CD8-binding polypeptides and uses thereof

Abstract

The present invention relates to the field of biologics. Specifically, the present invention relates to certain CD8-binding polypeptides and uses thereof.

Description

[Technical field]

[0001] The present invention relates to the field of biologics. In particular, the present invention relates to certain CD8-binding polypeptides and uses thereof. [Background technology]

[0002] Tumors have been diagnosed using emission computed tomography (ECT), which includes single-photon emission computed tomography (SPECT) and positron emission tomography (PET), and allows high-resolution tumor imaging and quantitative analysis via imaging.

[0003] In humans, CD8 is mainly expressed on cytotoxic T lymphocytes, but is also expressed on dendritic cells, natural killer cells, etc. CD8 molecules exist as homodimers formed by CD8α and heterodimers formed by CD8α and CD8β, with the αβ heterodimer being the more common.

[0004] The ability to monitor CD8+ tumor-infiltrating lymphocytes (TILs) in vivo is of great significance in evaluating the efficacy of immunotherapy and aiding in the development of more effective immune cell-targeted single-agent or combination therapies. "Immuno PET" imaging of tumor-infiltrating lymphocytes can provide a specific and sensitive method to help patients select specific immunotherapy regimens and determine whether the treatment is effective.

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

[0006] The present invention includes at least the following embodiments.

[0007] Embodiment 1: A CD8 binding polypeptide comprising at least one immunoglobulin single variable domain capable of specifically binding to CD8α, wherein the at least one immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 of any one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77 and 81.

[0008] Embodiment 2: A CD8 binding polypeptide according to embodiment 1, which is at least one immunoglobulin single variable domain comprising a CDR1, a CDR2 and a CDR3 selected from: (1) CDR1 of SEQ ID NO:2, CDR2 of SEQ ID NO:3, and CDR3 of SEQ ID NO:4; (2) CDR1 of SEQ ID NO:6, CDR2 of SEQ ID NO:7, and CDR3 of SEQ ID NO:8; (3) CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12; (4) CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; (5) CDR1 of SEQ ID NO: 18, CDR2 of SEQ ID NO: 19, and CDR3 of SEQ ID NO: 20; (6) CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24; (7) CDR1 of SEQ ID NO: 26, CDR2 of SEQ ID NO: 27, and CDR3 of SEQ ID NO: 28; (8) CDR1 of SEQ ID NO: 30, CDR2 of SEQ ID NO: 31, and CDR3 of SEQ ID NO: 32; (9) CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35 and CDR3 of SEQ ID NO: 36 CDR3 of; (10) CDR1 of SEQ ID NO: 38, CDR2 of SEQ ID NO: 39, and CDR3 of SEQ ID NO: 40; (11) CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; (12) CDR1 of SEQ ID NO: 46, CDR2 of SEQ ID NO: 47, and CDR3 of SEQ ID NO: 48; (13) CDR1 of SEQ ID NO: 50, CDR2 of SEQ ID NO: 51, and CDR3 of SEQ ID NO: 52; (14) CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; (15) CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO: 60; (16) CDR1 of SEQ ID NO: 62, CDR2 of SEQ ID NO: 63, and CDR3 of SEQ ID NO: 64; (17) CDR1 of SEQ ID NO: 66, CDR2 of SEQ ID NO: 67, and CDR3 of SEQ ID NO: 68; (18) CDR1 of SEQ ID NO: 70, CDR2 of SEQ ID NO: 71, and CDR3 of SEQ ID NO: 72; (19) CDR1 of SEQ ID NO: 74, CDR2 of SEQ ID NO: 75, and CDR3 of SEQ ID NO: 76; (20) CDR1 of SEQ ID NO: 78, CDR2 of SEQ ID NO: 79, and CDR3 of SEQ ID NO: 80.

[0009] Embodiment 3: A CD8 binding polypeptide according to embodiment 1 or 2, wherein the immunoglobulin single variable domain comprises an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% identical to the amino acid sequence of one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77 and 81.

[0010] Embodiment 4: A CD8 binding polypeptide according to any one of embodiments 1 to 3, wherein the immunoglobulin single variable domain comprises the amino acid sequence of one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77 and 81.

[0011] Embodiment 5: A CD8 binding polypeptide according to any one of embodiments 1 to 4, wherein the immunoglobulin single variable domain is a VHH.

[0012] Embodiment 6: A nucleic acid molecule encoding a CD8 binding polypeptide according to any one of embodiments 1 to 5.

[0013] Embodiment 7: An expression vector comprising the nucleic acid molecule of embodiment 6 operably linked to an expression control element.

[0014] Embodiment 8: A host cell capable of expressing a CD8 binding polypeptide, comprising the nucleic acid molecule according to embodiment 6 or transformed by an expression vector according to embodiment 7.

[0015] Embodiment 9: A method for producing a CD8 binding polypeptide according to any one of embodiments 1 to 5, comprising: a) culturing a host cell according to embodiment 8 under conditions allowing expression of a CD8 binding polypeptide. b) recovering the CD8 binding polypeptide expressed by the host cell from the culture obtained in step a); and c) Optionally, further purifying and / or modifying the CD8 binding polypeptide obtained in step b).

[0016] Embodiment 10: A conjugate molecule comprising a CD8 binding polypeptide according to any one of embodiments 1 to 5 and at least one detectable label attached to said CD8 binding polypeptide.

[0017] Embodiment 11: The conjugate molecule of embodiment 10, wherein the detectable label is selected from a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.

[0018] Embodiment 12: The detectable label comprises: 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67Ga, 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, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 The detectable label is selected from Sr or other γ-, β- or positive emitters, e.g. 68 Ga or 125 The conjugate molecule of embodiment 11, wherein said compound is I.

[0019] Embodiment 13: A conjugate molecule according to any one of embodiments 10 to 12, wherein the CD8 binding polypeptide is linked to a detectable label via a chelating agent.

[0020] Embodiment 14: The chelating agent is DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG 3 , MAG 2 ,HYNIC, DADT, E.C., N.S. 3 , H2d ed The conjugate molecule according to embodiment 13, which is selected from pa, HBED, DFO, PEPA or HEHA, and derivatives thereof.

[0021] Embodiment 15: A detectable label 68The conjugate molecule of embodiment 14, wherein said chelator is NOTA and said Ga is Ga.

[0022] Embodiment 16: A method of detecting the presence and / or amount of CD8 in a biological sample, comprising: a) contacting a biological sample and a control sample with a CD8 binding polypeptide of the invention or a conjugate molecule of the invention under conditions in which the CD8 binding polypeptide of any one of embodiments 1 to 5 or the conjugate molecule of any one of embodiments 10 to 15 can form a complex with CD8; and b) detecting the formation of a complex; Here, a difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of CD8 in the sample.

[0023] Embodiment 17: A detection agent for detecting CD8 positive cells, comprising a CD8 binding polypeptide according to any one of embodiments 1 to 5 or a conjugated molecule according to any one of embodiments 10 to 15, and optionally a physiologically acceptable carrier.

[0024] Embodiment 18: The detection agent of embodiment 17, wherein the detection agent is an imaging agent.

[0025] Embodiment 19: The detection agent of embodiment 18, wherein the imaging agent is an emission computed tomography (ECT) imaging agent, such as a single photon emission computed tomography (SPECT) imaging agent or a positron emission tomography (PET) imaging agent.

[0026] Embodiment 20: Use of a CD8 binding polypeptide according to any one of embodiments 1 to 5 or a conjugated molecule according to any one of embodiments 10 to 15 in the preparation of a detection agent for detecting CD8 positive cells.

[0027] Embodiment 21: The detection agent of embodiment 20, wherein the detection agent is an imaging agent.

[0028] Embodiment 22: The detection agent of embodiment 21, wherein the imaging agent is an ECT imaging agent, such as a SPECT imaging agent or a PET imaging agent.

[0029] Embodiment 23: A method for detecting the presence and / or amount of CD8 positive cells in a tissue, comprising: a) contacting the tissue with a conjugate molecule according to any one of embodiments 10 to 15 or a detection agent according to any one of embodiments 17 to 19; and b) Measuring the presence and / or quantity of CD8 positive cells in tissue.

[0030] Embodiment 24: The method of embodiment 23, wherein the tissue is selected from blood tissue, lymphatic tissue and tumor tissue.

[0031] Embodiment 25: The method of embodiment 23 or 24, wherein the CD8 positive cells are CD8 positive T cells.

[0032] Embodiment 26: The method according to any one of embodiments 23 to 25, wherein the presence and / or amount of CD8 positive cells in the tissue is determined by imaging the tissue.

[0033] Embodiment 27: The method according to any one of embodiments 23 to 25, wherein the presence and / or amount of CD8 positive cells in the tissue is determined by flow cytometry.

[0034] Embodiment 28: A method for detecting the presence and / or amount of CD8 positive cells in a tissue of a subject, comprising administering to the subject a conjugate molecule according to any one of embodiments 10 to 15 or a detection agent according to any one of embodiments 17 to 19.

[0035] Embodiment 29: The method of embodiment 28, wherein the tissue is a tumor tissue.

[0036] Embodiment 30: The method of embodiment 28 or 29, wherein the CD8 positive cells are CD8 positive T cells.

[0037] Embodiment 31: The method according to any one of embodiments 28 to 30, wherein the method further comprises imaging the subject, such as ECT imaging, for example, the ECT imaging is SPECT imaging or PET imaging.

[0038] Embodiment 32: A method for determining whether a tumor-bearing subject is suitable for anti-tumor therapy, comprising: 1) administering to a subject a conjugate molecule according to any one of embodiments 10 to 15 or a detection agent according to any one of embodiments 17 to 19; and 2) imaging the subject, such as with ECT imaging, to determine whether the subject's tumor contains CD8 positive cells; Here, if the presence of CD8 positive cells in a tumor is detected, for example, if the subject's tumor is infiltrated by CD8 positive cells, the subject is identified as suitable for anti-tumor therapy.

[0039] Embodiment 33: A method for predicting the response of a tumor-bearing subject to an antitumor therapy, comprising: 1) administering to a subject a conjugate molecule according to any one of embodiments 10 to 15 or a detection agent according to any one of embodiments 17 to 19; and 2) imaging the subject, such as with ECT imaging, to determine whether the subject's tumor contains CD8 positive cells; Here, if the presence of CD8 positive cell(s) in the tumor is detected, for example if the subject's tumor is infiltrated by CD8 positive cell(s), the subject may respond to anti-tumor therapy.

[0040] Embodiment 34: A method for treating a tumor in a subject, comprising: 1) administering to a subject a conjugate molecule according to any one of embodiments 10 to 15 or a detection agent according to any one of embodiments 17 to 19; and 2) imaging the subject, such as with ECT imaging, to determine whether the subject's tumor contains CD8 positive cells; If the presence of CD8 positive cells in the tumor is detected, for example, if the subject's tumor is infiltrated by CD8 positive cells, the subject is administered an anti-tumor therapy.

[0041] Embodiment 35: A method for monitoring the effectiveness of an anti-tumor therapy in a subject, comprising: 1) administering to a subject having a tumor and having been treated with an antitumor therapy a conjugate molecule according to any one of embodiments 10 to 15 or a detection agent according to any one of embodiments 17 to 19; and 2) Imaging the subject, such as ECT imaging, to determine the amount of CD8 positive cells in the subject's tumor.

[0042] Embodiment 36: The method of any one of embodiments 32 to 35, wherein the anti-tumor therapy is an immune checkpoint inhibitor therapy.

[0043] Embodiment 37: The method according to any one of embodiments 32 to 36, wherein the antitumor therapy is selected from the 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, an antagonist of another T-cell co-inhibitor or ligand, 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, an adjuvant that increases 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).

