WO2022017370A1 - Cd8结合多肽及其用途 - Google Patents

Cd8结合多肽及其用途 Download PDF

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WO2022017370A1
WO2022017370A1 PCT/CN2021/107312 CN2021107312W WO2022017370A1 WO 2022017370 A1 WO2022017370 A1 WO 2022017370A1 CN 2021107312 W CN2021107312 W CN 2021107312W WO 2022017370 A1 WO2022017370 A1 WO 2022017370A1
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seq
tumor
positive cells
subject
cancer
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PCT/CN2021/107312
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English (en)
French (fr)
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须涛
孙艳
王超
齐浩
杜海菁
杨艳玲
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苏州智核生物医药科技有限公司
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Priority to EP21845245.6A priority Critical patent/EP4186925A1/en
Priority to CN202180058970.5A priority patent/CN116723857A/zh
Priority to JP2023504174A priority patent/JP2023535708A/ja
Publication of WO2022017370A1 publication Critical patent/WO2022017370A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
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    • A61K51/1078—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody the antibody being against an immunoglobulin, i.e. being an (anti)-anti-idiotypic antibody
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    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • G01N2333/70517—CD8

Definitions

  • the present invention relates to the field of biomedicine.
  • the present invention relates to a specific CD8 binding polypeptide and uses thereof.
  • ECT Emission Computed Tomography
  • SPECT Single-Photon Emission Computed Tomography
  • PET Positron Emission Tomography
  • CD8 is mainly expressed on cytotoxic T lymphocytes, but also on dendritic cells, natural killer cells, etc.
  • the CD8 molecule can be a homodimer formed by CD8 ⁇ , or a heterodimer formed by CD8 ⁇ and CD8 ⁇ , with ⁇ heterodimer being more prevalent.
  • TILs tumor-infiltrating lymphocytes
  • Embodiment 1 A CD8 binding polypeptide comprising at least one immunoglobulin single variable domain capable of specifically binding CD8 ⁇ , the at least one immunoglobulin single variable domain comprising SEQ ID NOs: 5, 9, CDR1 , CDR2 and CDR3 in any of 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81.
  • Embodiment 2 The CD8-binding polypeptide of embodiment 1, wherein the at least one immunoglobulin single variable domain comprises CDRl, CDR2, and CDR3 selected from the group consisting of:
  • Embodiment 3 The CD8-binding polypeptide of embodiment 1 or 2, wherein the immunoglobulin single variable domain comprises a combination with SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41 , 45, 49, 53, 57, 61, 65, 69, 73, 77 and 81 have an amino acid sequence of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% Sequence identity of amino acid sequences.
  • Embodiment 4 The CD8 binding polypeptide of any one of embodiments 1-3, wherein the immunoglobulin single variable domain comprises SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, The amino acid sequence of one of 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, and 81.
  • Embodiment 5 The CD8-binding polypeptide of any one of embodiments 1-4, wherein the immunoglobulin single variable domain is a VHH.
  • Embodiment 6 A nucleic acid molecule encoding the CD8 binding polypeptide of any one of embodiments 1-5.
  • Embodiment 7 An expression vector comprising the nucleic acid molecule of embodiment 6 operably linked to an expression control element.
  • Embodiment 8 A host cell comprising the nucleic acid molecule of embodiment 6 or transformed with the expression vector of embodiment 7 and capable of expressing the CD8 binding polypeptide.
  • Embodiment 9 A method of producing the CD8-binding polypeptide of any of embodiments 1-5, comprising:
  • step b) recovering the CD8-binding polypeptide expressed by the host cell from the culture obtained in step a);
  • step b) optionally further purifying and/or modifying the CD8 binding polypeptide from step b).
  • Embodiment 10 A conjugate molecule comprising the CD8-binding polypeptide of any one of embodiments 1-5, and at least one detectable label conjugated to the CD8-binding polypeptide.
  • Embodiment 11 The conjugated molecule of Embodiment 10, wherein the detectable label is selected from the group consisting of radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.
  • Embodiment 12 Embodiment 11 Embodiment conjugated molecule, wherein the detectable label is selected from 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, 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 Sr or other ⁇ -, ⁇ -, or positron emitters, for example, the detectable label is 68 Ga or 125I .
  • Embodiment 13 The conjugated molecule of any one of embodiments 10-12, wherein the CD8-binding polypeptide is conjugated to the detectable label via a chelating agent.
  • Embodiment 14 The conjugated molecule of Embodiment 13, wherein the chelating agent is selected from the group consisting of DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar , MAG 3 , MAG 2 , HYNIC, DADT, EC, NS 3 , H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof.
  • the chelating agent is selected from the group consisting of DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar , MAG 3 , MAG 2 , HYNIC, DADT, EC, NS 3 , H2dedpa, HBED,
  • Embodiment 15 Embodiment 14 Embodiment conjugated molecule, wherein said detectable label is 68 Ga and the chelator is NOTA.
  • Embodiment 16 A method of detecting the presence and/or amount of CD8 in a biological sample, comprising:
  • the difference in complex formation between the biological sample and the control sample is indicative of the presence and/or amount of CD8 in the sample.
  • Embodiment 17 A detection agent for detecting CD8 positive cells comprising the CD8 binding polypeptide of any one of embodiments 1-5 or the conjugated molecule of any one of embodiments 10-15, and optionally a physiologically acceptable carrier.
  • Embodiment 18 The detection agent of Embodiment 17, which is a contrast agent.
  • Embodiment 19 The detection agent of Embodiment 18, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • an ECT contrast agent such as a SPECT contrast agent or a PET contrast agent.
  • Embodiment 20 Use of the CD8 binding polypeptide of any one of embodiments 1-5 or the conjugated molecule of any one of embodiments 10-15 in the manufacture of a detection agent for detecting CD8 positive cells.
  • Embodiment 21 The detection agent of Embodiment 20, which is a contrast agent.
  • Embodiment 22 The detection agent of Embodiment 21, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • an ECT contrast agent such as a SPECT contrast agent or a PET contrast agent.
  • Embodiment 23 A method for detecting the presence and/or amount of CD8 positive cells in a tissue, comprising
  • Embodiment 24 The method of Embodiment 23, wherein the tissue is selected from the group consisting of blood tissue, lymphoid tissue, and tumor tissue.
  • Embodiment 25 The method of embodiment 23 or 24, wherein the CD8 positive cells are CD8 positive T cells.
  • Embodiment 26 The method of any of embodiments 23-25, wherein the presence and/or amount of CD8 positive cells in the tissue is determined by imaging the tissue.
  • Embodiment 27 The method of any of embodiments 23-25, wherein the presence and/or amount of CD8 positive cells in the tissue is determined by flow cytometry.
  • Embodiment 28 A method for detecting the presence and/or amount of CD8 positive cells in a tissue in a subject comprising administering to the subject the conjugated molecule of any one of Embodiments 10-15 or Embodiment 17- The detection agent of any one of 19 contacts.
  • Embodiment 29 The method of embodiment 28, wherein the tissue is tumor tissue.
  • Embodiment 30 The method of embodiment 28 or 29, wherein the CD8 positive cells are CD8 positive T cells.
  • Embodiment 31 The method of any of Embodiments 28-30, wherein the method further comprises the step of imaging the subject, eg, ECT imaging, eg, the ECT imaging is SPECT imaging or PET imaging.
  • Embodiment 32 A method for determining whether a subject with a tumor is suitable for anti-tumor therapy, the method comprising
  • the subject is identified as being suitable for anti-tumor therapy if the presence of CD8 positive cells in the tumor is detected, eg, the subject's tumor is infiltrated by CD8 positive cells.
  • Embodiment 33 A method for predicting the response of a subject with a tumor to an anti-tumor therapy, the method comprising
  • the individual is likely to respond to anti-tumor therapy.
  • Embodiment 34 A method of treating a tumor in a subject, the method comprising:
  • the subject is administered an anti-tumor therapy if the presence of CD8 positive cells in the tumor is detected, eg, the subject's tumor is infiltrated by CD8 positive cells.
  • Embodiment 35 A method for monitoring the efficacy of an anti-tumor therapy in a subject, the method comprising
  • Embodiment 36 The method of any one of Embodiments 32-35, wherein the anti-tumor therapy is immune checkpoint inhibitor therapy.
  • Embodiment 37 The method of any one of Embodiments 32-36, wherein the anti-tumor therapy is selected from the group consisting of administering a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, TIGIT Inhibitors, CD47 inhibitors, GITR inhibitors, LAG3 inhibitors, antagonists of another T cell co-inhibitor or ligand, indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth Factor (VEGF) antagonists, Ang2 inhibitors, transforming growth factor beta (TGF ⁇ ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, CD20 inhibitors, antibodies against tumor-specific antigens, vaccines, agents that increase antigen presentation Adjuvants, bispecific antibodies, cytotoxins, chemotherapeutics, cyclophosphamide, radiotherapy, IL-6R inhibitors, IL-4R
  • Embodiment 38 The method of any one of Embodiments 32-37, wherein the tumor is a solid tumor.
  • Embodiment 39 The method of embodiment 38, wherein the solid tumor is selected from the group consisting of colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, Pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.
  • the solid tumor is selected from the group consisting of colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, Pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.
  • Embodiment 40 A method for isolating and/or purifying CD8 positive cells, the method comprising:
  • Embodiment 41 A method for isolating CD8 positive cells, the method comprising:
  • Embodiment 42 The method of embodiment 40 or 41, wherein the CD8 positive cells are CD8 positive T cells.
  • Embodiment 43 The method of any one of Embodiments 40-42, wherein the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Embodiment 44 The method of any of embodiments 40-44, wherein the CD8-binding polypeptide of any of embodiments 1-5 or the conjugated molecule of any of embodiments 10-15 is immobilized on a solid surface, eg Immobilized on gel or magnetic beads.
  • Embodiment 45 A kit comprising the CD8 binding polypeptide of any one of embodiments 1-5 or the conjugated molecule of any one of embodiments 10-15 or the detection agent of any one of embodiments 17-19.
  • Figure 1 FACS to examine the binding effect of candidate antibodies on the surface antigen of human PBMC CD8+ T cells.
  • Figure 8 In vivo distribution data of 68 Ga-labeled CD8 ⁇ single domain antibody.
  • Fig. 9 In vivo distribution target ratio of 68 Ga-labeled CD8 ⁇ single domain antibody.
  • Figure 10 In vivo PET/CT imaging of 68 Ga-labeled CD8 ⁇ single domain antibody.
  • Fig. 11 In vivo time biodistribution curve of 68 Ga-labeled CD8 ⁇ single domain antibody.
  • sequence as used herein (eg, in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) should be generally understood To include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding said sequence, unless a more limited interpretation is required herein.
  • domain refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • domains are responsible for individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or the domain.
  • immunoglobulin domain refers to a spherical region of an antibody chain (eg, a chain of a conventional 4-chain antibody or a chain of a heavy-chain antibody), or to a polypeptide consisting essentially of such spherical regions.
  • An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristics of an antibody molecule.
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer antigen specificity to antibodies by having an antigen-binding site.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an epitope without pairing with other immunoglobulin variable domains.
  • An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody”, eg an immunoglobulin single variable domain VH and VL (VH and VL domains).
  • Another example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") of Camelidae as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, are antigen-binding immunizations known as “heavy chain antibodies” (ie, "antibodies lacking light chains")
  • VHH domains also known as heavy chain single domain antibodies, VHHs, VHH domains, VHH antibody fragments, and VHH antibodies.
  • VHH antibodies are antigen-binding immunizations known as “heavy chain antibodies” (ie, "antibodies lacking light chains")
  • Variable domains of globulins Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 ( 1993)).
  • VHH domain is used to relate the variable domains to the heavy chain variable domains (which are referred to herein as "VH domains”) present in conventional 4-chain antibodies and to those present in conventional 4-chain antibodies
  • VH domains heavy chain variable domains
  • VL domains Light chain variable domains
  • the VHH domain binds specifically to an epitope without the need for additional antigen binding domains (in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain).
  • VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain.
  • VHH domain 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” are used interchangeably.
  • the amino acid residues used for the VHH domains of Camelidae can be based on the VH domains given by Kabat et al. Numbering by the general numbering method (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e. according to One or more positions of the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering).
  • Kabat numbering i.e. according to One or more positions of the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering.
  • the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • CDRs may include "extended CDRs” such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 in VL ( LCDR3); 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.
  • extended CDRs such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 in VL ( LCDR3)
  • HCDR1 50-65 or 49-65
  • HCDR3 93-102
  • HCDR3 94-102 or 95-102
  • the total number of amino acid residues in a VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domains and polypeptides containing them can be summarized as follows:
  • VHH domains (which have been naturally "designed” to functionally bind to antigen in the absence and interaction of light chain variable domains) can serve a single and relatively small function Antigen-binding structural units, domains or polypeptides. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suitable for practical application as single antigen binding proteins or immunoglobulin single variable domains, but require some
  • the form or another form is combined to provide a functional antigen binding unit (eg, in the form of conventional antibody fragments such as Fab fragments; or in the form of scFvs consisting of VH domains covalently linked to VL domains).
