8α antigen was performed, and by combining the mutants selected here, C37-S72R, C37-T85S, and C37-D109A, C37-RSA became the final mutant here.

[0270] By combining mutations related to affinity chromatography purification and affinity, and mutations related to expression level, we continued to analyze the negatively charged amino acids in the antibody sequence based on this, and comprehensively analyzed the entire sequence.By mutating the negatively charged amino acids, we improved the PI value of the entire antibody.

[0271] TIFF2024523560000005.tif234170

[0272] TIFF2024523560000006.tif113170

[0273] After a series of mutations and modifications to the C37 single domain antibody sequence, C37-F1 was finally identified as the final mutant at this stage, and its PI value was improved from 4.56 to 6.75 compared to the original C37 single domain antibody before modification.

[0274] 1.3 Humanization of the C37-F1 single domain antibody Camel antibodies have advantages over conventional antibodies, such as small molecular weight, excellent permeability in the body, and easy passage to reach the target site, so nanobodies are widely used as tools for diagnosing and detecting diseases. However, polyvalent antibodies may cause various degrees of immune reactions when used for a long period of time in clinical practice, which may affect the therapeutic effect, so humanization of the C37-F1 antibody is necessary. Humanization was performed using the protein surface amino acid humanization method and the VHH humanized universal framework grafting method.

[0275] The humanization step was based on sequence homology to obtain the universal humanized VHH framework h-NbBcIII0FGLA (PDB number: 3EAK) based on the nanobody NbBcIII0 antibody (PDB number: 3DWT) designed by Cecile Vincke et al., and humanized the protein surface amino acids based on the human source antibody, and selected the necessary humanized sites according to the specific circumstances of the C37-F1 nanobody sequence.

[0276] The C37-F1 antibody was humanized, and a total of five humanized variants of the antibody line C37-F1 were obtained. Table 2 shows the amino acid changes of these humanized variants and the nomenclature of the mutated antibodies, where the numbering of amino acid residues was compared with Kabat numbering, and the humanized variant C37H was finally selected as the humanized mutated antibody.

[0277] TIFF2024523560000007.tif56170

[0278] TIFF2024523560000008.tif156170

[0279] After mutation, the affinity and expression level of each mutant were analyzed, and the specific detection results are shown in Table 3.

[0280] TIFF2024523560000009.tif67170

[0281] 1.4 Terminal cystine modification of C37H antibody Since the C37H antibody sequence has a total of four lysines, which may increase batch-to-batch uncertainty when conjugating small molecules such as NOTA / DOTA, the C37H antibody sequence was modified with a terminal cystine to perform site-specific conjugation. A total of four forms of modification were made, each adding a single cysteine ​​C at its end and three forms of amino acid sequences, VDC, GGC, and GSC. When the construction was completed, it was transiently transfected, the expression level was calculated, and each was optimized and validated for the conjugation method to select the optimal terminal cystine modified form.

[0282] TIFF2024523560000010.tif122170

[0283] Example 2 Conjugation of NODAGA-GSC-C37H Single Domain Antibody Precursor The bifunctional chelator Maleimide-NODAGA was conjugated to the terminal cysteine ​​of the C37H-GSC single domain antibody, and the conjugated product was purified in a 10 kDa ultrafiltration tube, the buffer was replaced with pure water, and the conjugation efficiency was analyzed by SEC-HPLC by measuring the absorbance at 280 nm with a UV-Vis spectrophotometer. The in vitro affinity to CD8α protein was analyzed by ELISA. The specific method is as follows.

[0284] TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, Sigma) was dissolved in PBS, and the TCEP solution was mixed with C37H-GSC to cleave the disulfide bond formed by hemiglycine at the protein terminal. After ultrafiltration through an ultrafiltration tube, excess TCEP and free cysteine ​​were removed. The maleimide-NODAGA solution was mixed with C37H-GSC and reacted at 37°C. After the reaction, excess maleimide-NODAGA was removed through an ultrafiltration tube, and NODAGA-GSC-C37H single domain antibody was obtained as the product.