[0044] Embodiment 38: The method according to any one of embodiments 32 to 37, wherein the tumor is a solid tumor.

[0045] Embodiment 39: The method of embodiment 38, wherein the solid tumor is selected from 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, renal cancer, gastric cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.

[0046] Embodiment 40: A method for isolating and / or purifying CD8 positive cells, comprising: (a) Providing a cell population(s) suspected of containing CD8 positive cells. (b) identifying a subpopulation of the cell population, wherein the cells of the subpopulation bind to a CD8 binding polypeptide according to any one of embodiments 1 to 5 or a conjugate molecule according to any one of embodiments 10 to 15; and (c) isolating a subpopulation.

[0047] Embodiment 41: A method of isolating a CD8 positive cell(s), comprising: (a) Providing a cell population(s) suspected of containing CD8 positive cells. (b) contacting the cell population with a CD8 binding polypeptide according to any one of embodiments 1 to 5 or a conjugate molecule according to any one of embodiments 10 to 15, thereby allowing the CD8 positive cells to bind to the CD8 binding polypeptide according to any one of embodiments 1 to 5 or the conjugate molecule according to any one of embodiments 10 to 15. (c) removing cells that do not bind to the CD8 binding polypeptide according to any one of embodiments 1 to 5 or the conjugate molecule according to any one of embodiments 10 to 15; and (d) recovering CD8 positive cells that bind to the CD8 binding polypeptide according to any one of embodiments 1 to 5 or the conjugate molecule according to any one of embodiments 10 to 15.

[0048] Embodiment 42: The method of embodiment 40 or 41, wherein the CD8 positive cells are CD8 positive T cells.

[0049] Embodiment 43: The method according to any one of embodiments 40 to 42, wherein the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMCs).

[0050] Embodiment 44: The method according to any one of embodiments 40 to 44, wherein the CD8 binding polypeptide according to any one of embodiments 1 to 5 or the conjugated molecule according to any one of embodiments 10 to 15 is immobilized on a solid surface, for example on the surface of a gel or magnetic beads.

[0051] Embodiment 45: A kit comprising a CD8 binding polypeptide according to any one of embodiments 1 to 5, a conjugate molecule according to any one of embodiments 10 to 15, or a detection agent according to any one of embodiments 17 to 19. [Brief description of the drawings]

[0052] [Figure 1] Examination of the binding efficiency of candidate antibodies to human PBMC CD8+ T cell surface antigens by fluorescence-activated cell sorting (FACS). [Diagram 2] CD8 single domain antibody labeled with 125I. [Diagram 3] Uptake experiment of 125I-labeled CD8α single domain antibody into MC38-CD8 cells. [Figure 4] Saturation binding analysis of MC38-CD8 cells with 125I-labeled CD8 single domain antibodies. [Diagram 5] MC38-CD8 cell competitive binding analysis of 125I-SNA006a. [Figure 6] SPECT imaging with 125I-labeled CD8α single domain antibody. [Figure 7] Thin layer chromatography (TLC) analysis of 68Ga-labeled CD8α single domain antibody. [Figure 8] In-vivo distribution data of 68Ga-labeled CD8α single domain antibody. [Figure 9] In-vivo distribution target / background ratio of 68Ga-labeled CD8α single domain antibody. [Figure 10] In-vivo PET / CT imaging with 68Ga-labeled CD8α single domain antibody. [Figure 11] In-vivo time biodistribution curve of 68Ga-labeled CD8α single domain antibody. [Figure 12] In-vivo stability analysis of 68Ga-NOTA-SNA006a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] definition Unless otherwise indicated or expressly defined, all terms have their usual meaning in the art and will be apparent to those skilled in the art. For example, reference is made to standard handbooks such as "Molecular Cloning: A Laboratory Manual" (2nd.Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989) and the prior art cited herein. Also, unless otherwise stated, all methods, steps, techniques, and operations that are not specifically detailed can be performed in a manner known per se and will be apparent to those skilled in the art. For example, they are described in standard manuals, the general prior art mentioned above, and other references cited herein.

[0054] The terms "antibody" or "immunoglobulin", as used interchangeably herein, are used as general terms to include full-sized antibodies, whether heavy chain or traditional four-chain antibodies, their individual chains, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively), unless otherwise specified. Furthermore, the term "sequence" as used herein (e.g. terms such as "immunoglobulin sequence", "antibody sequence", "(single) variable domain sequence", "VHH sequence" or "protein sequence") shall be generally understood to include the nucleic acid or nucleotide sequence encoding it, as well as the relevant amino acid sequence, unless the context requires a more restrictive interpretation.

[0055] The term "domain" (of a polypeptide or protein) as used herein refers to a folded protein structure that has the ability to retain its tertiary structure independent of the rest of the protein. Generally, a domain is responsible for a distinct property of a protein and can often be added, removed or transferred to another functional protein without loss of function of the remaining domain and / or protein.

[0056] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional four-chain antibody or a chain of a heavy-chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by retaining the immunoglobulin fold that is characteristic of antibody molecules.

[0057] The term "immunoglobulin variable domain" as used herein means an immunoglobulin domain consisting essentially of four "framework regions", which are referred to in the art and herein as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively. These framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be depicted as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain that confers specificity to the antibody for the antigen by carrying the antigen binding site.

[0058] The term "immunoglobulin single variable domain" as used herein means an immunoglobulin variable domain that is capable of specifically binding to an epitope of an antigen without being paired with an additional variable immunoglobulin domain. An example of an immunoglobulin single variable domain in the sense of the present invention is a "domain antibody" such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Other examples of immunoglobulin single variable domains are the "VHH domains" (or simply "VHH") from camelids as defined below.

[0059] "VHH domains", also known as VHH, VHH domains, VHH antibody fragments, and VHH antibodies, were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., antibodies lacking light chains) (Hamas-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamas C, Songa EB, Bendahman N, Hamas R.:"Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domains" was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (herein referred to as "VH domains") and the light chain variable domains present in conventional four-chain antibodies (herein referred to as "VL domains"). VHH domains can specifically bind to an epitope without an additional antigen-binding domain (in contrast to VH and VL domains in conventional four-chain antibodies, where the epitope is recognized by the VL together with the VH domain). VHH domains are small, robust and efficient antigen recognition units formed by a single immunoglobulin domain.

[0060] In the context of the present invention, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment" and "VHH antibody" can be used interchangeably.

[0061] The amino acid residues of immunoglobulin single variable domains, e.g. VHHs, are numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of Proteins of Immunological interest", 5th ed., ed. Public Health Services, NIH Bethesda, Md., (1991)) as shown in FIG. 2, as applied to camelid VHH domains, as shown in Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999).

[0062] Alternative methods for numbering the amino acid residues of VH domains are known in the art and can be applied to VHH domains as well. For example, Chothia CDRs refer to the location of the structural loops (Chothia and Lesk, J. Mol.Biol. 196:901-917 (1987)). AbM CDRs represent a compromise between Kabat hypervariable regions and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on the analysis of available complex crystal structures. The residues of the CDRs according to each method are as follows:

[0063] JPEG0007688941000001.jpg43170

[0064] However, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each of the CDRs varies and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to the Kabat numbering may be unoccupied in the actual sequence or may contain more amino acid residues than the actual sequence Kabat numbering allows), which means that in general the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0065] For example, the CDRs may include "extended CDRs" such as 24-36 or 24-34 (LCDR1), 45-56 or 50-56 (LCDR2), 89-97 or 89-96 (LCDR3) in the VL, and 26-35 (HCDR1), 50-56 or 49-65 (HCDR2), 93-102, 94-102 or 95-102 (HCDR3) in the VH.

[0066] The total number of amino acid residues in a VHH domain is usually between 110 and 120, and often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0067] Further structural and functional properties of VHH domains and polypeptides containing them can be summarized as follows:

[0068] VHH domains (which are naturally "designed" to functionally bind antigens without the presence of and interaction with a light chain variable domain) can function as single, relatively small, functional antigen-binding structural units, domains, or polypeptides. Unlike the VH and VL domains of conventional four-chain antibodies, VHH domains are generally not practically usable alone as antigen-binding proteins or single variable domains of immunoglobulins, and must be provided as antigen-binding units that function in some way in combination (e.g., conventional antibody fragments such as Fab fragments, or scFvs in which the VH and VL domains are covalently linked).

[0069] Due to these unique properties, the use of VHH domains, either alone or as part of a larger polypeptide, offers a number of major advantages over the use of conventional VH and VL domains, scFvs or conventional antibody fragments (such as Fab- or F(ab')2 fragments): only a single domain is needed to bind the antigen with high affinity and selectivity, there is no need for two separate domains to be present, nor is there a need to ensure that these two domains are in the correct spatial conformation and arrangement (i.e. through the use of a specifically designed linker, as in scFvs). VHH domains can be expressed from a single gene and do not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats; VHH domains are highly soluble and do not tend to aggregate; VHH domains are highly stable against heat, pH, proteases and other denaturing agents or conditions, and therefore can be prepared, stored and transported without refrigeration, resulting in cost, time and environmental savings; VHH domains can be easily and relatively cheaply prepared, even on the scale required for production; VHH domains are relatively small (approximately 15 kDa, 10 times smaller than conventional IgG) compared to conventional four-chain antibodies and their antigen-binding fragments, which allows for better tissue penetration and administration in higher doses compared to conventional four-chain antibodies and their antigen-binding fragments; VHH domains can exhibit so-called cavity binding (especially due to the extended CDR3 loop compared to conventional VH domains), and therefore can access targets and epitopes that are inaccessible to conventional four-chain antibodies and their antigen-binding fragments.

[0070] Methods for obtaining VHH domains that bind specific antigens or epitopes have been previously described, for example, in R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., Affar Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17, 2009 June; and WO 94 / 04678.

[0071] Camelid-derived VHH domains can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with amino acid residues present at the corresponding positions in a VH domain from a conventional human four-chain antibody. (Herein also referred to as "sequence optimization", which in addition to humanization can encompass additional modification of the sequence with one or more mutations that confer improved properties to the VHH, such as removing potential sites for post-translational modification). Humanized VHH domains can include one or more fully human framework region sequences. Humanization can be performed using methods of humanizing the surface amino acids of a protein (resurfacing) and / or using humanized universal framework CDR grafting methods (CDR grafting into universal frameworks).

[0072] As used herein, the term "epitope" or the interchangeably used term "antigenic determinant" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Antigenic determinants usually comprise chemically active surface groups of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. For example, epitopes usually comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which may be "linear epitopes" or "conformational" epitopes. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across protein amino acid residues that are separated from each other.

[0073] Many epitope mapping techniques well known in the art can be used to identify the epitope of a given antigen.See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66, GE Morris, Ed. (1996).For example, linear epitopes can be determined, for example, by the following method: synthesizing multiple peptides simultaneously on a solid support, where these peptides correspond to portions of protein molecules, and reacting these peptides with antibodies while still attached to the support.These techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc.Natl.Acad.Sci.USA 81: 3998-4002; Geysen et al. (1986) Molec.Immunol.23:709-715. Similarly, conformational epitopes can be identified by determining the spatial distribution of amino acids by, e.g., x-ray crystallography or 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols, supra).

[0074] Conventional techniques known to those skilled in the art can be used to screen antibodies for competitive binding to the same epitope.For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to antigen.A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Application WO03 / 48731.Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and their antigen-binding fragments that compete with the antibody molecule of the present invention for binding to the same epitope on CD8.