  • VHH domains alone or as part of larger polypeptides—provides many advantages over the use of conventional VH and VL domains, scFvs, or conventional antibody fragments (eg, Fab- or F(ab')2-fragments)
  • VHH domains e.g., Fab- or F(ab')2-fragments
  • 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 (formatting); VHH structure Domains are highly soluble and have no tendency to aggregate; VHH domains are highly stable to heat, pH, proteases, and other denaturing agents or conditions, and can therefore be prepared, stored or shipped without refrigeration equipment, resulting in cost, time, and environmental savings VHH domains are easy and relatively inexpensive to prepare, even at the scale required for production; VHH domains are relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (approximately 15 kDa or 1/1/2 the size of conventional IgG) 10), and therefore show higher tissue permeability and can be administered in higher doses than conventional 4-chain antibodies and their antigen-binding fragments; VHH domains may exhibit so-called cavity-binding properties (especially due to the longer CDR3 loops), thereby reaching targets and
  • VHH domains derived from the family Camelidae can be modified by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in the VH domain of a conventional 4-chain antibody in humans.
  • Humanization also referred to herein as “sequence optimization", in addition to humanization, “sequence optimization” may also encompass other modifications of the sequence by one or more mutations that provide VHH improving properties, such as removal of potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be accomplished using methods of protein surface amino acid resurfacing (resurfacing) and/or humanized universal framework CDR grafting (CDR grafting to a universal framework).
  • epitopes typically contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear” "Epitope” or "Conformational” Epitope. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed.
  • Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • linear epitopes can be determined by, for example, synthesizing simultaneously a large number of peptides on a solid support, where the peptides correspond to portions of a protein molecule, and allowing these peptides to interact with the support while still attached to the support. Antibody response.
  • These techniques are known in the art and are described, for example, in US Pat. No. 4,708,871; Geysen et al (1984) Proc. Natl. Acad. Sci.
  • conformational epitopes can be identified by determining the spatial conformation of amino acids, such as by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See eg Epitope Mapping Protocols (supra).
  • Antibodies can be screened competitively for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in International Patent Application WO03/48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the invention for binding to the same epitope on CD8 can be obtained using conventional techniques known to those skilled in the art.
  • the term "specificity” refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (eg, an immunoglobulin single variable domain of the invention) can bind.
  • the specificity of an antigen binding protein can be determined based on its affinity and/or avidity.
  • the affinity expressed by the dissociation equilibrium constant (KD) of the antigen and the antigen-binding protein, is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the greater the affinity between the epitope and the antigen-binding protein.
  • KD dissociation equilibrium constant
  • affinity can also be expressed as an association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner depending on the particular antigen of interest.
  • Affinity is a measure of the strength of binding between an antigen-binding protein (eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing the same) and a relevant antigen. Affinity is related to both: the affinity between its antigen-binding site on an antigen-binding protein, and the number of associated binding sites present on the antigen-binding protein.
  • CD8 binding protein means any protein capable of specifically binding CD8 ⁇ protein.
  • a CD8 binding protein may include an antibody against CD8 ⁇ , eg an antibody as defined herein.
  • CD8 binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafting molecules.
  • IgSF immunoglobulin superfamily antibodies
  • An exemplary amino acid sequence of CD8 ⁇ is set forth in SEQ ID NO:1.
  • a “CD8 binding protein” of the invention may comprise at least one CD8 binding immunoglobulin single variable domain such as a VHH.
  • "CD8 binding molecules" of the invention may comprise 2, 3, 4 or more CD8 binding immunoglobulin single variable domains such as VHHs.
  • the CD8-binding proteins of the present invention may also comprise, in addition to the CD8-binding immunoglobulin single variable domain, a linker and/or a moiety having effector functions, such as a half-life extending moiety (eg, an immunoglobulin single variable structure that binds serum albumin) domain), and/or fusion partner (eg serum albumin) and/or conjugated polymer (eg PEG) and/or Fc region.
  • the "CD8 binding proteins" of the invention also encompass bispecific antibodies containing immunoglobulin single variable domains that bind different antigens.
  • CD8 binding proteins of the present invention will be such as to a Biacore or KinExA assay measured Fortibio or preferably 10-7 to 10-10 moles / liter (M), more preferably from 10-8 to 10-10 mol / l, even More preferably a dissociation constant (KD) of 10 -9 to 10 -10 or lower, and/or at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , more preferably An association constant (KA) of at least 10 <10 >M< "1 > binds the antigen to be bound (ie CD8 protein).
  • KD dissociation constant
  • Any KD value greater than 10 <"4 >M is generally considered to be indicative of nonspecific binding.
  • Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and/or competitive binding assays (e.g., radioimmunoassay ( RIA), enzyme immunoassay (EIA) and sandwich competitive assay.
  • SPR surface plasmon resonance
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as well known and agreed in the art.
  • amino acid difference refers to an insertion, deletion or substitution of a specified number of amino acid residues at a position in the reference sequence compared to another sequence.
  • substitutions will preferably be conservative amino acid substitutions, meaning that an amino acid residue is replaced by another amino acid residue that is chemically similar in structure and which affects the function, activity or other biological properties of the polypeptide Little or essentially no effect.
  • conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably one amino acid residue within the following groups (i)-(v) replaced by another amino acid residue within the same group: (i) lesser Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) Polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Val by Ile or Leu.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are identical or represent 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, amino acid sequence identity is typically measured using sequence analysis software. For example, identity can be determined using the BLAST program of the NCBI database.
  • sequence identity For determination of sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York , 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • a polypeptide or nucleic acid molecule is considered “isolated” when another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component) is isolated.
  • a polypeptide or nucleic acid molecule is considered “isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more.
  • An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique, eg, a suitable chromatographic technique, such as polyacrylamide gel electrophoresis.
  • subject means a mammal, especially a primate, especially a human.
  • CD8-binding polypeptides of the present invention 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.
  • the CD8-binding polypeptide is isolated.
  • the CD8-binding polypeptide specifically binds CD8 ⁇ .
  • the at least one immunoglobulin single variable domain comprises SEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57 , 61, 65, 69, 73, 77, 81 CDR1, CDR2 and CDR3 in the VHH shown in any of .
  • the CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs or Contact CDRs. In some embodiments, the CDRs are Kabat CDRs.
  • the at least one immunoglobulin single variable domain comprises CDR1 , CDR2 and CDR3 selected from the group consisting of:
  • said immunoglobulin single variable domain comprises the The amino acid sequence of one of , 61, 65, 69, 73, 77 and 81 has an amino acid sequence of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity.
  • the immunoglobulin single variable domain comprises SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, The amino acid sequence of one of 61, 65, 69, 73, 77 and 81.
  • the immunoglobulin single variable domain is a VHH. In some embodiments, the immunoglobulin single variable domain is humanized.
  • the present invention relates to nucleic acid molecules encoding the CD8-binding polypeptides of the present invention.
  • the nucleic acid of the present invention may be RNA, DNA or cDNA.
  • nucleic acid molecules encoding CD8-binding polypeptides of the invention comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 82-91.
  • the nucleic acid of the present invention may also be in the form of, may be present in and/or be part of a vector, such as a plasmid, cosmid or YAC.
  • the vector may in particular be an expression vector, ie a vector that provides for expression of the CD8 binding polypeptide in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
  • the expression vector typically contains at least one nucleic acid of the invention operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the general knowledge of those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the CD8-binding polypeptides of the invention, such as promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.
  • Nucleic acids of the invention may be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequences of the polypeptides of the invention given herein, and/or may be obtained from suitable natural sources are separated.
  • the present invention relates to host cells expressing or capable of expressing one or more CD8-binding polypeptides of the present invention and/or containing a nucleic acid or vector of the present invention.
  • Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include gram-negative bacterial strains (eg, Escherichia coli, Proteus, and Pseudomonas strains) and gram-positive bacterial strains (eg, Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains).
  • gram-negative bacterial strains eg, Escherichia coli, Proteus, and Pseudomonas strains
  • gram-positive bacterial strains eg, Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains.
  • Suitable fungal cells include cells of species of Trichoderma, Neurospora and Aspergillus; or Saccharomyces (eg Saccharomyces cerevisiae), Saccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Han Cells of Hansenula species.
  • Saccharomyces eg Saccharomyces cerevisiae
  • Saccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cell in the art for expressing heterologous proteins may also be used in the present invention.
  • the present invention also provides methods of producing the CD8-binding polypeptides of the present invention, said methods generally comprising the steps of:
  • CD8-binding polypeptides of the invention optionally further purification and/or modification of the CD8-binding polypeptides of the invention.
  • the CD8-binding polypeptides of the invention can be produced intracellularly (eg, in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, then isolated from the host cell and optionally further purified; or it can be extracellularly Produced (eg, in the medium in which the host cells are cultured), then isolated from the medium and optionally further purified.
  • CD8-binding polypeptides of the invention can also be obtained by other methods of protein production known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.
  • the present invention provides a conjugated molecule comprising a CD8-binding polypeptide of the present invention, and at least one detectable label conjugated to the CD8-binding polypeptide.
  • Such detectable labels include, but are not limited to, radionuclides, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, and enzymes.
  • Fluorescent agents useful for conjugation include, but are not limited to, isothionine fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescamine; chemiluminescent agents useful for conjugation Including, but not limited to, luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalate esters; bioluminescent agents useful for conjugation include, but are not limited to, luciferin, luciferase, and aequorin protein.
  • Paramagnetic ions useful for conjugation include, but are not limited to, 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 materials, Such as dam, diatrizoic acid, lipiodol, gallium citrate, iodocal acid, iodointaric acid, iodamide, cholic acid, iodoxamic acid, iodocouamide, iohexol, iopamidol, iodine Panoic acid, ioproxilic acid, iodocyfaric acid, iodoseric acid, meglu
  • the detectable label is a radionuclide.
  • Radionuclides are available for conjugating radionuclide between 20-4000KeV energy, including but not limited to, 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, 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 Sr, or other gamma-, beta-, or positron emitters.
  • the detectable label as 68 Ga or 125 I.
  • the CD8-binding polypeptide can be conjugated to the detectable label via a chelating agent.
  • CD8 binding polypeptides of the present invention In order to mark such as 68 Ga CD8 binding polypeptides of the present invention, with radionuclides, it is necessary to CD8 binding polypeptides of the present invention and a reagent having a long tail, which is attached with a plurality of long tail integration groups for binding the ions.
  • Such tails may be, for example, polylysine, polysaccharides, or other polymers of derivatized or derivatized chains with pendant groups that can bind chelating groups, such as ethylenediaminetetraacetic acid (EDTA) , Diethylenetriaminepentaacetic acid (DTPA), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid), NOTA, TETA, NETA, porphyrin , polyamines, crown ethers, thiosemicarbazides, polyoximes, and similar groups known to be useful for this purpose.
  • chelating agent is attached to the antibody using standard chemical methods.
  • the detectable label is conjugated to a CD8-binding polypeptide of the invention via a chelating agent.
  • chelating agents include, but are not limited to, DTPA, EDTA, NOTA, DOTA, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG 3, MAG 2, HYNIC, DADT, EC, NS 3 , H2dedpa, HBED, DFO, PEPA or HEHA and derivatives thereof, etc.
  • the detectable label is 68 Ga and the chelator is NOTA.
  • the present invention provides radionuclide one inventive method of preparing the conjugated molecules 68 Ga-labeled, which comprises 1) reacting CD8 binding polypeptides of the present invention is conjugated with a chelating agent to generate the CD8 binding polypeptide with a chelating agent conjugate; and 2) step 1) product with radionuclides such as 68 Ga contacts, whereby a radionuclide such as 68 Ga CD8 markers of the present invention, the chelating agent by chelation binding polypeptide.
  • the chelator is NOTA
  • the CD8-binding polypeptide is generated by reacting the CD8-binding polypeptide with p-SCN-Bn-NOTA or p-NH2-Bn-NOTA and the Conjugates of NOTA.
  • the present invention provides a method of conjugating molecules I-labeled A 125 of the present invention is prepared, which comprises a) reacting CD8 binding polypeptide of the present invention is reacted with 125 I in the presence of chloramine T; and 2) Sodium bisulfite was used to terminate the reaction.
  • the present invention provides a method for detecting the presence and/or amount of CD8 in a biological sample, comprising:
  • the biological sample is an ex vivo sample.
  • the present invention provides a detection agent for detecting CD8-positive cells, comprising a CD8-binding polypeptide of the present invention or a conjugated molecule of the present invention, and optionally a physiologically acceptable carrier.
  • the detection agent is used to detect the presence and/or amount of CD8 positive cells in a tissue in a subject.
  • the tissue is tumor tissue.
  • the CD8-positive cells are CD8-positive T cells.
  • the CD8-binding polypeptides of the present invention or the conjugated molecules of the present invention are particularly suitable for in vivo imaging, eg, for Emission Computed Tomography (ECT).
  • ECT Emission Computed Tomography
  • the CD8-binding polypeptide of the present invention or the conjugated molecule of the present invention can be applied to single-photon emission computed tomography (SPECT) and positron emission tomography (Positron Emission Tomography, PET).
  • SPECT single-photon emission computed tomography
  • PET positron emission tomography
  • ECT provides high-resolution imaging in diagnosis and allows quantitative analysis of the images.