[0285] Characterization was performed by SEC-HPLC (Figure 2) and LC-MS (Figure 3), and the results revealed that after the antibody was conjugated to a small molecule, the molecular weight increased slightly and the peak position advanced slightly in the SEC-HPLC results. Mass spectrum results showed that the molecular weight of GSC-C37H was 14031, and after reducing one molecule of cystine (molecular weight: 119), one molecule of NODAGA (molecular weight: 497) was conjugated, and finally, the molecular weight of NODAGA-GSC-C37H was 14409, which was in line with the theory.

[0286] The activity of the conjugated NODAGA-GSC-C37H was measured by ELISA. The specific method was to coat the plate with 5 μg / well of CD8α-Fc fusion protein overnight at 4 ° C. After blocking with 3% BSA at 37 ° C, gradient diluted samples (unconjugated C37H-GSC single domain antibody as standard) were added and reacted at 37 ° C for 1 hour. Next, anti-his-HRP (purchased from Abcam) was added and reacted at room temperature for 1 hour. Then, a color development solution was added and the absorbance was read at a wavelength of 450 nm. The ELISA activity was summarized in Table 4 and Figure 4, and the results show that GSC-C37H did not affect the binding ability to antigen after conjugation to NODAGA after reduction.

[0287] TIFF2024523560000011.tif47170

[0288] Example 3 68 Ga-NODAGA-GSC-C37H radiolabel Eckert & Ziegler IGG100 Germanium in 0.1M sterile HCl 68 / gallium 68 (Ge 68 / Ga 68) Rinse the generator, 68 A Ga eluent was prepared, and an equal volume of 0.2 M sodium acetate solution was added, followed by 1 / 4 volume of 0.1 M sodium acetate buffer (pH 5.3) containing the above NODAGA-GSC-C37H antibody, adjusting the pH of the reaction system to 4.5-4.7, and reacting for 10 minutes at room temperature. Unreacted ionic gallium was removed using a PD-10 column and filtered through a 0.22 um filter membrane. The product ( 68 The quality of the Ga-NODAGA-GSC-C37H single domain antibody injection solution is controlled by analysis of pH, Radio-TLC, Radio-HPLC, radioactivity, radiochemical purity, etc. 68 / gallium 68 (Ge-68 / Ga-68) Rinse the generator 68 It was also possible to prepare the Ga eluent, and other reaction and quality control conditions were the same.

[0289] A small amount of sample was taken and measured by Radio-TLC and Radio-HPLC. The peaks of the spectrum were integrated to calculate the radiochemical purity (RCP). The analytical results are shown in Figures 5A-5B. 68 This shows that Ga successfully labeled the single domain antibody. Figure 5A shows the results of Radio-TLC of the radiolabeled sample, and Figure 5B shows the results of Radio-HPLC of the radiolabeled sample.

[0290] The results of Radio-HPLC were consistent with those of Radio-TLC. 68 Ga moves faster with a mobility coefficient of about 0.9, whereas 68 The transfer coefficient of Ga-NODAGA-GSC-C37H was found to be approximately 0.2. 68 Ga peaks in about 1 minute, 68 Ga-NODAGA-GSC-C37H peaked at approximately 18 min, and the protein peak times were essentially consistent, revealing that the labeling efficiencies for the two methods were greater than 95%.

[0291] Example 4 HSC-NPG humanized animal PET / CT imaging experiment 4.1. HSC-NPG animal model used in the study In vivo studies were performed using 6- to 8-week-old HSC-NPG mice. For xenografts, 100 μl of MC38-CD8 cells / PBS was subcutaneously implanted into the right front leg of the mouse, and 100 μl of MC38 cells / PBS was subcutaneously implanted into the left front leg of the mouse. The cell seeding density was approximately 5-6 × 10 6 Implantation was performed under isoflurane anesthesia. Under these conditions, more than 90% of the injected animals developed usable tumors (100-300 mm) after 1-2 weeks. 3 ) (MC38-CD8 or MC38) was obtained.