[0075] In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (such as the immunoglobulin single variable domain of the present invention) molecule can bind. The specificity of an antigen-binding protein can be determined based on its affinity and / or avidity. The affinity (KD), expressed as the equilibrium constant for the dissociation of an antigen with an antigen-binding protein, is a measure of the binding strength between an epitope on the antigen-binding protein and an antigen-binding site. That is, the smaller the value of KD, the stronger the binding strength of the epitope with the antigen-binding protein (alternatively, affinity may be expressed as the affinity constant (KA), which is 1 / KD). As will be clear to the skilled artisan, affinity can be determined in a manner known per se depending on the specific antigen of interest. Avidity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptide comprising it) and the antigen in question. Avidity is related to both the affinity between an epitope on an antigen-binding molecule and its antigen-binding site and the number of suitable binding sites present on the antigen-binding protein.

[0076] As used herein, the term "CD8 binding protein (CD8 binding polypeptide)" refers to any protein capable of binding to CD8α protein. CD8 binding proteins can include antibodies against CD8α, such as those defined herein. CD8 binding proteins also cover immunoglobulin superfamily antibodies (IgSF) and CDR-grafted molecules. An exemplary amino acid sequence of CD8α is shown in SEQ ID NO:1.

[0077] A "CD8 binding protein" of the invention may comprise at least one immunoglobulin single variable domain, such as a VHH, that binds to CD8. In some embodiments, a "CD8 binding molecule" of the invention may comprise two, three, four or more immunoglobulin single variable domains, such as a VHH, that bind to CD8. A CD8 binding protein of the invention, other than an immunoglobulin single variable domain that binds to CD8, may comprise a linker and / or a moiety with effector function, such as a half-life extending moiety (such as an immunoglobulin single variable domain that binds serum albumin) and / or a fused ligand (such as serum albumin) and / or a conjugated polymer (such as PEG) and / or an Fc region. In some embodiments, a "CD8 binding protein" of the invention further contemplates bispecific antibodies comprising immunoglobulin single variable domains that bind different antigens.

[0078] In general, the CD8 binding proteins of the present invention have a binding affinity to the antigen (i.e., CD8 protein) of at least 100% of the antigen, preferably at least 100% of the antigen, as measured by Biacore or KinExA or Fortibio assays. -7 ~10 -10 mol / L (M), more preferably 10 -8 ~10 -10 mol / L, more preferably 10 -9 ~10 -10 with a dissociation constant (KD) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , more preferably at least 10 10 M -1 It binds with an association constant (KA) of 10 -4KD values ​​greater than M are generally considered to indicate non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined by any suitable method known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays described herein (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays).

[0079] Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code, as is commonly known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion or substitution of the indicated number of amino acid residues at the position of the reference sequence compared to the second sequence. In the case of a substitution, such a substitution is preferably an amino acid substitution, which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure and has little or essentially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, and conservative amino acid substitutions are preferably those in which one amino acid within the following groups (i) to (v) is replaced with 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. (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: 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.

[0080] "Sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the sequences. "Sequence similarity" refers to the percentage of identical or conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, the identity of amino acid sequences is usually measured using sequence analysis software. For example, the identity can be determined using the BLAST program of the NCBI database. For example, the identity of sequences can be determined by the following methods. Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, DW, eds., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds, M Stockton Press, New York, 1991.

[0081] A polypeptide or nucleic acid molecule is considered to be "substantially isolated" if it has been isolated from at least one other component that is normally associated with it in the natural biological source or culture medium (culture medium) from which the polypeptide or nucleic acid molecule is obtained (such as other proteins / polypeptides, other nucleic acids, other biological components or macromolecules, or at least one contaminant, impurity or trace component) compared to the source and / or culture medium (culture medium). In particular, a polypeptide or nucleic acid molecule is considered to be "substantially isolated" if it has been purified at least twice, in particular at least 10 times, more in particular at least 100 times, and up to 1000 times or more. A "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous, as determined by a suitable technique (e.g., a suitable chromatographic technique such as polyacrylamide gel electrophoresis).

[0082] As used herein, the term "subject" means mammals, particularly primates, especially humans.

[0083] CD8-binding polypeptides of the present invention The present invention provides a CD8 binding polypeptide comprising at least one immunoglobulin single variable domain capable of specifically binding to CD8. In some embodiments, the CD8 binding polypeptide is isolated. In some embodiments, the CD8 binding polypeptide specifically binds to CD8α.

[0084] In some embodiments, at least one immunoglobulin single variable domain comprises the CDR1, CDR2 and CDR3 of a VHH as set forth in any one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77 and 81. The CDRs may be Kabat, AbM, Chothia or Contact CDRs. In some embodiments, the CDRs are Kabat CDRs. In some embodiments, at least one immunoglobulin single variable domain comprises a CDR1, a CDR2 and a CDR3 selected from: (1) CDR1 of SEQ ID NO:2, CDR2 of SEQ ID NO:3, and CDR3 of SEQ ID NO:4; (2) CDR1 of SEQ ID NO:6, CDR2 of SEQ ID NO:7, and CDR3 of SEQ ID NO:8; (3) CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12; (4) CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; (5) CDR1 of SEQ ID NO: 18, CDR2 of SEQ ID NO: 19, and CDR3 of SEQ ID NO: 20; (6) CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24; (7) CDR1 of SEQ ID NO: 26, CDR2 of SEQ ID NO: 27, and CDR3 of SEQ ID NO: 28; (8) CDR1 of SEQ ID NO: 30, CDR2 of SEQ ID NO: 31, and CDR3 of SEQ ID NO: 32; (9) CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35 and CDR3 of SEQ ID NO: 36 CDR3 of; (10) CDR1 of SEQ ID NO: 38, CDR2 of SEQ ID NO: 39, and CDR3 of SEQ ID NO: 40; (11) CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; (12) CDR1 of SEQ ID NO: 46, CDR2 of SEQ ID NO: 47, and CDR3 of SEQ ID NO: 48; (13) CDR1 of SEQ ID NO: 50, CDR2 of SEQ ID NO: 51, and CDR3 of SEQ ID NO: 52; (14) CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; (15) CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO: 60; (16) CDR1 of SEQ ID NO: 62, CDR2 of SEQ ID NO: 63, and CDR3 of SEQ ID NO: 64; (17) CDR1 of SEQ ID NO: 66, CDR2 of SEQ ID NO: 67, and CDR3 of SEQ ID NO: 68; (18) CDR1 of SEQ ID NO: 70, CDR2 of SEQ ID NO: 71, and CDR3 of SEQ ID NO: 72; (19) CDR1 of SEQ ID NO: 74, CDR2 of SEQ ID NO: 75, and CDR3 of SEQ ID NO: 76; (20) CDR1 of SEQ ID NO: 78, CDR2 of SEQ ID NO: 79, and CDR3 of SEQ ID NO: 80.

[0085] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence that is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% identical to the amino acid sequence of one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, and 81. In some embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of one of SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, and 81.

[0086] In some embodiments, the immunoglobulin single variable domain is a VHH. In some embodiments, the immunoglobulin single variable domain is humanized.

[0087] Nucleic acids, vectors and host cells In another aspect, the present invention relates to a nucleic acid molecule encoding a CD8 binding polypeptide of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. The nucleic acid molecule encoding the CD8 binding polypeptide of the present invention can be selected by the skilled artisan according to need or by conventional means. In some embodiments, the nucleic acid molecule encoding the CD8 binding polypeptide of the present invention comprises a nucleotide sequence selected from SEQ ID NOs: 82-91.

[0088] The nucleic acids of the invention may also be in the form of a vector, present in a vector and / or be part of a vector such as a plasmid, cosmid, YAC, etc. The vector may in particular be an expression vector, i.e. a vector that provides for expression of a CD8 binding polypeptide in vitro and / or in vivo (i.e. in a suitable host cell, host organism, and / or expression system). An expression vector typically comprises at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (e.g. promoters, enhancers, terminators, etc.). The selection of elements and their sequences for expression in a particular host is within the knowledge of the skilled artisan. Specific examples of control elements and other elements useful or necessary for expression of a CD8 binding polypeptide of the invention include, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0089] The nucleic acids of the present invention may be prepared or obtained in a known manner (e.g., by automated DNA synthesis and / or recombinant DNA techniques) based on the information on the amino acid sequences of the polypeptides of the present invention disclosed herein, and / or may be isolated from suitable natural sources.

[0090] In another aspect, the invention comprises a host cell which expresses or is capable of expressing one or more of the CD8 binding polypeptides of the invention and / or which comprises a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial, fungal or mammalian cells.

[0091] Suitable bacterial cells include cells of gram-negative bacterial strains (eg, Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (eg, Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0092] Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0093] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0094] However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins can also be used in the present invention.

[0095] The present invention further provides a method for producing a CD8 binding polypeptide of the present invention, said method comprising the steps of: - culturing a host cell of the invention under conditions allowing expression of a CD8 binding polypeptide of the invention; - recovering from the culture the CD8 binding polypeptide expressed by the host cell; and - optionally further purifying and / or modifying the CD8 binding polypeptide of the invention.

[0096] The CD8 binding polypeptides of the invention may be produced intracellularly (e.g., in the cytoplasm, periplasm, or in inclusion bodies) as described above and then isolated from the host cell and optionally further purified; or may be produced extracellularly (e.g., in the medium in which the host cell is cultured) and then isolated from the medium and optionally further purified.

[0097] Methods and reagents for recombinant production of polypeptides are known in the art, e.g., certain suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques suitable for producing the CD8 binding polypeptides of the invention are well known to those of skill in the art.

[0098] However, the CD8 binding polypeptides of the present invention can also be obtained by other methods for producing proteins known in the art, such as chemical synthesis, including solid phase synthesis or liquid phase synthesis.

[0099] Conjugate molecules In another aspect, the present invention provides a conjugate molecule comprising a CD8 binding polypeptide of the present invention and at least one detectable label attached to said CD8 binding polypeptide.

[0100] Detectable labels include, but are not limited to, radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and the like.

[0101] Fluorescent agents that can be used for conjugation include, but are not limited to, fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorexamine. Chemiluminescent agents that can be used for conjugation include, but are not limited to, luminol, isoraminol, aromatic acridinium esters, imidazoles, acridine salts, and oxalates. Bioluminescent agents that can be used for conjugation include, but are not limited to, luciferin, luciferase, jellyfish photoprotein, and the like. Paramagnetic ions that can be used for conjugation include, but are not limited to, the following: 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 radiopaque substances such as dam, diatrizoate, etiolidin, gallium citrate, iocarmic acid, locetamic acid, iodoamide, odipamide, lodoxamic acid, iopromide, iohexol, iopamidol, iopanoic acid, ioprosem acid, iosephalic acid, ioceric acid, iocarmic acid, iocephalic ... Acid), iotasur, iothalamic acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, dionosyl and hydroxytalos. Enzymes that can be used for conjugation include, but are not limited to, horseradish peroxidase.

[0102] Preferably, the detectable label is a radionuclide. Radionuclides that can be used for conjugation include, but are not limited to, radionuclides having energies between 20 KeV and 4000 KeV. 110 In, 111 In, 177 Lu, 18F, 52 Fe, 62 Cu, 64 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, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 In some embodiments, the detectable label can be: Sr or other gamma-, beta- or positive emitters. 68 Ga or 125 I.

[0103] Methods for binding detectable labels to polypeptides are well known to those of skill in the art. For example, in some embodiments, CD8 binding polypeptides can be conjugated to detectable labels via chelators.