  • the SPECT imaging may further include SPECT/CT imaging
  • the PET imaging may further include PET/CT imaging, which may provide better imaging effects.
  • the detection agent is a contrast agent.
  • the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • the present invention provides use of a CD8-binding polypeptide of the present invention or a conjugated molecule of the present invention in the manufacture of a detection agent for detecting CD8-positive cells.
  • the detection agent is used to detect the presence and/or amount of CD8 positive cells in a tissue in a subject.
  • the tissue is tumor tissue.
  • the CD8-positive cells are CD8-positive T cells.
  • the detection agent is a contrast agent.
  • the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.
  • the present invention provides a method for detecting the presence and/or amount of CD8 positive cells in a tissue, comprising
  • the tissue is blood tissue. In some embodiments, the tissue is lymphoid 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.
  • the present invention provides a method for detecting CD8 positive cells in a subject comprising administering to the subject a conjugated molecule of the present invention or a diagnostic agent of the present invention.
  • the methods are used to detect the presence and/or amount of CD8 positive cells in a tissue in a subject.
  • the tissue is tumor tissue.
  • the CD8-positive cells are CD8-positive T cells.
  • the method further comprises the step of imaging the subject, eg, ECT imaging.
  • the ECT imaging is SPECT imaging.
  • the ECT imaging is PET imaging. Imaging techniques and devices for scanning by SPECT or PET are well known in the art and any such known ECT imaging techniques and devices may be used.
  • the invention provides a method for determining whether a subject with a tumor is suitable for anti-tumor therapy, the method comprising
  • the subject is identified as being suitable for anti-tumor therapy if the presence of CD8 positive cells in the tumor is detected, eg, the subject's tumor is infiltrated by CD8 positive cells.
  • the present invention provides a method for predicting the response of a subject with a tumor to an anti-tumor therapy, the method comprising
  • CD8 positive cells eg CD8 positive T cells
  • CD8 positive T cells can be a predictive marker of response to antitumor therapy or a prognostic marker of survival.
  • baseline CD8-positive cell tumor infiltration is a prognostic indicator of survival in breast, head/neck, and ovarian cancers.
  • tumor infiltration of CD8-positive cells detected during anti-PD-1 therapy or anti-PDL-1 therapy is a predictive marker for treatment response.
  • the present invention provides a method of treating a tumor in a subject, the method comprising:
  • the present invention provides a method for monitoring the efficacy of an anti-tumor therapy in a subject, the method comprising
  • the anti-tumor therapy is immune checkpoint inhibitor therapy.
  • anti-tumor therapy includes administration of PD-1 inhibitors (eg, REGN2810, BGB-A317, nivolumab, pilizumab, and pembrolizumab), PD-L1 inhibitors (eg, atezolizumab, avelumab, durvalumab, etc.), CTLA-4 inhibitors (eg, ipilimumab), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR Inhibitors, LAG3 inhibitors, antagonists of another T cell co-inhibitor or ligand (eg, antibodies to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3 - Dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonist
  • IDO indoleamine-2,3
  • 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, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer and myeloma.
  • the present invention also provides a method for isolating and/or purifying CD8 positive cells, the method comprising:
  • the CD8-positive cells are CD8-positive T cells.
  • the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMC).
  • CD8 positive cells can be isolated by flow cytometry.
  • the present invention also provides a method for isolating CD8 positive cells, the method comprising:
  • the CD8-positive cells are CD8-positive T cells.
  • the cell population comprising CD8 positive cells is human peripheral blood mononuclear cells (PBMC).
  • a CD8-binding polypeptide of the invention or a conjugated molecule of the invention is immobilized on a solid surface, eg, on a gel or magnetic bead surface.
  • kits comprising a CD8-binding polypeptide of the present invention or a conjugated molecule of the present invention or a diagnostic agent of the present invention.
  • kits are used to practice the methods of the present invention.
  • Kits generally include a label indicating the intended use of the contents of the kit.
  • label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the amino acid sequence of CD8 ⁇ -Fc fusion protein is:
  • Amino acids 1-21 are the signal peptides of CD8 ⁇ receptors
  • 22-136 are immunoglobulin (Ig) domains
  • 137-181 are the Ig domains of CD8 receptors
  • 182-209 are transmembrane domains
  • 210-235 are cytoplasmic domain.
  • the region marked with a single horizontal line is the Fc region, and the region marked with a double horizontal line is the linker.
  • the CD8 ⁇ -Fc fusion protein used for immunization was expressed by CHO cells and purified by Protein A affinity chromatography. Choose an alpaca to immunize. After the immunization, the lymphocytes from the peripheral blood of the alpaca were extracted and RNA was extracted according to the instructions of Trizol, and the RNA concentration was determined by Nanodrop. use The III First-Strand Synthesis System for RT-PCR kit reverse-transcribes the extracted RNA into cDNA according to the instructions. Nucleic acid fragments encoding the variable regions of heavy chain antibodies were amplified by nested PCR.
  • the VHH fragment was purified by a DNA product purification kit, and the vector and fragment were digested with restriction endonuclease sifi at 50°C overnight.
  • the digested fragment was recovered by gel cutting and cloned into the phage display vector pComb3XSS.
  • the product was then electro-transformed into E. coli electro-competent cells TG1, and a phage display library of heavy chain single-domain antibody against CD8 ⁇ was constructed and assayed.
  • the size of the library volume was calculated to be 5.04 ⁇ 10 9 by serial dilution plating. To test the insertion rate of the library, 48 clones were randomly selected for identification. The results show that the insertion rate has reached 100% and the size is correct.
  • Plates were coated with 10 ⁇ g/well of CD8 ⁇ -cHis fusion protein and placed at 4°C overnight. The next day, the plate was blocked with 3% BSA at 37°C for 2 hours, and the plate was washed 5 times with PBST (PBS containing 0.05% Tween 20), and then 100 ⁇ l of phage (1.71 ⁇ 10 11 cfu, derived from the camel heavy chain single constructed in 1.1) was added. Domain Antibody Phage Display Library) for 1 hour at 37°C. It was then washed 10 times with PBST (0.05% Tween 20 in PBS) and 10 times with PBS solution to wash away unbound phage.
  • PBST PBS containing 0.05% Tween 20
  • 96 clones were randomly picked from the plates from the last round of panning for identification. 96 single colonies were randomly selected to be cultured separately to produce and purify phage. The plates were coated with CD8 ⁇ -Fc fusion protein overnight at 4°C, blocked with 3% BSA, reacted at 37°C for 1 hour, and then added to each well with the phage supernatant obtained after infection (the control group was blank phage), and incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxide-labeled anti-M13 secondary antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added, and the reaction was performed at room temperature for 1 hour.
  • TMB chromogenic solution was added, and the absorption value was read at a wavelength of 450 nm.
  • the plate was coated with Fc protein overnight at 4°C, blocked with 3% BSA, and reacted at 37°C for 1 hour.
  • the phage supernatant obtained after infection was added (the control group was blank phage), and the reaction was performed at 37° C. for 1 hour.
  • anti-M13 secondary antibody labeled with horseradish peroxide purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • TMB color developing solution was added, and read the absorption value at 450nm wavelength.
  • the protein sequence of each clone was analyzed according to the sequence alignment software DNAMAN.
  • the clones with the same CDR1, CDR2, and CDR3 sequences were regarded as the same antibody strain, and the clones with different CDR sequences were regarded as different antibody strains. Finally, 20 candidate sequences were obtained.
  • 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.
  • the coding sequence of the candidate single-domain antibody obtained by the sequencing analysis in Example 1 was subcloned into the expression vector PET22b, and the correct recombinant plasmid identified by sequencing was transformed into the expression-type host strain BL21, which was coated with 100 micrograms per milliliter. Plate ampicillin in LB solid medium overnight at 37 °C. A single colony was selected for inoculation and cultured overnight. On the second day, the overnight strain was transferred and amplified, cultured on a shaker at 37°C until the OD value reached about 0.6, induced by 0.5mM IPTG, and cultured overnight at a shaker at 28°C. The next day, the bacteria were harvested by centrifugation, and the bacteria were sonicated to obtain crude antibody extracts. The antibody protein was then purified by nickel ion affinity chromatography. Finally, antibody proteins with a purity of more than 90% were obtained.
  • Primers were designed according to the nucleotide sequence of the CD8 ⁇ single domain antibody obtained by screening, using the above prokaryotic expression plasmid as a template, the nucleotide sequence (including histidine tag) of each antibody was amplified by PCR, and then cloned into pCDNA4 (Invitrogen, Cat V86220) vector. The correctness of the obtained target cloned gene sequence was confirmed by gene sequence determination.
  • the recombinantly constructed single-domain antibody plasmid with histidine tag was transfected into HEK293 cells for antibody expression.
  • the recombinant expression plasmid was diluted with Freestyle293 medium and added to the PEI (Polyethylenimine) solution required for transformation.
  • the plasmid/PEI mixture was added to the HEK293 cell suspension, and cultured at 37°C, 10% CO 2 , and 90 rpm. After culturing for 5-6 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.
  • CD8 ⁇ -Fc protein was obtained by transient expression of HEK293 and purification by proteinA affinity chromatography.
  • the obtained CD8 ⁇ -Fc protein 0.5 ⁇ g/well was coated on the plate overnight at 4°C, and then a serial dilution series of the obtained anti-CD8 ⁇ single domain antibody protein was added, and the reaction was performed 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 developing solution was added, and the absorption value was read at a wavelength of 450 nm.
  • the software SotfMaxPro v5.4 was used for data processing and graph analysis.
  • the antibody-to-CD8 ⁇ binding curve and EC50 values were obtained (the EC50 values of all tested antibodies were between 0.989 and 4.11 ng/mL).
  • the relative activities of different anti-CD8 ⁇ single domain antibodies were compared to reflect the affinity of the antibodies to CD8 ⁇ .
  • C37, C42, C46, S5, and S36 had higher binding activities.
  • the mouse colon cancer MC38 cell line stably transfected with the human CD8 ⁇ full-length protein gene was constructed to obtain MC38 cells (MC38-CD8 ⁇ cells) stably expressing human CD8 ⁇ protein on the membrane. Cells were grown to >90% confluence before use. Flow cytometric analysis of these types of cells was performed using an anti-CD8 ⁇ single-domain antibody for quantitative analysis of indirect immunofluorescence staining. to determine the number of surface receptors for each cell.
  • the cells were collected and suspended to a concentration of 2.5 ⁇ 10 6 cells / mL. An aliquot of 200 ⁇ L of cells was mixed with 20 ⁇ g of primary antibody and incubated on ice for 30 min. After the cells were washed by centrifugation and resuspended, 5 ⁇ L of secondary antibody-PE conjugate was added, and the cells were incubated on ice for 30 minutes in the dark. Cells were washed twice, resuspended, and flow cytometric analysis was performed on a BD FACSCelestaTM. For each tube, to collect a minimum of 5 ⁇ 10 4 events. All assays are single color, PE is detected in FL1. Forward scatter (FS) and side scatter (SS) data demonstrated that all cell populations were tightly clustered.
  • FS Forward scatter
  • SS side scatter
  • Human CD8+ T cells were obtained by separating human peripheral blood with magnetic beads, and anti-CD8 ⁇ PE conjugate (R&D Systems, Cat#FAB1509P) was used as a positive control to analyze the binding of anti-CD8 ⁇ single domain antibody C37-his to CD8+ T cells.
  • the concentration of isolated cells was adjusted to 2.5 ⁇ 10 6 cells/mL. An aliquot of 150 ⁇ L of cells was mixed with 3 ⁇ g of primary antibody and incubated on ice for 30 min. After the cells were washed and resuspended, 5 ⁇ L of secondary antibody-PE conjugate was added and incubated on ice for 30 minutes in the dark. Cells were washed twice, resuspended, and flow cytometric analysis was performed on a BD FACSCelestaTM. For each tube, to collect a minimum of 5 ⁇ 10 4 events. All assays are single color, PE is detected in FL1. Forward scatter (FS) and side scatter (SS) data demonstrated that all cell populations were tightly clustered.
  • FS Forward scatter
  • SS side scatter
  • CD8 ⁇ single-domain antibody C37-chis has a good binding ability to the surface antigen of human PBMC CD8+ T cells.
  • the anti-CD8 ⁇ single domain antibody-biontin was immobilized on the SA biosensor. Then, CD8 ⁇ -chis at a concentration of 6.25-100 nM was bound to the Nanobody, followed by dissociation.
  • Four antibody binding kinetic variables including Kon, Koff and Kd were determined using Octet Data Analysis version 9.0 assessment to determine C37, C42, C46, S5.
  • the measured binding affinities of the anti-CD8 ⁇ antibodies are shown in the table.
  • the results show that C37-his protein has a significant affinity for CD8 ⁇ target protein, and its higher Ka and lower Kd values indicate that the antibody fusion protein can bind CD8 ⁇ antigen more quickly and is difficult to dissociate.
  • 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
  • the single domain antibody S5-cHis was named SNA006d.
  • the 125 I labeling experiment was carried out by the chloramine T method.