[0292] 4.2. Micro-PET / CT Imaging MC38-CD8+ / - tumor models were inoculated according to the method in 4.1. Scanning was performed with Micro-PET / CT (IRIS PET / CT, inviscan, Strasbourg, France) to measure the lesion %ID / g. Small animal PET / CT scans were performed continuously for 120 min to analyze the radioactive uptake in tumor, muscle, heart (blood), liver, spleen, lungs and kidneys at multiple time points, as shown in Figure 6.

[0293] FIG. 6 shows the results of PET / CT imaging in HSC-NPG humanized animals, with the left side showing a CD8-negative expressing MC38 transplanted tumor and the right side showing a CD8-positive expressing MC38-CD8 transplanted tumor.

[0294] As shown in Figure 6, the above PET tracers 68 After injection of Ga-NODAGA-GSC-C37H, all MC38-CD8 transplanted tumors were clearly visible, but no uptake was observed in CD8-negative expressing MC38 transplanted tumors after injection. Therefore, MC38-CD8 tumors have excellent differentiation potential compared to the contralateral negative control tumors. The radioactive signal was highest in the kidney; 68 It has been demonstrated that Ga-NODAGA-GSC-C37H was primarily metabolized in the kidney.

[0295] Compared to non-humanized animals, the HSC-NPG humanized animal model expresses human CD8 in tissues and organs such as the heart (blood), liver, spleen, and lungs. 68 Ga-NODAGA-GSC-C37H also showed some uptake in the corresponding tissues, such as liver, spleen, and lung. 68 The potential of Ga-NODAGA-GSC-C37H as a tracer for CD8 was further demonstrated.

[0296] Example 5 PBMC Humanized Animal Dose Probe Test 5.1. PBMC animal model used in the study.

[0297] In vivo studies were performed using 6- to 8-week-old PBMC mice. For xenotransplantation, 100 μl of MC38-CD8 cells / PBS was subcutaneously implanted into the right front leg of the mouse, and 100 μl of MC38 cells / PBS was subcutaneously implanted into the left front leg of the mouse. The cell seeding density was approximately 5–6 × 10 6 Implantation was performed under isoflurane anesthesia. Under these conditions, more than 90% of the injected animals developed usable tumors (100-300 mm) after 1-2 weeks. 3 ) (MC38-CD8 or MC38) was obtained.

[0298] 5.2. Selection of administration method and dose 68 The administration method of Ga-NODAGA-GSC-C37H was a dose probe test of PBMC humanized animals, with a total protein content of 0.3 μg, 1 μg, 5 μg, 30 μg, 150 μg, and 500 μg, including a total of six dose groups. The evaluation methods included micro-PET scans (ROI drawing of tissue distribution), ex vivo tissue distribution, and ex vivo blood PK. 68 Co-injection of cold and hot proteins was employed to estimate the optimal clinical dose of Ga-NODAGA-GSC-C37H and to identify the dose-efficacy relationship in a PBMC humanized animal model.

[0299] 5.3. Micro-PET Imaging MC38-CD8+ / - tumor models were inoculated according to the method in 5.1. Mice were anesthetized with isoflurane and placed on the PET bed, and drugs were administered to the mice via tail vein injection according to six dose groups (~100 μCi): 0.3 μg, 1 μg, 5 μg, 30 μg, 150 μg, and 500 μg. PET scans were performed continuously for 120 min, and radioactive absorption was analyzed in tumors, heart (blood), liver, spleen, lungs, muscles, and kidneys at multiple time points. ROIs were drawn, and biodistribution and activity time curves were calculated using MIM software.

[0300] FIG. 7 shows microPET imaging (1 hour post-dose) of a dose escalation study in PBMC animals, with CD8-negative expressing MC38 xenograft tumors on the left and CD8-positive expressing MC38-CD8 xenograft tumors on the right.