[0104] The CD8-binding polypeptide of the present invention 68To label with a radionuclide such as Ga, the CD8 binding polypeptide of the invention must be reacted with a reagent that has a long tail with multiple integration groups attached for binding to ions. Such tails include, for example, polylysine, polysaccharides, or other polymers with derivatized or derivatizable side chains that can be attached to chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA, TETA, NETA, porphyrins, polyamines, crown ethers, bisthiosemicarbazones, polyoximes, and similar groups that are useful for this purpose. Chelators can be attached to the antibody by standard chemical methods. In some embodiments, the detectable label is conjugated to the CD8 binding polypeptide of the invention via a chelator. The chelating agents used are DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, and MAG. 3 , MAG 2 ,HYNIC, DADT, E.C., N.S. 3 , H2d ed pa, HBED, DFO, PEPA or HEHA, and derivatives thereof.

[0105] In some specific embodiments, the detectable label is 68 Ga and the chelating agent is NOTA.

[0106] In another aspect, the present invention provides a method for producing a composition comprising: 68 A method for preparing a conjugate molecule of the invention labeled with a radionuclide, such as Ga, is provided, comprising: 1) conjugating a CD8 binding polypeptide of the invention with a chelating agent to produce a conjugate of the CD8 binding polypeptide and the chelating agent; and 2) treating the product obtained in step 1). 68The CD8-binding polypeptide is contacted with a radionuclide such as Ga, and the CD8-binding polypeptide is reacted with the chelating agent to form a chelating agent. 68 Labeling with a radionuclide such as Ga. In some embodiments, the chelator is NOTA, and in step 1), the conjugate of the CD8 binding polypeptide and NOTA is generated by reacting the CD8 binding polypeptide with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA.

[0107] In another aspect, the present invention provides a method for producing a composition comprising: 125 A method for preparing a conjugate molecule of the invention labeled with I, comprising: 1) reacting a CD8-binding polypeptide of the invention in the presence of chloramine T; 125 I; and 2) terminating the reaction with sodium metabisulfite.

[0108] Detection and diagnostic applications In another aspect, the present invention provides a method for detecting the presence and / or amount of CD8 in a biological sample, the method comprising: a) contacting the biological sample and the control sample with a CD8 binding polypeptide of the invention or a conjugated molecule of the invention under conditions in which the CD8 binding polypeptide of the invention or the conjugated molecule of the invention can form a complex with CD8; and b) detecting the formation of a complex; wherein a difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of CD8 in the sample. In some embodiments, the biological sample is an ex vivo sample.

[0109] In another aspect, the present invention provides a detection agent for detecting CD8 positive cells, comprising the CD8 binding polypeptide of the present invention or the conjugated molecule of the present invention, and optionally a physiologically acceptable carrier. In some embodiments, the detection agent is used to detect the presence and / or amount of CD8 positive cells in a tissue of a subject. In some embodiments, the tissue is a tumor tissue. In some embodiments, the CD8 positive cells are CD8 positive T cells.

[0110] The CD8 binding polypeptide of the present invention or the conjugate molecule of the present invention is particularly suitable for in vivo imaging, for example, suitable for emission computed tomography (ECT). For example, the CD8 binding polypeptide of the present invention or the conjugate molecule of the present invention can be applied to single photon emission computed tomography (SPECT) and positron emission tomography (PET) by various labels. In diagnosis, ECT allows high-resolution imaging and quantitative analysis by images. In addition, SPECT imaging can include SPECT / CT imaging, and PET imaging can include PET / CT imaging, so that better imaging effect can be obtained.

[0111] Thus, in some embodiments, the detection agent is an imaging agent, hi some embodiments, the diagnostic agent is an ECT imaging agent, such as a SPECT imaging agent or a PET imaging agent.

[0112] In another aspect, the present invention provides the use of the CD8 binding polypeptide of the present invention or the conjugated molecule of the present invention in the preparation of a detection agent for detecting CD8 positive cells. In some embodiments, the detection agent is used to detect the presence and / or amount of CD8 positive cells in a tissue of a subject. In some embodiments, the tissue is a tumor tissue. In some embodiments, the CD8 positive cells are CD8 positive T cells. In some embodiments, the detection agent is an imaging agent. In some embodiments, the imaging agent is an ECT imaging agent, such as a SPECT imaging agent or a PET imaging agent.

[0113] In another aspect, the present invention provides a method for detecting the presence and / or amount of CD8 positive cells in a tissue, the method comprising: a) contacting the tissue with a conjugate molecule of the invention or a detection agent of the invention; and b) determining the presence and / or quantity of CD8 positive cells in the tissue.

[0114] In some embodiments, the tissue is blood tissue. In some embodiments, the tissue is lymphatic tissue. In some embodiments, the tissue is tumor tissue. In some embodiments, the CD8 positive cells are CD8 positive T cells. In some embodiments, the presence and / or amount of CD8 positive cells in the tissue is determined by imaging the tissue. In some embodiments, the presence and / or amount of CD8 positive cells in the tissue is determined by flow cytometry.

[0115] In another aspect, the present invention provides a method for detecting CD8 positive cells in a subject, comprising administering the conjugate molecule of the present invention or the detection agent of the present invention to the subject.In some embodiments, the method is used to detect the presence and / or amount of CD8 positive cells in the tissue of a subject.In some embodiments, the tissue is a tumor tissue.In some embodiments, the CD8 positive cells are CD8 positive T cells.

[0116] In some embodiments, the method further comprises imaging the subject, such as ECT imaging. In some embodiments, the ECT imaging is SPEC imaging. In some embodiments, the ECT imaging is PET imaging. SPECT and PET imaging techniques and equipment are well known in the art, and such known ECT imaging techniques and equipment may be used.

[0117] In another aspect, the present invention provides a method for determining whether a tumor-bearing subject is suitable for anti-tumor therapy, the method comprising: 1) administering a conjugate molecule of the present invention or a detection agent of the present invention to a subject; and 2) imaging the subject, such as by ECT imaging, to determine whether the subject's tumor contains CD8 positive cells; Here, if the presence of CD8 positive cells in a tumor is detected, for example, if the subject's tumor is infiltrated by CD8 positive cells, the subject is identified as suitable for anti-tumor therapy.

[0118] In another aspect, the present invention provides a method for predicting the response of a tumor-bearing subject to an anti-tumor therapy, comprising: 1) administering a conjugate molecule of the present invention or a detection agent of the present invention to a subject; and 2) imaging the subject, such as by ECT imaging, to determine whether the subject's tumor contains CD8 positive cells; Here, if the presence of CD8 positive cells in a tumor is detected, for example, if the subject's tumor is infiltrated by CD8 positive cells, the subject may respond to an anti-tumor therapy.

[0119] The presence of CD8+ cells, such as CD8+ T cells, can be a predictive marker of antitumor therapy efficacy and a prognostic marker of survival. For example, baseline CD8+ cell tumor infiltration is a prognostic indicator of survival in breast, head and neck, and ovarian cancers. Additionally, CD8+ cell tumor infiltration detected during anti-PD-1 or anti-PDL-1 therapy is a predictive marker of treatment efficacy.

[0120] In another aspect, the present invention provides a method for treating a tumor in a subject, the method comprising: 1) administering a conjugate molecule of the present invention or a detection agent of the present invention to a subject; and 2) imaging the subject, such as by ECT imaging, to determine whether the subject's tumor contains CD8 positive cells; If the presence of CD8 positive cells in a tumor is detected, for example if the subject's tumor is infiltrated by said CD8 positive cells, the subject is administered an anti-tumor therapy.

[0121] In another aspect, the present invention provides a method for monitoring the effectiveness of an anti-tumor therapy in a subject, the method comprising: 1) administering a conjugate molecule of the present invention or a detection agent of the present invention to a subject having a tumor and being treated with an antitumor therapy; and 2) Imaging the subject, such as by ECT imaging, to determine the amount of CD8 positive cells in the subject's tumor.

[0122] In some embodiments of the various aspects of the invention, the anti-tumor therapy is an immune checkpoint inhibitor therapy.In some embodiments, the anti-tumor therapy comprises administering a PD-1 inhibitor (e.g., REGN2810, BGB-A317, Nivolumab, Pirizumab, and Pamuzumab), a PD-L1 inhibitor (e.g., Atuzumab, Avelumab, and Duvalimab), a CTLA-4 inhibitor (e.g., Ipilimumab), 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 (e.g., CD-28, 2B4, LY108, LAIR1, ICOS, CD160 or VISTA antibodies), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists, Ang2 inhibitors (Neva monoclonal antibody), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., eroticinib and cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3)), Carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1 and CA19-9, vaccines (e.g. BCG vaccine, cancer vaccines), adjuvants to enhance antigen presentation (e.g. granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g. CD3×CD20 bispecific antibody and PSMA×CD3 bispecific antibody), cytotoxic agents, chemotherapeutic agents (e.g. Dacarbazine, Temozolomide, Cyclophosphamide, Docetaxel, Donomycin, Cisplatin, Carboplatin, Gemcitabine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel and Vincristine), Cyclophosphamide, Radiation therapy, IL-6R inhibitors (e.g. Sarelumab), IL-4R inhibitors (e.g. Dupirumab), IL-10 inhibitors, Cytokines (e.g. IL-2, IL-7, IL-21 and IL-15), Antibody Drug Conjugates (ADC) (e.g. Anti-CD19-DM4 ADC and Anti-DS6-DM4 ADC).

[0123] In some embodiments of the various aspects of the invention, the tumor is a solid tumor, including, but not limited to, 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, renal cancer, gastric cancer, esophageal cancer, head and neck cancer, salivary gland cancer, and myeloma.

[0124] Isolation and purification of CD8 positive cells In another embodiment, the present invention further provides a method for isolating and / or purifying CD8 positive cells comprising: (a) providing a cell population suspected of containing CD8 positive cells; (b) identifying a subpopulation of the cell population, wherein cells of the subpopulation bind to a CD8 binding polypeptide of the invention or a conjugated molecule of the invention; and (c) isolating a subpopulation.

[0125] In some embodiments, the CD8 positive cells are CD8 positive T cells. In some embodiments, the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMCs).

[0126] In some embodiments, CD8 positive cells can be isolated through flow cytometry. In another aspect, the present invention further provides a method for isolating CD8 positive cells comprising: (a) providing a cell population suspected of containing CD8 positive cells; (b) contacting the cell population with a CD8 binding polypeptide of the invention or a conjugated molecule of the invention, whereby CD8 positive cells bind to the CD8 binding polypeptide of the invention or the conjugated molecule of the invention; (c) removing cells that do not bind to the CD8 binding polypeptide of the invention or the conjugated molecule of the invention; and (d) recovering the CD8 positive cells bound to the CD8 binding polypeptide of the present invention or the conjugate molecule of the present invention.

[0127] In some embodiments, the CD8 positive cells are CD8 positive T cells. In some embodiments, the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMCs).

[0128] In some embodiments, the CD8 binding polypeptide of the invention or the conjugated molecule of the invention is immobilized on a solid surface, for example on the surface of a gel or a magnetic bead.

[0129] kit In another embodiment, the present invention provides a kit comprising a CD8 binding polypeptide of the present invention or a conjugated molecule of the present invention or a detection agent of the present invention. The kit is used to carry out the method of the present invention. Typically, the kit includes a tag indicating the expected use of the contents. The term "tag" includes any written or recorded material that is written on or provided with the kit or is otherwise provided with the kit. EXAMPLES

[0130] The present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the described examples.