  • MC38-CD8 cells (CD8 positive) and MC38 cells (negative control) were plated in 4 6-well plates, 1 ⁇ 10 6 cells per well, 3 mL medium/well, 37°C, 5% CO 2 overnight culture; cells were removed , incubate at 4°C for 30min; take out all 6-well plates, each experimental group is divided into total binding group and non-specific binding group, each group has three parallel samples, so one six-well plate corresponds to one group of experiments.
  • the 3-well medium was supplemented with medium containing 25 ⁇ M of unlabeled protein as a control sample for non-specific adsorption.
  • the cell uptake fraction (B) of the junction and the pellet was counted as the cell uptake fraction of the junction and the pellet, the sum of the radioactivity counts of the supernatant and pellet was the total radioactivity (T), and the B/T of the group minus the corresponding non-specific binding was the uptake of the 10 6 cells in the group
  • the radioimmunoassay activity fraction (calculated in %), the results are shown in Figure 3.
  • 125 I-labeled CD8 ⁇ single-domain antibody has a higher percentage of binding to MC38-CD8 cells, while the corresponding CD8-negative cells have no uptake; and 125 I-labeled CD8 ⁇ single-domain antibody binds to MC38-CD8 cells All can be blocked by unlabeled CD8 single-domain antibody; the specific binding ability of 125I- labeled CD8 single-domain antibody to MC38-CD8 cells was proved.
  • the percent binding of 125 I-SNA006a on 10 6 MC38-CD8 cells was calculated to be -50%, and the lowest bound 125 I-SNA006d was also greater than 10%. It showed that 125 I-labeled SNA006a, b, c, and d had higher uptake, and SNA006-a, b had higher affinity for CD8 than SNA006-c, d.
  • MC38-CD8 cells were cultured in a 50 mL petri dish. Before the experiment, the culture medium was removed, and the cells were washed twice with 0.01 M sterile PBS. After the cells were digested with trypsin, they were washed twice with PBS and finally prepared with 5% BSA. cell suspension. Cells were added to a 96-well suction filter plate at approximately 10 5 cells/well.
  • Receptor binding reaction using the radioligand saturation method, it is divided into two groups: total binding and non-specific binding. There were 12 dose points with increasing concentrations in the total binding group, and 12 dose points in the non-specific binding group. Each dose point was provided with 4 groups of parallel samples. The total reaction volume was 200 ⁇ L, corresponding to 1000-fold excess of cold antibody.
  • the binding of 125 I-labeled CD8 single-domain antibody to MC38-CD8 cells can be blocked by unlabeled CD8 single-domain antibody, which proves the specific binding ability of 125 I-labeled CD8 single-domain antibody to MC38-CD8 cells.
  • 125 I-SNA006a its K D value of 0.46nM, and 125 I-SNA006c of 3.8nM, 125 I-SNA006d of 1.5nM.
  • the affinity of 125 I-SNA006a was significantly better than that of 125 I-SNA006-c and d.
  • MC38-CD8 cells were cultured in a 50 mL petri dish. Before the experiment, the culture medium was removed, and the cells were washed twice with 0.01 M sterile PBS. After the cells were digested with trypsin, they were washed twice with PBS and finally prepared with 5% BSA. cell suspension. Cells were added to a 96-well suction filter plate at approximately 10 5 cells/well.
  • the 96-well cell filter plates were saturated with binding buffer and air-dried before use. Add about 10 5 cells to each well of the suction filter plate, and CD8 single-domain antibody of increasing concentration, and then add about 2 ⁇ 10 5 counts/min of 125 I-CD8 single-domain antibody to each well, and react with the binding buffer. The volume was adjusted to 200 ⁇ L.
  • the filter membrane at the bottom of the suction filter plate was dried, the filter membrane at the bottom was collected and measured with a gamma counter, calculated and plotted with Prism 7.0 (GraphPad Software, Inc.), and the median inhibitory concentration IC50 was calculated by nonlinear regression analysis. Four parallel samples were set at each experimental point, and the experiment was repeated twice. The results presented in this experiment are the mean plus the standard deviation.
  • the binding of 125I -SNA006a to the CD8 receptor of MC38-CD8 cells can also be inhibited by the "cold" protein of SNA006a in a concentration-dependent manner, which was obtained by Prism 7.0 (GraphPad Software, Inc.) software fitting has an IC50 value of ⁇ 3.0 nM.
  • the competitive binding assay described herein demonstrates the high affinity and specificity of SNA006a for CD8 antigen binding.
  • Embodiment 5 125I- labeled CD8 ⁇ single domain antibody and its SPECT/CT imaging
  • the tumor tissue was collected, and CD8 immunohistochemical staining was performed to analyze the expression level of CD8, and compared with the overall CD8 expression level of the tumor detected by PET/CT.
  • the MC38-CD8+/- tumor model was inoculated according to the method in 5.1, the mice were anesthetized with isoflurane and placed in the SPECT/CT bed, and ⁇ 10 ⁇ g of radiolabeled single domain antibody ( ⁇ 100 ⁇ Ci/10 ⁇ g) was administered to the mice by tail vein injection.
  • SPECT scans were performed at 1h, 2h, and 3h, respectively, to analyze the radioactivity uptake of tumors and tissues and organs at multiple time points. The results are shown in Figure 6.
  • the MC38-CD8 xenografts were clearly visible within 120 min after the injection of 125 I-CD8 single domain antibody.
  • the right MC38-CD8+ tumor had good contrast compared to the contralateral negative control tumor MC38-CD8-, and the 125I -CD8 single domain antibody had a significant concentration in the kidney, indicating that its main Metabolized by the kidneys. Since the above-mentioned iodine labeling methods inevitably produce deiodination, the SPECT results will cause radioactive accumulation in the thyroid and stomach. Moreover, the energy of 125 I is weak, and its application in SPECT imaging will cause a certain amount of tissue self-absorption.
  • the purpose of applying 125 I-labeled CD8 single-domain antibody to SPECT imaging is to screen out the best candidate antibodies for subsequent 68 Ga-labeled imaging agents.
  • the results showed that 125 I-labeled SNA006a, SNA006c, and SNA006d had higher uptake in tumor sites, and the imaging effect of SNA006a was significantly better than that of SNA006c and SNA006d.
  • the in vivo screening of SPECT was consistent with the corresponding in vitro experimental results, which further proved that SNA006a has the most CD8 tracking potential among all the aforementioned single domain antibodies.
  • Example 6 precursors for the synthesis of single domain antibodies NOTA-CD8 ⁇ and 68 Ga-NOTA-CD8 ⁇ single domain antibodies radiolabeled
  • the bifunctional chelator p-SCN-NOTA was coupled to CD8 single domain antibody, and the conjugated product was purified by PD-10 column and the buffer was changed to normal saline. Conjugation efficiency was analyzed by MS. In vitro cell assays (tumor cell uptake and affinity detection) were used to investigate the targeting of the conjugated product to tumor cells, and its in vitro affinity with CD8 ⁇ protein was analyzed by ELISA.
  • PD-10 column Using PD-10 column (GE), the buffer of CD8 ⁇ -cHis single domain antibody (single domain antibody includes C37, C46, S5) was replaced with NaHCO 3 buffer at a certain pH, p-SCN-Bn-NOTA (Macrocyclics, Cat. No. B-605) was dissolved in DMSO at a concentration of 25 mg/mL. The p-SCN-Bn-NOTA solution was added to the CD8 ⁇ single domain antibody at twice the moles of lysine on the single domain antibody, and reacted at room temperature for a certain period of time. The conjugated product was purified using a PD-10 column (GE) and concentrated by ultrafiltration.
  • p-SCN-Bn-NOTA Microcyclics, Cat. No. B-605
  • the product is quality controlled by analyzing pH, Radio-TLC, Radio-HPLC, radioactivity, radiochemical purity, etc.
  • 0.05M sterile HCl can also be used to rinse the ITG germanium 68/gallium 68 (Ge-68/Ga-68) generator to prepare 68 Ga eluent, add 1/2 volume of 0.2M sodium acetate solution, other reactions and The quality control conditions were all the same.
  • ITLC-SG was pre-cut to 1cm x 12cm strips and penciled to mark each end of the strips 1cm from each other. Pour the spreading agent in the spreading tank, cover the tank, and allow it to equilibrate. ITLC strips 1cm from the bottom of the drop of the pencil line 68 Ga-CD8 single domain antibody injection. The ITLC strip was placed in the spreading slot and allowed to spread 10 cm from the loading point (ie to the top pencil line mark). ITLC using ITLC scanner radiation measured by calculating the radiochemical purity (RCP) of the chromatographic peak integration in FIG., The analysis results shown in Figure 7, show that 68 Ga positron successful single domain antibodies labeled CD8.
  • RCP radiochemical purity
  • Example 7 In vivo distribution of 68 Ga-NOTA-CD8 ⁇ single domain antibody
  • the tumor tissue was collected, and CD8 immunohistochemical staining was performed to analyze the expression level of CD8, and compared with the overall CD8 expression level of the tumor detected by PET/CT.
  • the MC38-CD8+/- tumor model was inoculated according to the method in 7.1, and ⁇ 10 ⁇ g of radiolabeled single domain antibody ( ⁇ 100 ⁇ Ci/10 ⁇ g) was administered to mice by tail vein injection. Data were collected at 1, 1.5 hours, and 3 mice were euthanized at each time point. Tissues of interest such as blood, kidney, liver, spleen, lung, heart, intestine, stomach, muscle, skin, brain, bone, and CD8+/- tumors were dissected and counted on a gamma counter to collect data. Take the injected dose as the total injected dose. For each organ, the % injected dose (%ID) was determined based on the total injected dose, and the organ was weighed for determining the % injected dose per gram (%ID/g).
  • %ID % injected dose
  • Figure 8 shows the biodistribution data in intact male mice bearing MC38-CD8 tumor 68 Ga-NOTA-CD8 ⁇ single domain antibody uptake in vivo.
  • Data distribution in intact male mice with subcutaneous tumors (n 3) 68 Ga- NOTA-CD8 vitro biological uptake.
  • Data were collected 1, 1.5 hours after intravenous administration.
  • Data are presented as mean %ID/g ⁇ standard deviation (SD).
  • SD standard deviation
  • the error of the ratio is calculated as the geometric mean of the standard deviation.
  • the data are shown in Figure 8, where the error bars represent the standard deviation of the group.
  • Figure 9 shows the results for tumor tissue and blood uptake ratios and tumor tissue and contralateral normal muscle uptake ratios for these experiments.
  • CD8 single domain antibody showed good tumor uptake in expression of the target tumor MC38-CD8, the peak tumor / muscle ratio, showing good 68 Ga-NOTA-SNA006a a single domain antibody specific for the tumor.
  • This marker 68 Ga-NOTA-SNA006a could be a good tumor imaging agent.
  • Example 8 In vivo PET imaging of 68 Ga-NOTA-CD8 ⁇ single domain antibody
  • the MC38-CD8+/- tumor model was inoculated according to the method in 6.1.
  • Micro-PET/CT IRIS PET/CT, inviscan, France
  • Serial small animal PET/CT scans were performed at 120 minutes to analyze the radioactivity uptake of tumors, muscles and kidneys at multiple time points, as shown in Figure 10.
  • Image reconstruction was performed using a three-dimensional ordered subset expectation maximization algorithm based on a Monte Carlo model (Monte-Carlo based 3D OSEM).
  • the radioactivity was calculated by the region of interest (ROI) method in the tumor, muscle, liver and other organs, and the obtained value was divided by the injected dose to obtain the PET tracer uptake value (%ID/g) of each tissue (assumed The tissue density is 1 g/ml). The calculation results are shown in Fig. 10, respectively.
  • Embodiment 9 68 Ga-NOTA-SNA006a single domain antibody stability experiment in vivo
  • mice Of normal mice from the tail vein injection of 68 Ga-NOTA-SNA006a 74MBq ( 1mCi / 100 ⁇ L PBS). 0.5h and 1h after injection, urine samples were collected, all samples were dissolved in a certain amount of 50% acetonitrile aqueous solution, centrifuged at 8000 rpm for 15 min, the supernatant was taken and passed through a 0.22 ⁇ m filter membrane, and the filtrate was analyzed by Radio-TLC. analyze.
  • Radio-TLC results are shown in Figure 12, and no obvious degradation or 68 Ga shedding was observed, indicating that under these conditions, the radiolabeled antibody is structurally stable and should be stable in vivo.