[0301] The results in Figure 7 reveal that after tail vein injection of 0.3-500 μg radiolabeled single domain antibody (~100 μCi / 0.3-500 μg) into mice, all MC38-CD8 transplanted tumors were clearly visible, but no uptake of CD8-negative expressing MC38 transplanted tumors was observed after injection. Thus, MC38-CD8 tumors have excellent differentiation potential compared to the contralateral negative control tumors. The radioactive signal is highest in the kidney; 68 It has been demonstrated that Ga-NODAGA-GSC-C37H was primarily metabolized in the kidney. Compared to non-humanized animals, the PBMC humanized animal model expresses human CD8 in tissues and organs, including the heart (blood), liver, spleen, and lungs. 68 Ga-NODAGA-GSC-C37H also showed some uptake in the corresponding tissues, such as liver, spleen, and lung.

[0302] On the other hand, as the total protein dose increased, the uptake in CD8+ tumors gradually decreased and showed a significant correlation, as well as the uptake in CD8+ humanized tissues and organs such as liver, spleen, and lungs. 68 It was demonstrated that Ga-NODAGA-GSC-C37H specifically bound to CD8+.

[0303] 5.4. In vivo distribution MC38-CD8+ / - tumor model was inoculated according to the method of 5.1. After the mice were anesthetized with isoflurane, the drugs were administered to the mice via tail vein injection according to six dose groups (~100 μCi): 0.3 μg, 1 μg, 5 μg, 30 μg, 150 μg, and 500 μg. Data was collected every 2 hours, and three mice were euthanized at each time point. Target tissues, including blood, kidney, liver, spleen, lung, heart, intestine, stomach, muscle, skin, brain, bone, and CD8+ / - tumor, were dissected and counted in a gamma counter to collect data. The injection volume was taken as the total injection volume. For each organ, the % injected volume (%ID) was determined based on the total injection volume, and the organs were weighed to determine the % injected volume per gram (%ID / g). After micro-PET imaging, the biodistribution results of the ROIs drawn by the MIM software were compared with those detected by a gamma counter ex vivo.

[0304] Figure 8A shows the biodistribution results of ROIs drawn with the MIM software after micro-PET imaging at 1 hour post-injection. Figure 8B shows the biodistribution results of ROIs drawn with the MIM software after micro-PET imaging at 2 hours post-injection. Figure 8C shows the biodistribution results of dissected ex vivo samples at 2 hours post-injection (n=3).

[0305] As shown in Figures 8A-8C, the PET scan data at 1 hour and 2 hours after administration were similar to the ex vivo tissue distribution results at 2 hours after administration. In the two detection methods, the uptake values ​​of each tissue showed similar trends with dose changes, and in particular, the uptake in CD8+ tumor tissue and in the liver, spleen, and lungs showed essentially the same trends with dose changes.

[0306] After administration of 0.3–500 μg radiolabeled single domain antibody (~100 μCi / 0.3–500 μg) via tail vein injection into mice, uptake was high in MC38-CD8 transplanted tumors, but no uptake was observed in CD8-negative expressing MC38 transplanted tumors following injection.

[0307] On the other hand, as the total protein dose increased, the uptake in CD8+ tumors gradually decreased and showed a significant correlation, as well as the uptake in CD8+ humanized tissues and organs such as liver, spleen, and lungs. 68 It was demonstrated that Ga-NODAGA-GSC-C37H specifically binds to CD8+. At a dose of 0.3μg, CD8+ positive tumors were not higher than at a dose of 1μg, and at a dose of 150μg, CD8+ positive tumors were completely blocked from uptake compared to at a dose of 500μg. The biodistribution study of the two detection methods cross-validated that the optimal dose of administration in the animal model was 1μg, and the minimum blocking dose was 150μg.