[0131] Example 1 Screening of heavy chain single domain antibodies against CD8α The amino acid sequence of the CD8α-Fc fusion protein is as follows: JPEG0007688941000002.jpg48170

[0132] Amino acids 1 to 21 are the signal peptide of the CD8α receptor, amino acids 22 to 136 are the immunoglobulin (Ig) domain of the CD8α receptor, amino acids 137 to 181 are the Ig domain of the CD8α receptor, amino acids 182 to 209 are the transmembrane domain of the CD8α receptor, and amino acids 210 to 235 are the cytoplasmic domain of the CD8α receptor.

[0133] In the figure, the region marked with a single line is the Fc region, and the region marked with two lines is the linker.

[0134] 1.1 Building the library

[0135] CD8α-Fc fusion protein for immunization was expressed in CHO cells and purified by protein A affinity chromatography. Alpacas were selected and immunized. After immunization, lymphocytes were extracted from alpaca peripheral blood, RNA was extracted therefrom according to the Trizol instructions, and the RNA concentration was measured by Nanodrop. The extracted RNA was reverse transcribed into cDNA using the SuperScript® III First-Strand Synthesis System for RT-PCR kit according to the instructions. Nucleic acid fragments encoding the variable regions of heavy chain antibodies were amplified by nested PCR.

[0136] The VHH fragments were purified using a DNA product purification kit, and the vector and fragments were digested with the restriction enzyme sifi at 50°C overnight. The digested fragments were excised and collected, cloned into the vector pComb3XSS, and phage display was performed. The products were electrotransformed into E. coli electrocompetent cells TG1 to construct and validate a heavy chain single domain antibody phage display library against CD8α. The volume of the library was 5.04 × 10 9 To detect the insertion rate of the library, 48 clones were randomly selected and identified, and the insertion rate was found to be 100% and the size was consistent.

[0137] 1.2 Panning of heavy chain single domain antibodies against CD8α

[0138] The plate was coated with 10 μg / well of CD8α-cHis fusion protein and incubated overnight at 4° C. The next day, the plate was blocked with 3% BSA for 2 hours at 37° C., washed five times with PBST (PBS containing 0.05% Tween 20), and then incubated with 100 μL of phages (1.71 × 10 11 cfu, derived from the camel heavy chain single domain antibody phage display library constructed in 1.1) were added and allowed to react at 37°C for 1 hour. The plate was then washed 10 times with PBST (PBS, 0.05% Tween 20) solution, and then 10 times with PBS solution to wash away unbound phages. Then, 100μL of Gly-Hcl (PH=2.5) was added to each well and allowed to react at 37°C for 6-8 minutes, dissociating the phages that specifically bind to CD8α and transferring them to a sterile centrifuge tube, and the buffer was neutralized by quickly adding 1 / 10 volume of Tris-Hcl (PH=9.0) to the sterile centrifuge tube. 10μL of the neutralized solution was taken for gradient dilution, the titer was measured, and the panning recovery rate was calculated. The neutralized phages were infected with logarithmic growth phase Escherichia coil TG1 to produce and purify the phages for the next screening. The same screening was repeated several times, changing the panning conditions each time. Therefore, to achieve the goal of screening CD8-specific antibodies from the antibody library using phage display technology, positive clones were enriched. The affinity elution conditions are shown in the table below.

[0139] Table 1. Affinity panning conditions JPEG0007688941000003.jpg26170

[0140] 1.3 Screening for specific single positive clones using phage enzyme-linked immunosorbent assay (ELISA)

[0141] After 3-4 rounds of panning, 96 clones were randomly selected from the plate used for the last panning and identified. 96 single colonies were randomly picked up and cultured to produce and purify phages. The plate was coated with CD8α-Fc fusion protein overnight at 4°C, then blocked with 3% BSA and reacted at 37°C for 1 hour. Then, the phage supernatant obtained after infection was added to each well (blank phage was used as the control) and incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-labeled anti-M13 secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour. After washing, tetramethylbenzidine (TMB) coloring solution was added and the absorbance at 450 nm was read. Meanwhile, the plate was coated with Fc protein overnight at 4°C, then blocked with 3% BSA and reacted at 37°C for 1 hour. After washing, the phage supernatant obtained after infection (blank phage was used as a control) was added and reacted at 37°C for 1 hour. After washing, hydrogen peroxide-labeled anti-M13 secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added and reacted at room temperature for 1 hour. Then, TMB color development solution was added and the absorbance value at 450 nm was read. If the ratio of the OD value divided by the OD value of the blank control is 4 or more, the candidate antibody is judged to be able to bind to the CD8α-Fc protein. On the other hand, since the ratio of the CD8α-Fc binding OD value of the above antibody capable of binding to the CD8α-Fc antigen protein divided by the Fc protein binding OD value is 5 or more, it is considered to be a candidate that can specifically bind to the CD8α portion rather than the Fc portion. As a result, it was found that some of the screened antibodies can specifically bind to CD8α but not Fc. The clone candidate bacteria were transferred to LB liquid culture medium containing 100 μg / mL ampicillin, and plasmid extraction and base sequence determination were performed.

[0142] Table 2. Identification results of CD8α-Fc tag antigen by phage ELISA JPEG0007688941000004.jpg54170JPEG0007688941000005.jpg50170

[0143] Table 3. Identification of Fc-tagged antigens by phage ELISA JPEG0007688941000006.jpg54170JPEG0007688941000007.jpg64170

[0144] The protein sequence of each clone was analyzed according to the sequence alignment software DNAMAN. Clones with the same sequences of CDR1, CDR2, and CDR3 were all considered to be the same antibody, and clones with different CDR sequences were considered to be different antibodies. Finally, 20 candidate sequences were obtained.

[0145] NO.2:C2, NO.20:C20, NO.24:C24, NO.27:C27, NO.29:C29, NO.30:C30, NO.37:C37, NO.42:C42, NO.45:C45, NO.46:C46, NO.53:S5, NO.54:S6, NO.55:S7, NO.61:S13, NO.66:S18, NO.70:S22, NO.71:S23, NO.77:S29, NO.32:S80, NO.36:S84.

[0146] Table 4. Amino acid sequences of 20 single domain antibody candidates JPEG0007688941000008.jpg247170JPEG0007688941000009.jpg224170JPEG0007688941000010.jpg198170Note: The underlined parts are the CDR regions of the antibody sequence.

[0147] Example 2 Preliminary evaluation and identification of heavy chain single domain antibodies against CD8α 2.1 Expression and purification of heavy chain single domain antibodies in the host bacterium Escherichia coli

[0148] The coding sequence of the candidate single domain antibody obtained by sequence analysis in Example 1 was subcloned into the expression vector PET22b, and the recombinant plasmid with the correctly specified sequence was transformed into the expression host strain BL21, and the plasmid was inoculated on a plate of LB solid medium containing 100 μg / ml ampicillin at 37 ° C overnight. A single colony was selected and inoculated and cultured overnight. The following day, the strain that had been left overnight was transferred and amplified. The strain was cultured with shaking at 37 ° C until the OD value reached about 0.6, then induced with 0.5 mM IPTG and cultured with shaking at 28 ° C overnight. The following day, the strain was collected by centrifugation and disrupted by ultrasonication to obtain a crude antibody extract. The antibody protein was then purified by nickel ion affinity chromatography. Finally, an antibody protein with a purity of more than 90% was obtained.

[0149] 2.2 Preparation of CD8α antibody protein using mammalian cells

[0150] Primers were designed for the nucleotide sequences of the screened CD8α single domain antibodies, and the nucleotide sequences of each antibody (including histidine tags) were amplified by PCR using the above-mentioned prokaryotic expression plasmids as templates, and cloned into pCDNA4 (Invitrogen, Cat V86220) vector. The correctness of the sequences of the obtained target clone genes was determined by gene sequencing. Nucleotide sequence of C2-cHis antibody (SEQ ID NO:82): JPEG0007688941000011.jpg56170

[0151] Nucleotide sequence of C24-cHis antibody (SEQ ID NO:83): JPEG0007688941000012.jpg56170

[0152] Nucleotide sequence of C27-cHis antibody (SEQ ID NO:84): JPEG0007688941000013.jpg55170

[0153] Nucleotide sequence of C29-cHis antibody (SEQ ID NO:85): JPEG0007688941000014.jpg56170

[0154] Nucleotide sequence of C37-cHis antibody (SEQ ID NO:86): JPEG0007688941000015.jpg56170

[0155] Nucleotide sequence of C42-cHis antibody (SEQ ID NO:87): JPEG0007688941000016.jpg55170

[0156] Nucleotide sequence of C46-cHis antibody (SEQ ID NO:88): JPEG0007688941000017.jpg57170

[0157] Nucleotide sequence of S5-cHis antibody (SEQ ID NO:89): JPEG0007688941000018.jpg58170

[0158] Nucleotide sequence of S13-cHis antibody (SEQ ID NO:90): JPEG0007688941000019.jpg55170

[0159] Nucleotide sequence of S36-cHis antibody (SEQ ID NO:91): JPEG0007688941000020.jpg54170

[0160] The above-mentioned single domain antibody plasmid with a histidine tag constructed by recombinantly was transfected into HEK293 cells to express the antibody. The recombinant expression plasmid was diluted with Freestyle293 culture medium, and a polyethyleneimine (PEI) solution required for transformation was added. The mixture of the plasmid and PEI was added to the HEK293 cell suspension, and incubated at 37°C and 10% CO. 2After culturing for 5 to 6 days, the transient expression culture supernatant was collected, and the histidine-tagged single domain antibody fusion protein was purified by Ni+ resin gel affinity chromatography.

[0161] 2.3 ELISA activity detection of different anti-CD8α single domain antibodies against human CD8α protein

[0162] CD8 α-Fc protein was transiently expressed in HEK293 and purified by protein A affinity chromatography. The obtained CD8α-Fc protein was plate-coated at a concentration of 0.5 μg / well overnight at 4°C, and then a gradient dilution series of the obtained anti-CD8α 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, a color development solution was added and the absorbance at 450 nm was read. Data processing and plot analysis were performed using the software SotfMaxPro v5.4. CD8α binding curves and EC50 values ​​of the antibodies (EC50 values ​​of all tested antibodies were 0.989 ng / mL to 4.11 ng / mL) were obtained by four-parameter fitting. Among the 10 selected antibodies (see Table 5 for details), the relative activity of different anti-CD8α single domain antibodies was compared using C2-cHis as a control to reflect the affinity of the antibodies to CD8α. Among the 10 antibodies tested, C37, C42, C46, ​​S5, and S36 have relatively high binding activity.

[0163] Table 5. ELISA results of 10 antibodies for CD8α binding JPEG0007688941000021.jpg59170

[0164] Example 3 In-vitro activity analysis of CD8α single domain antibody 3.1 Examination of the binding activity of anti-CD8α single domain antibodies to cell surface CD8α by FACS

[0165] We constructed a mouse colon cancer MC38 cell line stably transfected with the human CD8α full-length protein gene, and obtained MC38 cells (MC38-CD8α cells) that stably express human CD8α protein on the cell membrane. Before use, the cells were cultured until they reached 90% or more convergence. Flow cytometry was performed on these cells using an anti-CD8α single domain antibody, and indirect immunofluorescence staining was quantitatively analyzed to determine the amount of receptor on each cell surface.

[0166] Harvest the cells and culture 2.5 x 10 6 The cells were suspended at a concentration of 1000 cells / mL. 200 μL of cell sample was mixed with 20 μL of primary antibody, and the resulting mixture was incubated on ice for 30 min. The cells were centrifuged, washed, and resuspended, after which 5 μL of secondary antibody-PE conjugate was added, and the above mixture was incubated on ice for 30 min. The cells were washed twice, resuspended, and flow cytometry was performed on a BD FACSCelesta™. At least 5 × 10 4 Events were collected. All analyses were monochromatic, with PE detection in FL1. Forward scatter (FS) and side scatter (SS) data demonstrated that all cell populations were tightly clustered.