Abstract

一种特异性CD8结合多肽及其用途。

Description

CD8结合多肽及其用途 技术领域
本发明涉及生物医药领域。具体而言，本发明涉及一种特异性CD8结合多肽及其用途。
背景技术
发射型计算机断层成像术(Emission Computed Tomography，ECT)已用于肿瘤的诊断。ECT包括单光子发射计算机断层成像术(Single-Photon Emission Computed Tomography，SPECT)和正电子发射断层成像术(Positron Emission Tomography，PET)，其提供了高分辨率的肿瘤成像并且可通过图像进行定量分析。
在人类中，CD8主要在细胞毒性T淋巴细胞上表达，但也在树突状细胞、自然杀伤细胞等表达。CD8分子可以是由CD8α形成的同二聚体，或是由CD8α和CD8β形成的异二聚体，其中αβ异二聚体更普遍。
在体内监测CD8阳性肿瘤浸润淋巴细胞(TILs)的能力对于评估免疫治疗的反应和协助开发更有效的免疫细胞靶向单一和联合治疗具有重要意义。肿瘤浸润性T细胞的“免疫PET”显像可以提供一种特异、敏感的方法，帮助患者选择特定的免疫治疗方案，并确定治疗是否有效。
本领域需要能够用于检测表达CD8的细胞的检测剂，特别是适合于ECT检测的基于CD8抗体的检测剂。
发明简述
本发明至少包含以下实施方案：
实施方案1.一种CD8结合多肽，其包含至少一个能够特异性结合CD8α的免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77、81中任一个中的CDR1、CDR2和CDR3。
实施方案2.实施方案1的CD8结合多肽，其中所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
(1)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2，SEQ ID NO:4所示的CDR3；
(2)SEQ ID NO:6所示的CDR1，SEQ ID NO:7所示的CDR2，SEQ ID NO:8所示的CDR3；
(3)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2，SEQ ID NO:12所 示的CDR3；
(4)SEQ ID NO:14所示的CDR1，SEQ ID NO:15所示的CDR2，SEQ ID NO:16所示的CDR3；
(5)SEQ ID NO:18所示的CDR1，SEQ ID NO:19所示的CDR2，SEQ ID NO:20所示的CDR3；
(6)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；
(7)SEQ ID NO:26所示的CDR1，SEQ ID NO:27所示的CDR2，SEQ ID NO:28所示的CDR3；
(8)SEQ ID NO:30所示的CDR1，SEQ ID NO:31所示的CDR2，SEQ ID NO:32所示的CDR3；
(9)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:37所示的CDR3；
(10)SEQ ID NO:38所示的CDR1，SEQ ID NO:39所示的CDR2，SEQ ID NO:40所示的CDR3；
(11)SEQ ID NO:42所示的CDR1，SEQ ID NO:43所示的CDR2，SEQ ID NO:44所示的CDR3；
(12)SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2，SEQ ID NO:48所示的CDR3；
(13)SEQ ID NO:50所示的CDR1，SEQ ID NO:51所示的CDR2，SEQ ID NO:52所示的CDR3；
(14)SEQ ID NO:54所示的CDR1，SEQ ID NO:55所示的CDR2，SEQ ID NO:56所示的CDR3；
(15)SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2，SEQ ID NO:60所示的CDR3；
(16)SEQ ID NO:62所示的CDR1，SEQ ID NO:63所示的CDR2，SEQ ID NO:64所示的CDR3；
(17)SEQ ID NO:66所示的CDR1，SEQ ID NO:67所示的CDR2，SEQ ID NO:68所示的CDR3；
(18)SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2，SEQ ID NO:72所示的CDR3；
(19)SEQ ID NO:74所示的CDR1，SEQ ID NO:75所示的CDR2，SEQ ID NO:76所示的CDR3；
(20)SEQ ID NO:78所示的CDR1，SEQ ID NO:79所示的CDR2，SEQ ID NO:80所示的CDR3。
实施方案3.实施方案1或2的CD8结合多肽，其中所述免疫球蛋白单一可变结构 域包含与SEQ ID NO：5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77和81之一的氨基酸序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
实施方案4.实施方案1-3中任一项的CD8结合多肽，其中所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77和81之一的氨基酸序列。
实施方案5.实施方案1-4中任一项的CD8结合多肽，其中所述免疫球蛋白单一可变结构域是VHH。
实施方案6.核酸分子，其编码实施方案1-5中任一项的CD8结合多肽。
实施方案7.表达载体，其包含与表达调控元件可操作地连接的实施方案6的核酸分子。
实施方案8.宿主细胞，其包含实施方案6的核酸分子或以实施方案7的表达载体转化，并能够表达所述CD8结合多肽。
实施方案9.产生实施方案1-5中任一项的CD8结合多肽的方法，包括：
a)在允许所述CD8结合多肽表达的条件下培养实施方案8的宿主细胞；
b)从得自步骤a)的培养物回收由所述宿主细胞表达的CD8结合多肽；及
c)任选进一步纯化和/或修饰得自步骤b)的CD8结合多肽。
实施方案10.一种缀合分子，其包含实施方案1-5任一项的CD8结合多肽，以及与所述CD8结合多肽缀合的至少一种可检测标记。
实施方案11.实施方案10的缀合分子，其中所述可检测标记选自放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
实施方案12.实施方案11的缀合分子，其中所述可检测标记选自 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb、 83Sr或其它γ-、β-、或正电子发射体，例如，所述可检测标记为 68Ga或 125I。
实施方案13.实施方案10-12中任一项的缀合分子，其中所述CD8结合多肽通过螯合剂与所述可检测标记缀合。
实施方案14.实施方案13的缀合分子，其中所述螯合剂选自DTPA、EDTA、NOTA、DOTA、TRAP、TETA、NETA、CB-TE2A、Cyclen、Cyclam、Bispidine、TACN、ATSM、SarAr、AmBaSar、MAG 3、MAG 2、HYNIC、DADT、EC、NS 3、H2dedpa、HBED、DFO、PEPA或者HEHA及其衍生物。
实施方案15.实施方案14的缀合分子，其中所述可检测标记是 68Ga且所述螯合剂为NOTA。
实施方案16.一种检测生物学样品中CD8的存在和/或量的方法，包括：
a)在实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子与 CD8之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的CD8结合多肽或本发明的缀合分子；
b)检测复合物的形成，
其中所述生物学样品与对照样品之间复合物形成的差异指示样品中CD8的存在和/或量。
实施方案17.一种用于检测CD8阳性细胞的检测剂，其包含实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子，以及任选的生理学上可接受的载体。
实施方案18.实施方案17的检测剂，其是造影剂。
实施方案19.实施方案18的检测剂，其中所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
实施方案20.实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子在制备用于检测CD8阳性细胞的检测剂中的用途。
实施方案21.实施方案20的检测剂，其是造影剂。
实施方案22.实施方案21的检测剂，其中所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
实施方案23.一种用于检测组织中CD8阳性细胞的存在和/或量的方法，包括
a)使所述组织与实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂接触；和
b)确定组织中CD8阳性细胞的存在和/或量。
实施方案24.实施方案23的方法，其中所述组织选自血液组织、淋巴组织、和肿瘤组织。
实施方案25.实施方案23或24的方法，其中所述CD8阳性细胞是CD8阳性T细胞。
实施方案26.实施方案23-25中任一项的方法，其中通过对所述组织成像确定组织中CD8阳性细胞的存在和/或量。
实施方案27.实施方案23-25中任一项的方法，其中通过流式细胞术确定组织中CD8阳性细胞的存在和/或量。
实施方案28.一种用于检测对象体内的组织中的CD8阳性细胞的存在和/或量的方法，包括给所述对象施用实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂接触。
实施方案29.实施方案28的方法，其中所述组织是肿瘤组织。
实施方案30.实施方案28或29的方法，其中所述CD8阳性细胞是CD8阳性T细胞。
实施方案31.实施方案28-30中任一项的方法，其中所述方法还包括对所述对象进行成像例如ECT成像的步骤，例如所述ECT成像是SPECT成像或PET成像。
实施方案32.一种用于确定患有肿瘤的对象是否适于抗肿瘤疗法的方法，所述方法包含
1)给所述对象施用实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂，和
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则将所述对象鉴别为适于抗肿瘤疗法。
实施方案33.一种用于预测患有肿瘤的对象对抗肿瘤疗法的反应的方法，所述方法包含
1)向所述对象施用实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂，和
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则个体可能对抗肿瘤疗法起反应。
实施方案34.一种治疗对象中的肿瘤的方法，所述方法包括：
1)向所述对象施用实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂，和
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则给所述对象施用抗肿瘤疗法。
实施方案35.一种用于监测抗肿瘤疗法在对象中的功效的方法，所述方法包含
1)给患有肿瘤且使用抗肿瘤疗法治疗的对象施用实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂；
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤中CD8阳性细胞的量。
实施方案36.实施方案32-35中任一项的方法，其中所述抗肿瘤疗法是免疫检查点抑制剂疗法。
实施方案37.实施方案32-36中任一项的方法，其中抗肿瘤疗法选自施用PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、TIM3抑制剂、BTLA抑制剂、TIGIT抑制剂、CD47抑制剂、GITR抑制剂、LAG3抑制剂、另一T细胞共抑制剂或配体的拮抗剂、吲哚胺-2,3-双加氧酶(IDO)抑制剂、血管内皮生长因子(VEGF)拮抗剂、Ang2抑制剂、转化生长因子β(TGFβ)抑制剂、表皮生长因子受体(EGFR)抑制剂、CD20抑制剂、针对肿瘤特异性抗原的抗体、疫苗、增加抗原呈递的佐剂、双特异性抗体、细胞毒素、化疗剂、 环磷酰胺、放疗、IL-6R抑制剂、IL-4R抑制剂、IL-10抑制剂、细胞因子以及抗体-药物缀合物(ADC)。
实施方案38.实施方案32-37中任一项的方法，其中所述肿瘤是实体肿瘤。
实施方案39.实施方案38的方法，其中所述实体肿瘤选自结肠直肠癌、卵巢癌、前列腺癌、乳腺癌、脑癌、宫颈癌、膀胱癌、肛门癌、子宫癌、结肠癌、肝癌、胰腺癌、肺癌、子宫内膜癌、骨癌、睾丸癌、皮肤癌、肾癌、胃癌、食管癌、头颈癌、唾液腺癌以及骨髓瘤。
实施方案40.一种用于分离和/或纯化CD8阳性细胞的方法，所述方法包括：
(a)提供疑似包含CD8阳性细胞的细胞群；
(b)鉴定所述细胞群的亚群，所述亚群的细胞结合实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子；和
(c)分离所述亚群。
实施方案41.一种用于分离CD8阳性细胞的方法，所述方法包括：
(a)提供疑似包含CD8阳性细胞的细胞群；
(b)使所述细胞群与实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子接触，从而允许CD8阳性细胞结合至实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子；
(c)去除未结合至实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子的细胞；和
(c)回收结合至实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子的CD8阳性细胞。
实施方案42.实施方案40或41的方法，其中所述CD8阳性细胞是CD8阳性T细胞。
实施方案43.实施方案40-42中任一项的方法，其中所述包含CD8阳性细胞的细胞群是人外周血单个核细胞(PBMC)。
实施方案44.实施方案40-44中任一项的方法，其中实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子固定化于固体表面上，例如固定化于凝胶或磁珠表面。
实施方案45.一种试剂盒，其包含实施方案1-5任一项的CD8结合多肽或实施方案10-15任一项的缀合分子或实施方案17-19任一项的检测剂。
附图说明
图1、FACS考察候选抗体对人PBMC CD8+T细胞表面抗原的结合效果。
图2、 125I标记CD8单域抗体。
图3、 125I标记CD8α单域抗体MC38-CD8细胞摄取实验。
图4、 125I标记CD8单域抗体MC38-CD8细胞饱和结合分析。
图5、 125I-SNA006a MC38-CD8细胞竞争结合分析。
图6、 125I标记CD8α单域抗体的SPECT成像。
图7、 68Ga标记CD8α单域抗体TLC色谱分析。
图8、 68Ga标记CD8α单域抗体的体内分布数据。
图9、 68Ga标记CD8α单域抗体的体内分布靶本比。
图10、 68Ga标记CD8α单域抗体的体内PET/CT成像。
图11、 68Ga标记CD8α单域抗体的体内时间生物分布曲线。
图12、 68Ga-NOTA-SNA006a体内稳定性分析。
发明详述