[0308] 5.5. Pharmacokinetics (PK) for Ex Vivo Blood Collection MC38-CD8+ / - tumor model was inoculated according to the method of 5.1. After anesthetizing the mice with isoflurane, the drugs were administered to the mice via tail vein injection according to six dose groups (~100 μCi): 0.3 μg, 1 μg, 5 μg, 30 μg, 150 μg, and 500 μg. 2, 5, 10, 15, 30, 60, 90, and 120 minutes after administration, 0.5 to 0.8 mL of blood was collected from the medial canthus and weighed. The blood samples were gamma-counted using a gamma counter, and ID% / g was calculated.

[0309] FIG. 9A is a trend graph showing the time course of blood radioactivity ID% / g, and after 0.3-500 μg radiolabeled single domain antibody (~100 μCi / 0.3-500 μg) was administered to mice via tail vein injection, the blood half-life was short and clearance was rapid. 68 The results suggest that Ga-NODAGA-GSC-C37H had a rapid metabolic clearance rate, similar to other nanobody-like radiopharmaceuticals. The general trend was that as the total protein dose increased, the radioactive blood half-life increased and the clearance rate slowed. Also, at low doses, there was high initial uptake, short half-life, and rapid clearance, whereas at high doses the opposite was true.

[0310] Figure 9B shows the %ID / g TAC curves of blood uptake of different doses of radioactive material, with the half-life of the 0.3μg low dose group being 20-30 minutes, close to that of early-stage non-humanized animals. At doses >1ug, the difference in half-life was not significant.

[0311] Example 6 68 Ga-NODAGA-GSC-C37H stability experiment 6.1 68 Analysis of in vitro serum stability of Ga-NODAGA-GSC-C37H Take 300 μL human serum and 500 μL 68 After adding 74 MBq (1 mCi / 100 μL PBS) of Ga-NODAGA-GSC-C37H and mixing, the mixture was placed at 37°C and detected by Radio-TLC and Radio-HPLC at 0 hours, 0.5 hours, 1 hour, 2 hours, and 4 hours.

[0312] As shown in Figures 10A to 10B, in serum at 37°C, 68 No significant increase in Ga was observed, and no significant decomposition peaks appeared, indicating that the radiolabeled antibody was structurally stable and serum stable in vitro.

[0313] 6.2 68 Analysis of in vivo stability of Ga-NODAGA-GSC-C37H Take a normal mouse, 68 Ga-NODAGA-GSC-C37H 74 MBq (1 mCi / 100μL PBS) was injected via the tail vein. Urine samples were collected 15 minutes, 1.5 hours, and 4 hours after injection. All samples were dissolved in a certain amount of 50% acetonitrile solution, centrifuged at 8000 rpm for 15 minutes, and the supernatant was passed through a 0.22μm filter membrane, and the filtrate was analyzed by Radio-TLC and Radio-HPLC.

[0314] As shown in Figures 11A to 11B, 4 hours after administration to mice, the free 68 No significant increase in Ga content was observed, and in the blood samples taken 30 minutes after administration, free 68No Ga content was observed, nor was there an increase in other degradation peaks, indicating that the radiolabeled antibody was present in the blood and urine in an intact state and was structurally stable.

Claims

Claim 1 A CD8α-binding polypeptide comprising at least one variable domain, wherein the amino acid sequence of the variable domain has one or more amino acids substituted, deleted or added as compared with the amino acid sequence represented by SEQ ID NO: 1, a CD8α-binding polypeptide. Claim 2 The CD8α-binding polypeptide according to claim 1, wherein the CD8α-binding polypeptide has one or more of the following characteristics: (i) capable of binding to a filler of a Protein A affinity chromatography column; (ii) capable of binding to CD8α with the same or higher affinity as compared with a reference antibody having the amino acid sequence represented by SEQ ID NO: 1; (iii) having an increased expression level as compared with a reference antibody having the amino acid sequence represented by SEQ ID NO: 1; and (iv) having an increased isoelectric point as compared with a reference antibody having the amino acid sequence represented by SEQ ID NO:

1. Claim 3 The CD8α-binding polypeptide according to claim 1, wherein the variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence represented by SEQ ID NO:

14. Claim 4 The CD8α-binding polypeptide according to claim 1, wherein the variable domain comprises CDR1 having the amino acid sequence represented by SEQ ID NO:

19. Claim 5 The CD8α-binding polypeptide according to claim 1, wherein the variable domain comprises CDR2 having the amino acid sequence represented by SEQ ID NO:

23. Claim 6 The CD8α-binding polypeptide according to claim 1, wherein the variable domain comprises CDR3 having the amino acid sequence represented by SEQ ID NO:

27. Claim 7 The CD8α-binding polypeptide according to claim 1, wherein the variable domain comprises CDR1 having the amino acid sequence represented by SEQ ID NO: 19, CDR2 having the amino acid sequence represented by SEQ ID NO: 23, and CDR3 having the amino acid sequence represented by SEQ ID NO:

27. Claim 8 ​ ​ The variable domain comprises a CDR1 having the amino acid sequence represented by SEQ ID NO: 17, a CDR2 having the amino acid sequence represented by SEQ ID NO: 20, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 24 The CD8α-binding polypeptide according to claim 7

9. The variable domain comprises a CDR1 having the amino acid sequence represented by SEQ ID NO: 17, a CDR2 having the amino acid sequence represented by SEQ ID NO: 20, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 28 The CD8α-binding polypeptide according to claim 7

10. The variable domain comprises a CDR1 having the amino acid sequence represented by SEQ ID NO: 18, a CDR2 having the amino acid sequence represented by SEQ ID NO: 23, and a CDR3 having the amino acid sequence represented by SEQ ID NO: 26 The CD8α-binding polypeptide according to claim 7

11. The variable domain, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1 has an amino acid substitution at one or more positions selected from the group consisting of (a) E30; (b) S60; (c) E62; (d) S72; (e) Q78; (f) I83; (g) T85; (h) D109, and (i) M112 The CD8α-binding polypeptide according to claim 1

12. The variable domain, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1 has one or more amino acid substitutions selected from the group consisting of (a) E30S, E30G, E30T or E30Y; (b) S60Y; (c) E62D; (d) S72R, S72H or S72K; (e) Q78H or Q78T; (f) I83M; (g) T85S; (h) D109A, D109V, D109L, D109I, D109G, D109S or D109T, and (i) M112L, M112A, M112V, M112I, M112G, M112S or M112L The CD8α-binding polypeptide according to claim 11

13. The variable domain, compared to a reference antibody having the amino acid sequence represented by SEQ ID NO: 1 (i) an amino acid substitution of S60Y / E62D / Q78H / I83M / T85S (ii) amino acid substitutions including S72R, T85S, D109A, S72R / D109A or S72R / T85S / D109A, and (iii) amino acid substitutions selected from one or more groups consisting of the amino acid substitution of E30S / M112L The CD8α-binding polypeptide according to claim 12.

14. The variable domain has, as compared with a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, one or more amino acid substitutions at one or more positions selected from the group consisting of (a) L2; (b) A14; (c) G56; (d) N57; (e) A75; (f) K87; (g) P88, and (h) K117 The CD8α-binding polypeptide according to claim 11.

15. The variable domain has, as compared with a reference antibody having the amino acid sequence represented by SEQ ID NO: 1, one or more amino acid substitutions selected from the group consisting of (a) L2V; (b) A14P; (c) G56R; (d) N57R; (e) A75S; (f) K87R; (g) P88A, and (h) K117Q The CD8α-binding polypeptide according to claim 14.

16. The variable domain has the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13 The CD8α-binding polypeptide according to claim 11.

17. The CD8α-binding polypeptide further comprises a cysteine modification at the C-terminus The CD8α-binding polypeptide according to claim 1.

18. The CD8α-binding polypeptide has the amino acid sequence represented by SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32 The CD8α-binding polypeptide according to claim 1.

19. Comprising the CD8α-binding polypeptide according to any one of claims 1 to 18 Immune complex.

20. Comprising the CD8α-binding polypeptide according to any one of claims 1 to 18 Composition.

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