[0167] CD8α expressing cells were evaluated in vitro by flow cytometry (Table 6). The positive rates of the 20 single domain antibodies in MC38-CD8α cell binding were all 93.6% or higher. Of the 20 single domains, the positive rates of six single domain antibodies, C2, C37, C42, C46, ​​S5, and S36, were all 99.5% or higher, and the positive control huOKT8 (huOKT8 is an expressed and purified protein based on the sequence of US20160024209A1) was 93.0%.

[0168] Table 6. Binding efficiency of antibodies to cell surface CD8α as determined by FACS JPEG0007688941000022.jpg101170

[0169] 3.2 Binding of anti-CD8α single domain antibodies to human PBMC CD8 + T cell surface antigens

[0170] Human peripheral blood was separated with magnetic beads to obtain human CD8+ T cells, and the binding of anti-CD8α single domain antibody C37-his to CD8+ T cells was analyzed using anti-CD8α PE conjugate (R&D Systems, Cat # FAB1509P) as a positive control.

[0171] The concentration of the isolated cells was 2.5 × 10 6 The concentration of cells was adjusted to 1000 cells / mL. 150 μL of cell sample was mixed with 3 μg of primary antibody, and the resulting mixture was incubated on ice for 30 min. After washing and resuspending the cells, 5 μL of secondary anti-PE conjugate was added, and the mixture was incubated on ice for 30 min in the dark. The cells were washed twice, resuspended, and flow cytometry was performed on a BD FACSCelesta™. At least 5 × 10 4 events were collected. All analyses were monochromatic, with PE detection in FL1. Forward scatter (FS) and side scatter (SS) data demonstrate that all cell populations are tightly clustered.

[0172] Flow cytometry was used to evaluate CD8α expression on T cells (Figure 1). The CD8α single domain antibody C37-chis has good binding affinity to the human PBMC CD8+ T cell surface antigen.

[0173] 3.3 Identification of the binding ability of anti-CD8α single domain antibodies to CD8α (ForteBio method)

[0174] Anti-CD8α single domain antibody-biontin was immobilized on the SA biosensor. Then, CD8α-chis at concentrations ranging from 6.25 to 100 nM was allowed to bind to the nanoantibody and dissociate. The binding kinetic parameters of the four antibodies C37, C42, C46, ​​and S5 were evaluated and determined using Octet Data Analysis version 9.0, including Kon, Koff, and Kd.

[0175] The measured binding affinity of the anti-CD8α antibody is shown in Table 7. As a result, the C37-his protein has a high affinity for the CD8α target protein, and its relatively high Ka value and relatively low Kd value indicate that this antibody fusion protein can bind to the CD8α antigen quickly and the CD8α antigen is not easily dissociated.

[0176] Table 7. Binding affinity of antibodies to CD8α JPEG0007688941000023.jpg30170

[0177] Example 4 125 In-vitro analysis of I-labeled CD8α single domain antibodies 4.1 Labeled CD8α single domain antibody 125 I

[0178] For ease of explanation, the single domain antibody C37-cHis was named SNA006a, the single domain antibody C42-cHis was named SNA006b, the single domain antibody C46-cHis was named SNA006c, and the single domain antibody S5-cHis was named SNA006d. 125 I labeling experiments were carried out using the chloramine T method.

[0179] A 10 mg / mL chloramine T solution and a sodium metabisulfite solution were prepared using 0.05 mol / L, pH=7.5 PBS as the solvent. 2 μL of protein was diluted in 50 μL of PB solution, and 1.5 mCi (10 μL) of Na metabisulfite was added to the 50 μL solution. 125I was added and mixed, and chloramine T solution was added to the mixed solution and mixed quickly and uniformly, and the above materials were reacted at room temperature for 5 minutes. 50 μL of sodium metabisulfite solution was added to the above solution obtained after the reaction and mixed to terminate the reaction. The labeling rate was confirmed by a Radio-TLC scanner. Unreacted 125 I was removed by a PD-10 column, during which the product buffer system was changed to normal saline. The labeling results are shown in Figure 2.

[0180] 4.2 125 Cell-binding assay of I-labeled CD8α single domain antibodies to MC38-CD8 cells

[0181] MC38-CD8 cells (CD8 positive) and MC38 cells (negative control) were plated in four 6-well plates at 1 × 10 6 Plate cells / well, 3 mL medium / well, and incubate at 37°C, 5% CO 2 The cells were cultured overnight under 5% CO. The cells were removed and incubated at 4°C for 30 minutes. All 6-well plates were removed and each experimental group was divided into a total binding group and a non-specific binding group. Three parallel samples were prepared for each group, so one 6-well plate corresponded to one experiment. 26 μM of culture medium not labeled with protein was added to three wells of culture medium to serve as a non-specific adsorption control sample. The culture medium was 125 The corresponding ( 125 The culture medium was then completely replaced with I-labeled SNA006a, b, c, d). The cells were incubated at 37°C and 5% CO 2 After incubation, the supernatant was collected, and the cells were washed with pre-chilled PBS containing 1% BSA and combined with the supernatant. The remaining cell pellet was resuspended in PBS containing SDS, and the radioactive cpm values ​​of the supernatant and pelleted cells were measured using a γ counter, and decay correction was performed.

[0182] The precipitation count is the cell-bound portion of the bound group (B), and the sum of the radioactivity counts in the supernatant and precipitate is the total radioactivity (T). The B / T of this group minus the corresponding nonspecific binding is the 10% of this group, as shown in Figure 3. 6 is the fraction of radioimmunoreactivity bound to each cell (calculated as a %).

[0183] As shown in Figure 3, 125 I-labeled CD8α single domain antibodies bind highly to MC38-CD8 cells and fail to bind to the corresponding CD8-negative cells. 125 The binding of I-labeled CD8α single domain antibody to MC38-CD8 cells could be completely blocked by unlabeled CD8 single domain antibody. 125 This demonstrated the specific binding ability of the I-labeled CD8 single domain antibody to MC38-CD8 cells.

[0184] On the other hand, the calculation shows that 10 MC38-CD8 cells 6 For individuals 125 The binding rate of I-SNA006a was approximately 50%. 125 The minimum binding rate of I-SNA006d was also ≥10%. 125 I-labeled SNA006a, b, c, and d all showed high binding affinity, demonstrating that SNA006-a and b have higher affinity for CD8 than SNA006-c and d.

[0185] 4.3 125 Saturation binding assay of I-labeled CD8α single domain antibodies

[0186] Cell preparation: Saturation tests were performed in 96-well filter plates. MC38-CD8 cells were cultured in 50 mL culture dishes. Before the test, the culture medium was removed, the cells were washed twice with 0.01 M sterile PBS, digested with trypsin, washed twice again with PBS, and finally prepared into a cell suspension with the addition of 5% BSA. Approximately 10 5 The cells were added at a concentration of 100 x 100 cells / well.

[0187] Preparation of cold antibody solution for blocking specific binding: 10 mg / mL CD8 single domain antibody solution (dissolved in 0.01M PBS) was diluted 10-fold with 5% BSA to give a 1 mg / mL solution.

[0188] Receptor binding reaction: Radioligand saturation method was used. Total binding group and non-specific binding group were included. Total binding group had 12 doses of increasing concentration, non-specific binding group had 12 doses, each dose had 4 parallel samples, total reaction volume was 200μL, corresponding to 1000-fold excess of cold antibody.

[0189] The cells were incubated at 4°C for 2 h. Then, the supernatant was extracted and washed six times with PBS pre-cooled at 4°C, and then 200 μL of PBS was added to each well each time. Finally, the filter membrane was collected, and the radioactivity counts of each tube of samples and standards were measured by a gamma counter. Finally, saturation curve fitting was performed according to the single-point binding model using Prism 7.0 (GraphPad Software, Inc.) software to calculate the KD and Bmax.

[0190] MC38-CD8 cells and 125 The saturation curves of the radioligand binding analysis with the I-CD8 single domain antibody are shown in FIG.

[0191] 125 The measured binding affinities of the I-CD8 single domain antibodies are shown in Table 8. 125 Binding of I-labeled CD8 single domain antibodies to MC38-CD8 cells can be blocked by unlabeled CD8 single domain antibodies, 125 The specific binding ability of I-labeled CD8 single domain antibody to MC38-CD8 cells was demonstrated. 125 The KD value of I-SNA006a was 0.46 nM. 125 I-SNA006cK D The value is 3.8 nM. 125 I-SNA006dK D The value is 1.5 nM. 125 The affinity of I-SNA006a is 125The results show that the CD8 single domain antibody has significant affinity for the CD8 target protein, especially I-SNA006c and d. 125 I-SNA006a was found to have the strongest affinity.

[0192] Table 8. Binding affinity of CD8 single domain antibodies to CD8 JPEG0007688941000024.jpg22170

[0193] 4.4 125 Competitive binding assay of I-labeled CD8α single domain antibody

[0194] Cell preparation: Saturation tests were performed in 96-well filter plates. MC38-CD8 cells were cultured in 50 mL culture dishes. Before the test, the culture medium was removed, the cells were washed twice with 0.01 M sterile PBS, digested with trypsin, washed twice again with PBS, and finally prepared into a cell suspension with the addition of 5% BSA. Approximately 10 5 The cells were added at a concentration of 100 x 100 cells / well. The 96-well cell aspiration filter plate was saturated with binding buffer and allowed to dry in air prior to use. Approximately 10 5 After addition of cells and increasing concentrations of CD8 single domain antibodies, 125 I-CD8 single domain antibody was added at approximately 2 × 10 5 was added to each well at a rate of counts / min, and the reaction volume was adjusted to 200 μL with binding buffer.

[0195] After reacting for 2 hours at 4°C, the reaction solution in the suction filter plate was discharged using a vacuum pump and an auxiliary device for the suction filter plate, and 200 μL of PBS buffer (0.01 M, pH = 7.4) was added to each well and discharged, and the cells were washed six times. After drying the filter membrane at the bottom of the suction filter plate, the filter membrane at the bottom was collected and measured using a gamma counter. Calculations and plots were performed using Prism 7.0 (GraphPad Software, Inc.), and the half-life inhibitory concentration IC50 was calculated by nonlinear regression analysis. Four parallel samples were set for each experimental point, and the experiment was repeated twice. The results obtained in this experiment were the average value plus the standard deviation.

[0196] As shown in Figure 5, 125 The binding of I-SNA006a to the MC38-CD8 cell receptor was found to be inhibited by the SNA006a "cold" protein in a concentration-dependent manner. The IC50 value obtained by Prism 7.0 (GraphPad Software, Inc.) software fitting was approximately 3.0 nM. The competitive binding essays described herein demonstrate the high affinity and high specificity of SNA006a for the CD8 antigen.

[0197] Example 5 125 I-labeled CD8α single domain antibody and its SPECT / CT imaging 5.1 125 Animal models for studying I-labeled CD8α single domain antibodies

[0198] Female nude mice aged 6–8 weeks were used for in-vivo studies. Nude mice were raised in a specific pathogen-free (SPF) environment, fed ad libitum, and exposed to a standard 12-h light-dark cycle. Xenografts were performed by subcutaneously implanting 100 μL of cells (MC38-CD8 or MC38) / PBS into the right front leg of nude mice. The cell inoculation density was approximately 5–6 × 10 6 Implantation was performed under isoflurane anesthesia. Under these conditions, more than 90% of the injected animals developed adequate tumors (100-300 mm) after 1-2 weeks. 3) (MC38-CD8 or MC38) was obtained.