定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。参考例如标准手册，如Sambrook等人,“Molecular Cloning:A Laboratory Manual”(第2版),第1-3卷,Cold Spring Harbor Laboratory Press(1989)；Lewin,“Genes IV”，Oxford University Press,New York,(1990)；及Roitt等人,“Immunology”(第2版),Gower Medical Publishing,London,New York(1989)，以及本文中引用的一般现有技术；此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。亦参考例如标准手册、上述一般现有技术及其中引用的其他参考文献。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、“抗体序列”、“单一可变结构域序列”、“VHH序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用，术语(多肽或蛋白的)“结构域”是指折叠蛋白结构，其能够独立于蛋白的其余部分维持其三级结构。一般而言，结构域负责蛋白的单个的功能性质，且在许多情况下可添加、移除或转移至其他蛋白而不损失蛋白的其余部分和/或结构域的功能。
如本文所用的术语“免疫球蛋白结构域”是指抗体链(例如常规4链抗体的链或重链抗体的链)的球形区域，或是指基本上由这类球形区域组成的多肽。免疫球蛋白结构域的特征在于其维持抗体分子的免疫球蛋白折叠特征。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互 补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
如本文所用的术语“免疫球蛋白单一可变结构域”是指能够在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原表位的免疫球蛋白可变结构域。本发明含义中的免疫球蛋白单一可变结构域的一个实例为“结构域抗体”，例如免疫球蛋白单一可变结构域VH及VL(VH结构域及VL结构域)。免疫球蛋白单一可变结构域的另一实例为如下文定义的骆驼科的“VHH结构域”(或简称为“VHH”)。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH结构域、VHH抗体片段和VHH抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域(Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:“Naturally occurring antibodies devoid of light chains”；Nature 363,446-448(1993))。使用术语“VHH结构域”以将所述可变结构域与存在于常规4链抗体中的重链可变结构域(其在本文中称为“VH结构域”)以及存在于常规4链抗体中的轻链可变结构域(其在本文中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规4链抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
在本发明的上下文中，术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、以及“VHH抗体”可互换使用。
例如Riechmann及Muyldermans,J.Immunol.Methods 231,25-38(1999)的图2中所示，对于骆驼科的VHH结构域所应用的氨基酸残基，可以根据Kabat等人给出的VH结构域的一般编号法来编号(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。
本领域中已知对VH结构域的氨基酸残基进行编号的替代方法，所述替代方法还可以类似地应用于VHH结构域。例如，Chothia CDR指的是结构环的位置(Chothia and Lesk,J.Mol.Biol.196:901-917(1987))。AbM CDR代表的是Kabat超变区和Chothia结构环的折中，并且在Oxford Molecular's AbM抗体建模软件中使用。“接触(Contact)”CDR则基于对可获得的复合物晶体结构的分析。来自每种方法的CDR的残基描述如下：
环 Kabat AbM Chothia Contact
LCDR1 L24-L34 L24-L34 L26-L32 L30-L36
LCDR2 L50-L56 L50-L56 L50-L52 L46-L55
LCDR3 L89-L97 L89-L97 L91-L96 L89-L96
HCDR1(Kabat编号) H31-H35B H26-H35B H26-H32 H30-H35B
HCDR1(Chothia编号) H31-H35 H26-H35 H26-H32 H30-H35
HCDR2 H50-H65 H50-H58 H53-H55 H47-H58
HCDR3 H95-H102 H95-H102 H96-H101 H93-H101
然而应注意，如本领域中对于VH结构域及VHH结构域所公知的，各CDR中的 氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)。这意味着一般而言，根据Kabat的编号可能对应或可能不对应于实际序列中氨基酸残基的实际编号。
例如，CDR可以包括“扩展的(extended)CDR”，例如：在VL中的24-36或24-34(LCDR1)，46-56或50-56(LCDR2)以及89-97或89-96(LCDR3)；在VH中的26-35(HCDR1)，50-65或49-65(HCDR2)以及93-102、94-102或95-102(HCDR3)。
VHH结构域中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本文所述的目的。
VHH结构域及含有其的多肽的其他结构特性及功能性质可总结如下：
VHH结构域(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分VHH结构域与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于这些独特性质，使用VHH结构域—单独或作为较大多肽的一部分—提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势：仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；VHH结构域可自单一基因表达且不需要翻译后折叠或修饰；VHH结构域可容易地改造成多价及多特异性格式(格式化)；VHH结构域高度可溶且无聚集趋势；VHH结构域对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；VHH结构域易于制备且相对廉价，甚至在生产所需的规模上亦如此；VHH结构域与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；VHH结构域可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的VHH的方法，先前已公开于以下文献中：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.,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17 June,2009和WO94/04678。
源自骆驼科的VHH结构域可通过以人常规4链抗体VH结构域中相应位置处存在的一个或多个氨基酸残基置换原始VHH序列的氨基酸序列中的一个或多个氨基酸残基 而经“人源化”(本文中亦称为“序列优化”，除人源化外，“序列优化”也可涵盖通过提供VHH改良性质的一个或多个突变对序列进行的其他修饰，例如移除潜在的翻译后修饰位点)。人源化VHH结构域可含有一个或多个完全人框架区序列。人源化可以使用蛋白表面氨基酸人源化(resurfacing)的方法和/或人源化通用框架CDR移植法(CDR grafting to a universal framework)完成。
如本文所用，术语“表位”或可互换使用的术语“抗原决定簇”指抗体的互补位所结合的抗原上的任何抗原决定簇。抗原决定簇通常包含分子的化学活性表面基团，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸，其可以是“线性”表位或“构象”表位。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
可使用本领域中熟知的许多表位定位技术鉴别给定抗原的表位。参见例如Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。举例而言，线性表位可通过例如以下方法来确定：在固体支持物上同时合成大量肽，其中这些肽对应于蛋白质分子的各部分，且使这些肽在仍然与支持物连接的情况下与抗体反应。这些技术在本领域中为已知的且描述于例如美国专利第4,708,871号；Geysen等人(1984)Proc.Natl.Acad.Sci.USA 81:3998-4002；Geysen等人(1986)Molec.Immunol.23:709-715中。类似地，构象表位可通过诸如通过例如x射线结晶学及2维核磁共振确定氨基酸的空间构形加以鉴别。参见例如Epitope Mapping Protocols(同上)。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本发明的抗体分子竞争结合CD8上的相同表位的抗体及其抗原结合片段。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本发明的免疫球蛋白单一可变结构域)可结合的不同类型抗原或表位的数目。可基于抗原结合蛋白的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合蛋白之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合蛋白(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合蛋白上的抗原结合位点之间的亲和力，以及存在于抗原结合蛋白上的相关结合位 点的数目。
如本文所用，术语“CD8结合蛋白(CD8结合多肽)”意指任何能够特异性结合CD8α蛋白的蛋白。CD8结合蛋白可以包括针对CD8α的抗体，例如如本文定义的抗体。CD8结合蛋白还涵盖免疫球蛋白超家族抗体(IgSF)或CDR移植分子。示例性的CD8α的氨基酸序列如SEQ ID NO:1所示。
本发明的“CD8结合蛋白”可以包含至少一个结合CD8的免疫球蛋白单一可变结构域如VHH。在一些实施方案中，本发明的“CD8结合分子”可以包含2、3、4或更多个结合CD8的免疫球蛋白单一可变结构域如VHH。本发明的CD8结合蛋白除结合CD8的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。在一些实施方案中，本发明的“CD8结合蛋白”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
通常，本发明的CD8结合蛋白将以如于Biacore或KinExA或Fortibio测定中测量的优选10 -7至10 -10摩尔/升(M)、更优选10 -8至10 -10摩尔/升、甚至更优选10 -9至10 -10或更低的解离常数(KD)，和/或以至少10 7M -1、优选至少10 8M -1、更优选至少10 9M -1，更优选至少10 10M -1的缔合常数(KA)结合所要结合的抗原(即CD8蛋白)。任何大于10 -4M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如表面等离子体共振术(SPR)测定、Scatchard测定和/或竞争性结合测定(例如放射免疫测定(RIA)、酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。在比较两个氨基酸序列时，术语“氨基酸差异”是指与另一序列相比，在参考序列某一位置处指定数目氨基酸残基的插入、缺失或取代。在取代的情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸是指氨基酸残基被化学结构类似的另一氨基酸残基置换，且其对多肽的功能、活性或其他生物性质影响较小或基本上无影响。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(i)-(v)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(i)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(ii)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(iii)极性带正电残基：His、Arg及Lys；(iv)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(v)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。“序列相似性”指示相同或代表保守氨基酸取代的氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“分离的”多肽或核酸分子优选基本上为均质的。
如本文所用的术语“对象”意指哺乳动物，尤其灵长类动物，尤其是人。
本发明的CD8结合多肽
本发明提供了一种CD8结合多肽，其包含至少一个能够特异性结合CD8的免疫球蛋白单一可变结构域。在一些实施方案中，所述CD8结合多肽是分离的。在一些实施方案中，所述CD8结合多肽特异性结合CD8α。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77、81中任一所示的VHH中的CDR1、CDR2和CDR3。所述CDR可以是Kabat CDR、AbM CDR、Chothia CDR或Contact CDR。在一些实施方案中，所述CDR是Kabat CDR。
在一些实施方案中，所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
(1)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2，SEQ ID NO:4所示的CDR3；
(2)SEQ ID NO:6所示的CDR1，SEQ ID NO:7所示的CDR2，SEQ ID NO:8所示的CDR3；
(3)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2，SEQ ID NO:12所示的CDR3；
(4)SEQ ID NO:14所示的CDR1，SEQ ID NO:15所示的CDR2，SEQ ID NO:16所 示的CDR3；
(5)SEQ ID NO:18所示的CDR1，SEQ ID NO:19所示的CDR2，SEQ ID NO:20所示的CDR3；
(6)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；
(7)SEQ ID NO:26所示的CDR1，SEQ ID NO:27所示的CDR2，SEQ ID NO:28所示的CDR3；
(8)SEQ ID NO:30所示的CDR1，SEQ ID NO:31所示的CDR2，SEQ ID NO:32所示的CDR3；
(9)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:37所示的CDR3；
(10)SEQ ID NO:38所示的CDR1，SEQ ID NO:39所示的CDR2，SEQ ID NO:40所示的CDR3；
(11)SEQ ID NO:42所示的CDR1，SEQ ID NO:43所示的CDR2，SEQ ID NO:44所示的CDR3；
(12)SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2，SEQ ID NO:48所示的CDR3；
(13)SEQ ID NO:50所示的CDR1，SEQ ID NO:51所示的CDR2，SEQ ID NO:52所示的CDR3；
(14)SEQ ID NO:54所示的CDR1，SEQ ID NO:55所示的CDR2，SEQ ID NO:56所示的CDR3；
(15)SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2，SEQ ID NO:60所示的CDR3；
(16)SEQ ID NO:62所示的CDR1，SEQ ID NO:63所示的CDR2，SEQ ID NO:64所示的CDR3；
(17)SEQ ID NO:66所示的CDR1，SEQ ID NO:67所示的CDR2，SEQ ID NO:68所示的CDR3；
(18)SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2，SEQ ID NO:72所示的CDR3；
(19)SEQ ID NO:74所示的CDR1，SEQ ID NO:75所示的CDR2，SEQ ID NO:76所示的CDR3；
(20)SEQ ID NO:78所示的CDR1，SEQ ID NO:79所示的CDR2，SEQ ID NO:80所示的CDR3。
在一些实施方案中，其中所述免疫球蛋白单一可变结构域包含与SEQ ID NO：5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77和81之一的氨基酸序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选 地至少99％序列相同性的氨基酸序列。在一些实施方案中，其中所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77和81之一的氨基酸序列。
在一些实施方案中，所述免疫球蛋白单一可变结构域是VHH。在一些实施方案中，所述免疫球蛋白单一可变结构域是人源化的。
核酸、载体、宿主细胞
在另一方面，本发明涉及编码本发明的CD8结合多肽的核酸分子。本发明的核酸可为RNA、DNA或cDNA。本领域技术人员可根据需要或常规手段选择编码本发明的CD8结合多肽的核酸分子。在一些具体实施方案中，编码本发明的CD8结合多肽的核酸分子包含选自SEQ ID NO:82-91的核苷酸序列。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供CD8结合多肽体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。针对在特定宿主中的表达对所述元件及其序列进行选择为本领域技术人员的常识。对本发明的CD8结合多肽的表达有用或必需的调控元件及其他元件的具体实例，例如启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
本发明的核酸可基于关于本文给出的本发明的多肽的氨基酸序列的信息通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
在另一方面中，本发明涉及表达或能够表达一种或多种本发明的CD8结合多肽和/或含有本发明的核酸或载体的宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichia pastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
本发明还提供产生本发明的CD8结合多肽的方法，所述方法通常包含以下步骤：
-在允许表达本发明的CD8结合多肽的条件下培养本发明的宿主细胞；及
-从培养物回收由所述宿主细胞表达的CD8结合多肽；及
-任选进一步纯化和/或修饰本发明的CD8结合多肽。
本发明的CD8结合多肽可在如上所述细胞中以细胞内方式(例如在细胞质中、在周质中或在包涵体中)产生，接着从宿主细胞分离且任选进一步纯化；或其可以细胞外方式(例如在培养宿主细胞的培养基中)产生，接着自培养基分离且任选进一步纯化。
用于重组产生多肽的方法及试剂，例如特定适合表达载体、转化或转染方法、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。类似地，适用于制造本发明的CD8结合多肽的方法中的蛋白分离及纯化技术为本领域技术人员所公知。
然而，本发明的CD8结合多肽也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成，包括固相或液相合成。
缀合分子
在另一方面，本发明提供一种缀合分子，其包含本发明的CD8结合多肽，以及与所述CD8结合多肽缀合的至少一种可检测标记。
所述可检测标记包括但不限于放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
可用于缀合的荧光剂包括但不限于异硫氨酸荧光素、罗丹明、藻红蛋白、藻蓝蛋白、别藻蓝蛋白、邻苯二醛和荧光胺；可用于缀合的化学发光剂包括但不限于鲁米诺、异鲁米诺、芳族吖啶酯、咪唑、吖啶盐和草酸酯；可用于缀合的生物发光剂包括但不限于荧光素、荧光素酶和水母发光蛋白。可用于缀合的顺磁离子包括但不限于铬(III)、锰(II)、铁(III)、铁(II)、钻(II)、镍(II)、铜(II)、钕(III)、钐(III)、镱(III)、钆(III)、钒(II)、铽(III)、镝(III)、钬(III)和铒(III)，或者是不透辐射的材料，如坝、泛影酸盐、乙碘油、柠檬酸镓、碘卡酸、碘因他酸、碘达胺、胆影酸、碘沙酸、碘古酰胺、碘海醇、碘帕醇、碘番酸、碘普西酸、碘西法酸、碘丝酸、碘砜葡胺、碘酞硫、碘替酸、碘他拉酸、碘曲西酸、碘克沙酸、羟泛影酸、碘泊酸盐、葡甲胺、甲泛葡胶、甲泛影盐、丙碘酣和氧化亚铊。可用于缀合的酶包括但不限于辣根过氧化物酶等。
优选地，所述可检测标记是放射性核素。可用于缀合的放射性核素是能量在20-4000KeV之间的放射性核素，包括但不限于 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb、 83Sr或其它γ-、β-、或正电子发射体。在一些实施方案中，所述可检测标 记为 68Ga或 125I。
将可检测标记于多肽缀合的方法是本领域技术人员熟知的。例如，在一些实施方案中，所述CD8结合多肽可以通过螯合剂与所述可检测标记缀合。
为了用放射性核素如 68Ga标记本发明的CD8结合多肽，有必要使本发明的CD8结合多肽和具有长尾的试剂反应，该长尾附着有多个用于结合离子的整合基团。这样的尾可以是例如聚赖氨酸、多聚糖或其它具有侧基的衍生的或可衍生的链的聚合物，该侧基可结合螯合基团，例如乙二胺四乙酸(EDTA)、二乙烯三胺五乙酸(DTPA)、DOTA(1,4,7,10-四氮杂环十二烷-1,4,7,10-四羧酸)、NOTA、TETA、NETA、卟啉、聚胺、冠状醚、双缩氨硫脲、聚肟以及己知可用于此目的类似基团。使用标准的化学方法将螯合剂连接到抗体上。在一些实施方案中，可检测标记通过螯合剂与本发明的CD8结合多肽缀合。所用的螯合剂包括但不限于DTPA、EDTA、NOTA、DOTA、TRAP、TETA、NETA、CB-TE2A、Cyclen、Cyclam、Bispidine、TACN、ATSM、SarAr、AmBaSar、MAG 3、MAG 2、HYNIC、DADT、EC、NS 3、H2dedpa、HBED、DFO、PEPA或者HEHA及其衍生物等。
在一些具体实施方案中，其中所述可检测标记是 68Ga且所述螯合剂为NOTA。
在另一方面，本发明提供一种制备本发明的放射性核素如 68Ga标记的缀合分子的方法，其包含1)使本发明的CD8结合多肽与螯合剂缀合以生成所述CD8结合多肽与螯合剂的缀合物；和2)使步骤1)的产物与放射性核素如 68Ga接触，由此放射性核素如 68Ga通过螯合剂的螯合作用标记本发明的CD8结合多肽。在一些实施方案中，所述螯合剂是NOTA，且步骤1)中通过使所述CD8结合多肽与p-SCN-Bn-NOTA或p-NH2-Bn-NOTA反应而生成所述CD8结合多肽与NOTA的缀合物。
在另一方面，本发明提供一种制备本发明的 125I标记的缀合分子的方法，其包含1)使本发明的CD8结合多肽在氯胺T存在下与 125I反应；和2)用偏重亚硫酸钠终止反应。
检测/诊断用途
在另一方面，本发明提供一种检测生物学样品中CD8的存在和/或量的方法，包括：
a)在本发明的CD8结合多肽或本发明的缀合分子与CD8之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的CD8结合多肽或本发明的缀合分子；
b)检测复合物的形成，
其中所述生物学样品与对照样品之间复合物形成的差异指示样品中CD8的存在和/或量。在一些实施方案中，所述生物学样品是离体样品。
在另一方面，本发明提供一种用于检测CD8阳性细胞的检测剂，其包含本发明的CD8结合多肽或本发明的缀合分子，以及任选的生理学上可接受的载体。在一些实 施方案中，所述检测剂用于检测对象体内的组织中的CD8阳性细胞的存在和/或量。在一些实施方案中，所述组织是肿瘤组织。在一些实施方案中，所述CD8阳性细胞是CD8阳性T细胞。
本发明的CD8结合多肽或本发明的缀合分子特别适合于体内成像，例如适用于发射型计算机断层成像术(Emission Computed Tomography，ECT)。例如，本发明的CD8结合多肽或本发明的缀合分子根据标记的不同可应用于单光子发射计算机断层成像术(Single-Photon Emission Computed Tomography，SPECT)和正电子发射断层成像术(Positron Emission Tomography，PET)。ECT在诊断中可提供高分辨率的成像并且可通过图像进行定量分析。所述SPECT成像还可以包括SPECT/CT成像，且所述PET成像还可以包括PET/CT成像，其可以提供更优的成像效果。
因此，在一些实施方案中，所述检测剂是造影剂。在一些实施方案中，所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
在另一方面，本发明提供了本发明的CD8结合多肽或本发明的缀合分子在制备用于检测CD8阳性细胞的检测剂中的用途。在一些实施方案中，所述检测剂用于检测对象体内的组织中的CD8阳性细胞的存在和/或量。在一些实施方案中，所述组织是肿瘤组织。在一些实施方案中，所述CD8阳性细胞是CD8阳性T细胞。在一些实施方案中，所述检测剂是造影剂。在一些实施方案中，所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
在另一方面，本发明提供一种用于检测组织中CD8阳性细胞的存在和/或量的方法，包括
a)使所述组织与本发明的缀合分子或本发明的检测剂接触；和
b)确定组织中CD8阳性细胞的存在和/或量。
在一些实施方案中，所述组织是血液组织。在一些实施方案中，所述组织是淋巴组织。在一些实施方案中，所述组织是肿瘤组织。在一些实施方案中，所述CD8阳性细胞是CD8阳性T细胞。在一些实施方案中，通过对所述组织成像确定组织中CD8阳性细胞的存在和/或量。在一些实施方案中，通过流式细胞术确定组织中CD8阳性细胞的存在和/或量。
在另一方面，本发明提供一种用于在对象中检测CD8阳性细胞的方法，包括给所述对象施用本发明的缀合分子或本发明的诊断剂。在一些实施方案中，所述方法用于检测对象体内的组织中的CD8阳性细胞的存在和/或量。在一些实施方案中，所述组织是肿瘤组织。在一些实施方案中，所述CD8阳性细胞是CD8阳性T细胞。
在一些实施方案中，所述方法还包括对所述对象进行成像例如ECT成像的步骤。在一些实施方案中，所述ECT成像是SPECT成像。在一些实施方案中，所述ECT成像是PET成像。用于通过SPECT或PET扫描的成像技术和装置是本领域公知的且可使用任何此类己知的ECT成像技术和装置。
在另一方面，本发明提供一种用于确定患有肿瘤的对象是否适于抗肿瘤疗法的方 法，所述方法包含
1)给所述对象施用本发明的缀合分子或本发明的检测剂，和
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则将所述对象鉴别为适于抗肿瘤疗法。
在另一方面，本发明提供一种用于预测患有肿瘤的对象对抗肿瘤疗法的反应的方法，所述方法包含
1)向所述对象施用本发明的缀合分子或本发明的检测剂，和
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则个体可能对抗肿瘤疗法起反应。
CD8阳性细胞例如CD8阳性T细胞的存在可以是抗肿瘤疗法反应的预测标记或存活的预后标记。举例来说，基线CD8阳性细胞肿瘤浸润是乳腺癌、头/颈癌和卵巢癌存活的预后指标。另外，在抗PD-1疗法或抗PDL-1疗法期间检测到的CD8阳性细胞的肿瘤浸润是对治疗反应的预测标记。
在另一方面，本发明提供一种治疗对象中的肿瘤的方法，所述方法包括：
1)向所述对象施用本发明的缀合分子或本发明的检测剂，和
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则给所述对象施用抗肿瘤疗法。
在另一方面，本发明提供一种用于监测抗肿瘤疗法在对象中的功效的方法，所述方法包含
1)给患有肿瘤且使用抗肿瘤疗法治疗的对象施用本发明的缀合分子或本发明的检测剂；
2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤中CD8阳性细胞的量。
在本发明各个方面的一些实施方案中，所述抗肿瘤疗法是免疫检查点抑制剂疗法。在一些实施方案中，抗肿瘤疗法包括施用PD-1抑制剂(例如，REGN2810、BGB-A317、纳武单抗、匹立珠单抗和派姆单抗)、PD-L1抑制剂(例如，阿特珠单抗、阿维鲁单抗、度伐单抗等)、CTLA-4抑制剂(例如，伊匹单抗)、TIM3抑制剂、BTLA抑制剂、TIGIT抑制剂、CD47抑制剂、GITR抑制剂、LAG3抑制剂、另一T细胞共抑制剂或配体的拮抗剂(例如，针对CD-28、2B4、LY108、LAIR1、ICOS、CD160或VISTA的抗体)、吲哚胺-2,3-双加氧酶(IDO)抑制剂、血管内皮生长因子(VEGF)拮抗剂、Ang2抑制剂(例如， 内瓦单抗)、转化生长因子β(TGFβ)抑制剂、表皮生长因子受体(EGFR)抑制剂(例如，埃罗替尼、西妥昔单抗)、CD20抑制剂(例如，抗CD20抗体，如利妥昔单抗)、针对肿瘤特异性抗原[例如，CA9、CA125、黑素瘤相关抗原3(MAGE3)、癌胚抗原(CEA)、波形蛋白、肿瘤-M2-PK、前列腺特异性抗原(PSA)、粘蛋白-1、MART-1和CA19-9]的抗体、疫苗(例如，卡介苗、癌症疫苗)、增加抗原呈递的佐剂(例如，粒细胞-巨噬细胞集落刺激因子)、双特异性抗体(例如，CD3×CD20双特异性抗体或PSMA×CD3双特异性抗体)、细胞毒素、化疗剂(例如，达卡巴嗪、替莫唑胺、环磷酰胺、多西他赛、道诺霉素、顺铂、卡铂、吉西他滨、甲氨蝶呤、米托蒽醌、奥沙利铂、太平洋紫杉醇和长春新碱)、环磷酰胺、放疗、IL-6R抑制剂(例如，沙瑞卢单抗)、IL-4R抑制剂(例如，杜匹鲁单抗)、IL-10抑制剂、细胞因子(如IL-2、IL-7、IL-21和IL-15)以及抗体-药物缀合物(ADC)(例如，抗CD19-DM4ADC和抗DS6-DM4ADC)。
在在本发明各个方面的一些实施方案中，所述肿瘤是实体肿瘤，包括但不限于结肠直肠癌、卵巢癌、前列腺癌、乳腺癌、脑癌、宫颈癌、膀胱癌、肛门癌、子宫癌、结肠癌、肝癌、胰腺癌、肺癌、子宫内膜癌、骨癌、睾丸癌、皮肤癌、肾癌、胃癌、食管癌、头颈癌、唾液腺癌以及骨髓瘤。
分离和/或纯化CD8阳性细胞
在另一方面，本发明还提供一种用于分离和/或纯化CD8阳性细胞的方法，所述方法包括：
(a)提供疑似包含CD8阳性细胞的细胞群；
(b)鉴定所述细胞群的亚群，所述亚群的细胞结合本发明的CD8结合多肽或本发明的缀合分子；和
(c)分离所述亚群。
在一些实施方案中，所述CD8阳性细胞是CD8阳性T细胞。在一些实施方案中，所述包含CD8阳性细胞的细胞群是人外周血单个核细胞(PBMC)。
在一些实施方案中，可以通过流式细胞术分离CD8阳性细胞。
在另一方面，本发明还提供一种用于分离CD8阳性细胞的方法，所述方法包括：
(a)提供疑似包含CD8阳性细胞的细胞群；
(b)使所述细胞群与本发明的CD8结合多肽或本发明的缀合分子接触，从而允许CD8阳性细胞结合至本发明的CD8结合多肽或本发明的缀合分子；