[0199] After the experiment, tumor tissue was collected and subjected to CD8 immunohistochemical staining to analyze the CD8 expression levels, which were compared with the CD8 expression levels in the entire tumor detected by PET / CT.

[0200] 5.2 125 In-vivo SPECT imaging using I-labeled CD8α single domain antibody

[0201] The MC38-CD8+ / - tumor model was inoculated according to the method in 5.1. Nude mice were anesthetized with isoflurane, then placed on the SPECT / CT bed, and approximately 10 μg of radiolabeled single domain antibody (approximately 100 μCi / 10 μg) was administered via the tail vein. SPECT scans were performed at 1 h and 2 h, respectively, and the radioactive uptake of tumors and tissue organs at multiple time points was analyzed. The results are shown in Figure 6.

[0202] 125 Within 120 minutes of injection of the I-CD8 single domain antibody, the MC38-CD8 implanted tumors were clearly visible. As shown in Figure 6, the right MC38-CD8+ tumor showed good contrast compared to the contralateral negative control tumor MC38-CD8-. 125 The I-CD8 single domain antibody was significantly concentrated in the kidney, indicating that it is primarily metabolized there. The above iodine labeling method inevitably leads to deiodination, which may result in radioactive material being concentrated in the thyroid and stomach as a result of SPECT. Furthermore, 125 The energy of I is weak and may cause self-absorption in certain tissues when applied to SPECT imaging. 125 The application of I-labeled CD8 single domain antibodies to SPECT imaging was 68 The aim of the study was to screen for the best candidate antibodies for use in Ga-labeled imaging agents. 125It was found that I-labeled SNA006a, SNA006c, and SNA006d showed high uptake at tumor sites, and the imaging effect of SNA006a was significantly better than that of SNA006c and SNA006d. The results of the SPECT in-vivo screening experiments were consistent with the corresponding in-vitro experiments, further proving that SNA006a has the strongest CD8 tracing ability among all the aforementioned single domain antibodies.

[0203] Example 6 Synthesis of NOTA-CD8α single domain antibody precursor and 68 Radiolabeling of Ga-NOTA-CD8α single domain antibody 6.1 Synthesis of NOTA-CD8α single domain antibody precursor

[0204] The bifunctional chelator p-SCN-NOTA was conjugated to a CD8 single domain antibody. The coupling product was purified on a PD-10 column and the buffer was replaced with normal saline. The absorbance at 280 nm was measured by UV spectrophotometer and the coupling efficiency was analyzed by ESI-Q-TOF-MS. The targeting of the coupling product to tumor cells was analyzed by in-vitro cell assay (tumor cell binding and affinity detection) and the in-vitro affinity to CD8α protein was analyzed by ELISA.

[0205] Using a PD-10 column (GE), the CD8α-cHis single domain antibody (including C37, C46, ​​and S5) was dissolved in a buffer of the indicated pH with NaHCO 3 The buffer solution was changed to p-SCN-Bn-NOTA (Macrocyclics, product no. B-605) and dissolved in DMSO at a concentration of 25 mg / mL. A solution of p-SCN-Bn-NOTA with twice the moles of lysine on the single domain antibody was added to the CD8α single domain antibody, and the above materials were reacted at room temperature for a certain period of time. The conjugated product was purified on a PD-10 column (GE) and concentrated by ultrafiltration.

[0206] The activity of the above CD8α-NOTA after labeling was measured by ELISA. Specifically, CD8α-Fc fusion protein was added to the coated plate at a concentration of 5 μg / well and left to stand overnight at 4°C. After blocking with 3% BSA at 37°C, gradient diluted samples (unbound CD8α single domain antibody was the standard substance) were added and reacted at 37°C for 1 hour. Then, anti-his-HRP (purchased from Abcam Company) was added and reacted at room temperature for 1 hour. Then, a color developing solution was added and the absorbance value at 450 nm was read. The ELISA activity is summarized in Table 9.

[0207] Table 9. Summary of ELISA activity measurement results for CD8α-cHis-NOTA JPEG0007688941000025.jpg21170

[0208] 6.2 68 Radioisotope analysis of Ga-NOTA-CD8α single domain antibody

[0209] 68 Ga solution ( 68 Germanium 68 / gallium 68 (Ge 68 / Ga 68) leacheate was prepared by eluting an Eckert & Ziegler IGG100 germanium 68 / gallium 68 (Ge 68 / Ga 68) generator with 0.1 M sterile HCl and adding an equal amount of 0.2 M sodium acetate solution. 1 / 4 volume of 0.1 M sodium acetate buffer solution containing the above NOTA-CD8 antibody and pH 5.3 was added to adjust the pH of the reaction system to 4.5-4.7, and the above material was reacted at room temperature for 10 min. The unreacted ionic gallium was removed with a PD-10 column while the buffer system of the product was exchanged with normal saline, and filtered with a 0.22 μm filter membrane. Quality control was performed by performing analyses such as pH, Radio-TLC, Radio-HPLC, radioactivity, and radiochemical purity. In addition, the ITG germanium-68 / gallium-68 (Ge-68 / Ga-68) generator was leached with 0.05 M sterile hydrochloric acid, and 1 / 2 the amount of 0.2 M sodium acetate solution was added. The other reaction conditions and quality control conditions were the same. 68 A Ga leachate was also prepared.

[0210] 6.3 Instant Thin Layer Chromatography (ITLC) Analysis

[0211] The ITLC-SG was cut into strips measuring 1 cm x 12 cm in advance, and the distance between the ends of the strips was labeled with a pencil. The developer was poured into the developing tank until the tank was covered, and the developer was balanced. 68 The Ga-CD8 single domain antibody injection solution was dropped onto the pencil line 1 cm from the bottom of the ITLC strip. The ITLC strip was placed into the developing chamber and developed 10 cm from the sampling point (to the top pencil mark). The LTIC was scanned with a synchrotron ITLC scanner and the radiochemical purity (RCP) was calculated by integrating the peaks on the chromatogram. The analysis results are shown in Figure 7. The CD8 single domain antibody was positron emission tomography (PET) spectroscopy. 68 It can be seen that labeling with Ga was successful.

[0212] Example 7 68 Biodistribution of Ga-NOTA-CD8α single domain antibody 7.1 68 Animal model for studying Ga-NOTA-CD8α single domain antibodies

[0213] Female nude mice aged 6–8 weeks were used for in-vivo studies. Nude mice were raised in a specific pathogen-free (SPF) environment, fed ad libitum, and exposed to a standard 12-h light-dark cycle. Xenografts were performed by subcutaneously implanting 100 μL of cells (MC38-CD8 or MC38) / PBS into the right front leg of nude mice. The cell inoculation density was approximately 5–6 × 10 6 Implantation was performed under isoflurane anesthesia. Under these conditions, more than 90% of the injected animals developed adequate tumors (100-300 mm) after 1-2 weeks. 3 ) (MC38-CD8 or MC38) was obtained.

[0214] After the experiment, tumor tissue was collected and subjected to CD8 immunohistochemical staining to analyze the CD8 expression levels, which were compared with the CD8 expression levels in the entire tumor detected by PET / CT.

[0215] 7.2 68 Biodistribution of Ga-NOTA-CD8α single domain antibody

[0216] MC38-CD8+ / - tumor model was inoculated according to the method in 7.1, and approximately 10 μg (approximately 100 μCi / 10 μg) of radiolabeled single domain antibody was administered to nude mice via the tail vein. Data were collected 1 and 1.5 hours later, and three nude 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 excised and counted in a gamma counter to collect data. The injected volume was used as the total injected volume. For each organ, the % injected volume (%ID) was calculated from the total injected volume, and the organs were weighed to calculate the % injected volume per gram (%ID / g).

[0217] FIG. 8. MC38-CD8 tumor-bearing intact male mice. 68 We present biodistribution data of the Ga-NOTA-CD8α single domain antibody following in vivo administration in intact male mice bearing subcutaneous tumors. 68 Ex-vivo biodistribution data (n=3) from Ga-NOTA-CD8 ingestion. Data were collected 1 and 1.5 hours after intravenous administration. Data are presented as mean %ID / g ± standard deviation (SD). Percentage error is calculated as the geometric mean of the standard deviations. Error bars represent the standard deviation for this group, as shown in Figure 8.

[0218] Figure 9 shows the uptake ratios of tumor tissue to blood and tumor tissue to contralateral normal muscle in these experiments. CD8 single domain antibodies show good tumor infiltration in MC38-CD8 tumors expressing the target. The tumor to muscle ratio peaked at a high level. 68 The Ga-NOTA-SNA006a single domain antibody shows high tumor specificity. 68 Ga-NOTA-SNA006a may be an effective agent for tumor imaging.

[0219] Example 8 68In-vivo PET imaging with Ga-NOTA-CD8α single domain antibody

[0220] MC38-CD8+ / - tumor models were inoculated according to the method in 6.1. MicroPET / CT (IRIS PET / CT, Inviscan, Strasbourg, France) scans were used to measure focal %ID / g. Sequential PET / CT scans of small animals were performed for 120 min to analyze radioactivity uptake in tumor, muscle, and kidney at multiple time points as shown in Figure 10. A 3D OSEM algorithm based on Monte Carlo method was used for image reconstruction. Radioactivity (MBq / mL) was calculated by the region of interest method (ROI) in tumor, muscle, liver, and other organs. The obtained values ​​were divided by the injected volume to obtain the uptake (%ID / g) of each tissue for the PET tracer (assuming a tissue density of 1 g / ml). The calculation results are shown in Figure 10.

[0221] The results in Figure 10 show that the PET tracer 68 At 120 min after Ga-NOTA-SNA006a injection, the MC38-CD8 transplanted tumors could be clearly identified, indicating that the CD8-negatively expressed MC38 transplanted tumors were not taken up after injection. Therefore, the MC38-CD8 tumors have good discrimination compared with the contralateral negative control tumors. The PET results showed that the radioactivity uptake at multiple time points in the tumor, muscle, and kidney was analyzed. The biodistribution ROI results were analyzed with activity-time curves as shown in Figure 11.

[0222] the result, 68 Ga-NOTA-SNA006a, 68 Ga-NOTA-SNA006c, 68 Ga-NOTA-SNA006d had high uptake at the tumor site. 68 The imaging effect of Ga-NOTA-SNA006a was found to be significantly better than that of c and d. The results of the in-vivo PET screening experiment were consistent with the corresponding in-vitro experimental results. 68This further demonstrated that Ga-NOTA-SNA006a had the strongest CD8 tracking ability among all the above single domain antibodies.

[0223] Example 9 68 In-vivo stability study of Ga-NOTA-CD8α single domain antibody

[0224] Normal mice were harvested and immunized via the tail vein. 68 Ga-NOTA-SNA006a (74 MBq, 1 mCi / 100 μL PBS) was administered. Urine samples were collected 0.5 and 1 hour after injection. All samples were dissolved in a certain amount of 50% acetonitrile and centrifuged at 8000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was analyzed by Radio-TLC.

[0225] The results of Radio-TLC are shown in Figure 12. 68 No loss of Ga was observed, indicating that the radiolabeled antibody is structurally stable under these conditions and has good stability in vivo.

Claims

1. comprising at least one immunoglobulin single variable domain capable of specifically binding to CD8α, wherein the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 selected from the following (1) to (20), and the immunoglobulin single variable domain is VHH, a CD8α-binding polypeptide: (1) CDR1 of SEQ ID NO: 26, CDR2 of SEQ ID NO: 27, and CDR3 of SEQ ID NO: 28; (2) CDR1 of SEQ ID NO: 30, CDR2 of SEQ ID NO: 31, and CDR3 of SEQ ID NO: 32; (3) CDR1 of SEQ ID NO: 38, CDR2 of SEQ ID NO: 39, and CDR3 of SEQ ID NO: 40; (4) CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; (5) CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 3, and CDR3 of SEQ ID NO: 4; (6) CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 8; (7) CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12; (8) CDR1 of SEQ ID NO: 14, CDR2 of SEQ ID NO: 15, and CDR3 of SEQ ID NO: 16; (9) CDR1 of SEQ ID NO: 18, CDR2 of SEQ ID NO: 19, and CDR3 of SEQ ID NO: 20; (10) CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24; (11) CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of SEQ ID NO: 36; (12) CDR1 of SEQ ID NO: 46, CDR2 of SEQ ID NO: 47, and CDR3 of SEQ ID NO: 48; (13) CDR1 of SEQ ID NO: 50, CDR2 of SEQ ID NO: 51, and CDR3 of SEQ ID NO: 52; (14) CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; (15) CDR1 of SEQ ID NO: 58, CDR2 of SEQ ID NO: 59, and CDR3 of SEQ ID NO: 60; (16) CDR1 of SEQ ID NO: 62, CDR2 of SEQ ID NO: 63, and CDR3 of SEQ ID NO: 64; (17) CDR1 of SEQ ID NO: 66, CDR2 of SEQ ID NO: 67, and CDR3 of SEQ ID NO: 68; (18) CDR1 of SEQ ID NO: 70, CDR2 of SEQ ID NO: 71, and CDR3 of SEQ ID NO: 72; (19) CDR1 of SEQ ID NO: 74, CDR2 of SEQ ID NO: 75, and CDR3 of SEQ ID NO: 76; (20) CDR1 of SEQ ID NO: 78, CDR2 of SEQ ID NO: 79, and CDR3 of SEQ ID NO:

80.

2. The CD8α-binding polypeptide according to claim 1, wherein the immunoglobulin single variable domain comprises an amino acid sequence having at least 90% sequence identity to one of the amino acid sequences of SEQ ID NOs: 29, 33, 41, 45, 5, 9, 13, 17, 21, 25, 37, 49, 53, 57, 61, 65, 69, 73, 77 and 81.

3. The CD8α-binding polypeptide according to claim 1 or 2, wherein the immunoglobulin single variable domain comprises one of the amino acid sequences of SEQ ID NOs: 29, 33, 41, 45, 5, 9, 13, 17, 21, 25, 37, 49, 53, 57, 61, 65, 69, 73, 77 and 81.

4. A nucleic acid molecule encoding the CD8α-binding polypeptide according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid molecule according to claim 4 operably linked to an expression control element.

6. A host cell capable of expressing a CD8α-binding polypeptide, comprising the nucleic acid molecule according to claim 4 or transformed by the expression vector according to claim 5.

7. A method for producing the CD8α-binding polypeptide according to any one of claims 1 to 3, comprising: a) culturing the host cell according to claim 6 under conditions allowing the expression of the CD8α-binding polypeptide; b) recovering the CD8α-binding polypeptide expressed by the host cell from the culture obtained in step a); and c) further purifying and / or modifying the CD8α-binding polypeptide obtained in step b).

8. A conjugate molecule comprising the CD8α-binding polypeptide according to any one of claims 1 to 3 and at least one detectable label bound to the CD8α-binding polypeptide.

9. The conjugate molecule according to claim 8, wherein the detectable label is selected from a radionuclide, a fluorescent agent, a chemiluminescent agent, a bioluminescent agent, a paramagnetic ion, and an enzyme.

10. Detectable labels are 110 In, 111 In, 177 Lu, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 68 Ge, 86 Y, 90 Y, 89 Zr, 120 I, 123 I, 124 I, 125 I, 131 I, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 55 Co, 72 As, 75 Br, 76 Br, 83 The conjugate molecule according to claim 9, selected from Sr or other γ-, β- or positive emitters.

11. The detectable label is 68 Ga or 125 I, and the conjugate molecule according to claim 9.

12. The conjugate molecule according to any one of claims 8 to 10, wherein the CD8α-binding polypeptide is bound to a detectable label via a chelating agent.

13. The chelating agent is selected from DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETTA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG 3 , MAG 2 , HYNI C, DADT, NS 3 , the conjugate molecule according to claim 12, selected from H2dedpa, HBED, DFO, PEPA or HEHA, and derivatives thereof.

14. The detectable label is 68 The conjugate molecule according to claim 13, wherein the detectable label is Ga and the chelating agent is NOTA.

15. A method for detecting the presence and / or amount of CD8α in a biological sample, comprising: a) contacting a biological sample and a control sample with a CD8α-binding polypeptide according to any one of claims 1 to 3 or a conjugate molecule according to any one of claims 8 to 14 under conditions such that the CD8α-binding polypeptide or the conjugate molecule can form a complex with CD8α; and b) detecting the formation of the complex; wherein a difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of CD8α in the sample. **Claim 16** A detection agent for detecting CD8α-positive cells, comprising a CD8α-binding polypeptide according to any one of claims 1 to 3 or a conjugate molecule according to any one of claims 8 to 14, and a physiologically acceptable carrier. **Claim 17** The detection agent according to claim 16, wherein the detection agent is a contrast agent. **Claim 18** The detection agent according to claim 17, wherein the contrast agent is an emission computed tomography (ECT) contrast agent. **Claim 19** The detection agent according to claim 18, wherein the contrast agent is a single photon emission computed tomography (SPECT) contrast agent or a positron emission tomography (PET) contrast agent. **Claim 20** Use of a CD8α-binding polypeptide according to any one of claims 1 to 3 or a conjugate molecule according to any one of claims 8 to 14 in the preparation of a detection agent for detecting CD8α-positive cells. **Claim 21** The use according to claim 20, wherein the detection agent is a contrast agent. **Claim 22** The use according to claim 21, wherein the contrast agent is an ECT contrast agent. **Claim 23** The use according to claim 22, wherein the contrast agent is an SPECT contrast agent or a PET contrast agent. **Claim 24** A conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19 for use in a method for detecting the presence and / or amount of CD8α-positive cells in a tissue, wherein the method comprises a) contacting the tissue with a conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19; and b) measuring the presence and / or amount of CD8α-positive cells in the tissue comprising the conjugate molecule or the detection agent. **Claim 25** The conjugate molecule or detection agent according to claim 24, wherein the tissue is selected from blood tissue, lymph tissue, and tumor tissue. **Claim 26** The conjugate molecule or detection agent according to claim 24 or 25, wherein the CD8α-positive cell is a CD8α-positive T cell.

27. The conjugate molecule or detection agent according to any one of claims 24 to 26, wherein the presence and / or amount of CD8α-positive cells in a tissue is determined by imaging the tissue.

28. The conjugate molecule or detection agent according to any one of claims 24 to 26, wherein the presence and / or amount of CD8α-positive cells in a tissue is determined by flow cytometry.

29. The conjugate molecule according to any one of claims 8 to 14 or the detection agent according to any one of claims 16 to 19, for use in a method of detecting the presence and / or amount of CD8α-positive cells in a subject's tissue, the method comprising administering the conjugate molecule according to any one of claims 8 to 14 or the detection agent according to any one of claims 16 to 19 to a subject.

30. The conjugate molecule or detection agent according to claim 29, wherein the tissue is a tumor tissue.

31. The conjugate molecule or detection agent according to claim 29 or 30, wherein the CD8α-positive cell is a CD8α-positive T cell.

32. The conjugate molecule or detection agent according to any one of claims 29 to 31, further comprising the step of imaging the subject by ECT imaging.

33. The conjugate molecule or detection agent according to claim 32, wherein the ECT imaging is SPECT imaging or PET imaging.

34. The conjugate molecule according to any one of claims 8 to 14 or the detection agent according to any one of claims 16 to 19 for use in a method of determining whether a subject having a tumor is suitable for an anti-tumor therapy, the method comprising: 1) administering the conjugate molecule according to any one of claims 8 to 14 or the detection agent according to any one of claims 16 to 19 to the subject; and 2) imaging the subject by ECT imaging to determine whether the subject's tumor contains CD8α-positive cells comprising, wherein when the presence of CD8α-positive cells in the tumor is detected, the subject is identified as being suitable for anti-tumor therapy. A conjugate molecule or detection agent.

35. A conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19 for use in a method for determining whether a subject having a tumor is suitable for an anti-tumor therapy, wherein the method comprises: 1) administering to the subject a conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19; and 2) imaging the subject by ECT imaging to determine whether the subject's tumor contains CD8α-positive cells. Including: Here, when the presence of CD8α-positive cells in the tumor is detected, the subject may respond to the anti-tumor therapy, conjugate molecule or detection agent.

36. A conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19 for use in a method for treating a tumor in a subject, wherein the method comprises: 1) administering to the subject a conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19; and 2) imaging the subject by ECT imaging to determine whether the subject's tumor contains CD8α-positive cells. Including: Here, when the presence of CD8α-positive cells in the tumor is detected, an anti-tumor therapy is applied to the subject, conjugate molecule or detection agent.

37. A conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19 for use in a method for monitoring the effectiveness of an anti-tumor therapy in a subject, wherein the method comprises: 1) administering to the subject having a tumor and being treated with an anti-tumor therapy a conjugate molecule according to any one of claims 8 to 14 or a detection agent according to any one of claims 16 to 19; and 2) imaging the subject by ECT imaging to determine the amount of CD8α-positive cells in the subject's tumor. A conjugate molecule or detection agent comprising:

38. A conjugate molecule or detection agent according to any one of claims 34 to 37, wherein the anti-tumor therapy is an immune checkpoint inhibitor therapy.

39. The anti-tumor therapy is radiotherapy or selected from the 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, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor β (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a vaccine, an adjuvant that increases antigen presentation, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, a cytokine, and an antibody-drug conjugate (ADC), the conjugate molecule or detection agent according to any one of claims 34 to 38.

40. The conjugate molecule or detection agent according to any one of claims 34 to 39, wherein the tumor is a solid tumor.

41. The conjugate molecule or detection agent according to claim 40, wherein the solid tumor is selected from colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain tumor, 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 multiple myeloma.

42. A method for isolating and / or purifying CD8α-positive cells, the method comprising: (a) providing a cell population suspected of containing CD8α-positive cells; (b) identifying a subpopulation of the cell population, wherein the cells of the subpopulation bind to the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14; and (c) isolating the subpopulation.

43. A method for isolating CD8α-positive cells, the method comprising: (a) providing a cell population suspected of containing CD8α-positive cells; (b) contacting the cell population with the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14, thereby enabling the CD8α-positive cells to bind to the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14; removing cells that do not bind to the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14; and recovering CD8α-positive cells that bind to the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14. **Claim 44** The method according to claim 42 or 43, wherein the CD8α-positive cells are CD8α-positive T cells. **Claim 45** The method according to any one of claims 42 to 44, wherein the cell population suspected of containing CD8α-positive cells is human peripheral blood mononuclear cells (PBMC). **Claim 46** The method according to any one of claims 42 to 45, wherein the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14 is immobilized on a solid surface. **Claim 47** The method according to any one of claims 42 to 45, wherein the CD8α-binding polypeptide according to any one of claims 1 to 3 or the conjugate molecule according to any one of claims 8 to 14 is immobilized on the surface of a gel or magnetic beads. **Claim 48** A kit comprising the CD8α-binding polypeptide according to any one of claims 1 to 3, the conjugate molecule according to any one of claims 8 to 14, or the detection agent according to any one of claims 16 to 19.

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