(c)去除未结合至本发明的CD8结合多肽或本发明的缀合分子的细胞；和
(c)回收结合至本发明的CD8结合多肽或本发明的缀合分子的CD8阳性细胞。
在一些实施方案中，所述CD8阳性细胞是CD8阳性T细胞。在一些实施方案中，所述包含CD8阳性细胞的细胞群是人外周血单个核细胞(PBMC)。
在一些实施方案中，本发明的CD8结合多肽或本发明的缀合分子固定化于固体表面上，例如固定化于凝胶或磁珠表面。
试剂盒
在另一方面，本发明提供一种试剂盒，其包含本发明的CD8结合多肽或本发明的缀合分子或本发明的诊断剂。所述试剂盒用于实施本发明的方法。试剂盒一般包括表明试剂盒内容物的预期用途的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
实施例
下面将通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1、针对CD8α的重链单域抗体的筛选
CD8α-Fc融合蛋白氨基酸序列为：
Figure PCTCN2021107312-appb-000001
氨基酸1-21为CD8α受体的信号肽，22-136为免疫球蛋白(Ig)结构域，137-181为CD8受体的Ig结构域，182-209为跨膜结构域，210-235为细胞质结构域。
图中单横线标记区域为Fc区域，双横线标记区域为linker。
1.1.文库的构建
免疫用的CD8α-Fc融合蛋白由CHO细胞表达，经Protein A亲和层析纯化得到。选取一只羊驼进行免疫。免疫结束后，提取羊驼外周血的淋巴细胞并按照Trizol使用说明提取RNA，并用Nanodrop测定RNA浓度。使用
Figure PCTCN2021107312-appb-000002
III First-Strand Synthesis System for RT-PCR试剂盒按照说明书将提取的RNA反转录成cDNA。用巢式PCR扩增编码重链抗体的可变区的核酸片段。
利用DNA产物纯化试剂盒纯化VHH片段，将载体与片段分别使用限制性内切酶sifi 50℃过夜酶切，对酶切后的片段进行切胶回收，将其克隆进入噬菌体展示用载体pComb3XSS中。产物随后电转化至大肠杆菌电转感受态细胞TG1中，构建针对CD8α的重链单域抗体噬菌体展示文库并对文库进行检定。通过梯度稀释铺板，计算库容的大小为5.04×10 9。为检测文库的插入率，随机选取48个克隆进行鉴定。结果显示插入率 已达到100％且大小正确。
1.2.针对CD8α的重链单域抗体淘选
用CD8α-cHis融合蛋白10μg/孔包被平板，4℃放置过夜。第二天用3％的BSA 37℃封闭2小时，PBST(PBS中含有0.05％吐温20)洗板5遍后，加入100μl噬菌体(1.71×10 11cfu，来自1.1所构建的骆驼重链单域抗体噬菌展示文库)，37℃下作用1小时。之后用PBST(PBS中含有0.05％吐温20)洗10遍，再用PBS溶液洗10遍，以洗掉不结合的噬菌体。之后每孔加入100uL的Gly-Hcl(PH＝2.5)37℃作用6-8min，将与CD8α特异性结合的噬菌体解离下并转移至一无菌的离心管中，迅速向其中加入1/10体积的Tris-Hcl(PH＝9.0)溶液中和缓冲液。取10uL进行梯度稀释，测定滴度并计算淘选回收率，并取中和后的噬菌体感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复几轮，每一轮淘选需改变淘选条件。由此，阳性的克隆被富集，达到了利用噬菌体展示技术筛取抗体库中CD8α特异抗体的目的。下表为亲和淘选的条件。
表1.亲和淘选条件
Figure PCTCN2021107312-appb-000003
1.3.用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
经过3-4轮淘选后，从最后一轮淘选的平板上随机挑取96个克隆进行鉴定。将随机挑选96个单菌落分别培养，生产并纯化噬菌体。用CD8α-Fc融合蛋白包被平板4℃过夜，3％BSA进行封闭，37℃反应1小时，随后每孔加入侵染后得到的噬菌体上清(对照组为空白噬菌体)，37℃孵育1h。PBST洗涤之后加入辣根过氧化物标记的抗M13二抗(购于北京义翘神州生物技术有限公司)，室温反应1小时。洗涤之后加入TMB显色液，450nm波长读取吸收值。同时用Fc蛋白包被平板4℃过夜，3％BSA进行封闭，37℃下反应1小时。洗涤之后加入侵染后得到的噬菌体上清(对照组为空白噬菌体)，37℃反应1小时。洗涤之后辣根过氧化物标记的抗M13二抗(购于北京义翘神州生物技术有限公司)，室温反应1小时。之后加入TMB显色液，450nm波长读取吸收值。其中当对CD8α-Fc蛋白的OD值除以空白对照OD值的比值>＝4时，判断候选抗体能结合CD8α-Fc蛋白；同时上述能结合CD8α-Fc抗原蛋白的抗体，其结合CD8α-Fc的OD值除以其结合Fc蛋白的OD值，比值>＝5时，则认为该候选抗体能特异性结合CD8α部分而非Fc部分。结果显示筛选出的抗体中，有能特异性结合CD8α，而不与Fc结合。将候选阳性克隆孔的菌转移至含有100μg/mL氨苄霉素的LB液体中培养以便提取质粒并进行测序。
表2.CD8α-Fc tag抗原phage ELISA鉴定
No. OD450 No. OD450 No. OD450 No. OD450 No. OD450 No. OD450
1 2.981 9 3.013 17 3.127 25 3.127 33 3.149 41 2.951
2 3.208 10 3.160 18 2.962 26 1.777 34 3.096 42 3.183
3 2.944 11 2.957 19 3.015 27 2.738 35 3.12 43 0.557
4 3.092 12 3.162 20 3.188 28 2.780 36 3.162 44 2.468
5 3.152 13 3.249 21 3.198 29 1.746 37 3.198 45 2.420
6 3.079 14 3.119 22 2.506 30 2.029 38 1.534 46 2.355
7 3.067 15 2.572 23 3.000 31 3.067 39 3.067 47 2.507
8 3.230 16 1.855 24 2.353 32 2.132 40 3.406 BLank 0.067
No. OD450 No. OD450 No. OD450 No. OD450 No. OD450 No. OD450
49 0.152 57 0.785 65 1.830 73 0.055 81 1.645 89 1.833
50 0.212 58 1.501 66 1.722 74 0.359 82 0.193 90 1.774
51 0.088 59 1.702 67 1.426 75 1.891 83 0.051 91 1.934
52 0.681 60 0.069 68 0.080 76 0.193 84 0.566 92 1.691
53 1.195 61 1.713 69 0.129 77 1.630 85 1.351 93 1.771
54 1.305 62 0.256 70 1.726 78 0.110 86 1.806 94 0.167
55 1.609 63 1.166 71 1.618 79 1.724 87 1.330 95 1.726
56 1.843 64 1.831 72 1.505 80 1.438 88 1.821 BLank 0.067
表3.Fc tag抗原phage ELISA鉴定
No. OD450 No. OD450 No. OD450 No. OD450 No. OD450 No. OD450
1 0.129 9 0.140 17 0.116 25 3.146 33 3.080 41 0.123
2 0.149 10 3.459 18 0.089 26 0.104 34 0.109 42 0.111
3 0.111 11 0.102 19 2.974 27 0.084 35 2.989 43 0.127
4 0.100 12 0.114 20 0.133 28 0.100 36 0.221 44 0.102
5 3.173 13 3.224 21 0.106 29 0.115 37 0.110 45 0.115
6 0.162 14 0.098 22 0.088 30 0.103 38 0.086 46 0.081
7 3.023 15 0.071 23 0.074 31 0.257 39 0.088 47 0.159
8 0.104 16 0.09 24 0.083 32 0.085 40 3.102 BLank 0.093
No. OD450 No. OD450 No. OD450 No. OD450 No. OD450 No. OD450
49 1.940 57 0.063 65 0.076 73 0.078 81 0.076 89 0.176
50 0.059 58 0.055 66 0.081 74 0.060 82 0.061 90 0.070
51 0.066 59 0.151 67 0.058 75 0.061 83 0.066 91 0.314
52 0.071 60 0.056 68 0.053 76 0.136 84 0.059 92 0.169
53 0.068 61 0.094 69 0.060 77 0.080 85 0.064 93 0.081
54 0.058 62 0.070 70 0.059 78 0.057 86 0.057 94 2.022
55 0.059 63 0.063 71 0.093 79 0.063 87 0.060 95 0.056
56 0.062 64 0.058 72 0.064 80 0.061 88 0.117 BLank 0.068
根据序列比对软件DNAMAN分析各个克隆的蛋白序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。最终获 得20条候选序列。
其中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。
表4. 20条候选单域抗体的氨基酸序列
Figure PCTCN2021107312-appb-000004
Figure PCTCN2021107312-appb-000005
Figure PCTCN2021107312-appb-000006
注：下划线部分为抗体序列的CDR区
实施例2、针对CD8α的重链单域抗体的初步评价鉴定
2.1.重链单域抗体在宿主菌大肠杆菌中表达、纯化
将实施例1测序分析所获得的候选单域抗体的编码序列亚克隆至表达载体PET22b中，并将测序鉴定正确的重组质粒转化到表达型宿主菌BL21中，其涂布在含有100微克每毫升氨苄青霉素的LB固体培养基的板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，37℃摇床培养至OD值达到0.6左右，0.5mM IPTG诱导，28℃摇床培养过夜。第二天，离心收菌，并将菌体超声破碎以获得抗体粗提液。然后镍离子亲和层析柱纯化抗体蛋白。最终得到纯度达90％以上的抗体蛋白。
2.2.用哺乳动物细胞制备CD8α抗体蛋白
针对筛选得到的CD8α单域抗体的核苷酸序列设计引物，以上述原核表达质粒为模板，通过PCR扩增各抗体的核苷酸序列(包含组氨酸标签)，然后克隆至pCDNA4(Invitrogen，Cat V86220)载体中。经基因序列测定确定所获得目的克隆基因序列的正确性。
C2-cHis抗体的核苷酸序列(SEQ ID NO:82):
Figure PCTCN2021107312-appb-000007
Figure PCTCN2021107312-appb-000008
C24-cHis抗体的核苷酸序列(SEQ ID NO:83):
Figure PCTCN2021107312-appb-000009
C27-cHis抗体的核苷酸序列(SEQ ID NO:84):
Figure PCTCN2021107312-appb-000010
C29-cHis抗体的核苷酸序列(SEQ ID NO:85):
Figure PCTCN2021107312-appb-000011
C37-cHis抗体的核苷酸序列(SEQ ID NO:86)：
Figure PCTCN2021107312-appb-000012
C42-cHis抗体的核苷酸序列(SEQ ID NO:87):
Figure PCTCN2021107312-appb-000013
C46-cHis抗体的核苷酸序列(SEQ ID NO:88):
Figure PCTCN2021107312-appb-000014
S5-cHis抗体的核苷酸序列(SEQ ID NO:89):
Figure PCTCN2021107312-appb-000015
S13-cHis抗体的核苷酸序列(SEQ ID NO:90):
Figure PCTCN2021107312-appb-000016
S36-cHis抗体的核苷酸序列(SEQ ID NO:91):
Figure PCTCN2021107312-appb-000017
将上述重组构建的带有组氨酸标签的单域抗体质粒转染HEK293细胞进行抗体表达。将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10％CO 2，90rpm中培养。培养5～6天后，收集瞬时表达培养上清液，组氨酸标签的单域抗体融合蛋白通过Ni+树脂凝胶亲和层析法进行纯化。
2.3.不同抗CD8α单域抗体对人CD8α蛋白的ELISA活性检测
CD8α-Fc蛋白由HEK293瞬时表达及proteinA亲和层析纯化获得。得到的CD8α-Fc蛋白0.5μg/孔4℃过夜包被平板，之后加入获得的抗CD8α单域抗体蛋白的梯度稀释系列，室温下反应1小时。洗涤之后加入anti-his辣根过氧化物酶标记抗体，室温反应1小时。洗涤之后加入显色液，450nm波长读取吸收值。应用软件SotfMaxPro v5.4进行数据处理和作图分析，通过四参数拟合，得到抗体对CD8α结合曲线及EC50值(所有供试抗体EC50值在0.989～4.11ng/mL之间)，在选取的10个抗体(详见表5)中，以C2-cHis为对照，比较不同抗CD8α单域抗体的相对活性，以反映抗体对CD8α的亲和能力。所测试的10个抗体中，C37、C42、C46、S5、S36具有较高的结合活性。
表5. 10种抗体对CD8α结合的ELISA结果
名称 EC50(ng/mL) 相对活性(％)
C2 2.199 100
C24 4.588 47.9
C27 2.280 96.4
C29 3.552 61.9
C2 3.18 100
C37 0.989 322
C42 1.18 269
C46 1.82 175
C2 4.11 100
S5 2.28 180
S13 2.89 142
S36 1.42 289
实施例3、CD8α单域抗体体外活性分析
3.1.通过FACS考察抗CD8α单域抗体对细胞表面CD8α结合效果
通过构建人CD8α全长蛋白基因的小鼠结肠癌MC38细胞稳定转染细胞株，获得在 膜上稳定表达人CD8α蛋白的MC38细胞(MC38-CD8α细胞)。在使用前将细胞培养至>90％汇合。使用抗CD8α单域抗体对这几种细胞进行流式细胞分析，用于定量分析间接免疫荧光染色。以确定对于每个细胞表面受体的数量。
收集细胞，并悬浮至2.5×10 6个细胞/mL的浓度。将200μL细胞的等分试样与20μg一抗混合，冰上孵育30分钟。细胞离心洗涤后重悬，加入5μL二抗-PE缀合物，冰上避光孵育30分钟。细胞洗涤两次，重悬，在BD FACSCelestaTM上进行流式细胞分析。对于每个管，收集最少5×10 4个事件。所有分析为单一颜色，在FL1中检测PE。向前散射(FS)和侧散射(SS)数据证明所有细胞群体紧密成群。
流式细胞术用于评价细胞的体外CD8α表达(表6)，20个单域抗体对MC38-CD8α细胞结合的阳性率均为93.6％以上，其中，C2、C37、C42、C46、S5、S36这六个单域抗体的阳性率均高于99.5％，阳性对照huOKT8(huOKT8为自行表达纯化蛋白，序列参考US20160024209A1)的阳性率为93.0％。
表6.FACS考察Antibody对细胞表面CD8α的结合效果
名称 阳性率(％)
C2 99.9
C20 98.9
C24 99.4
C27 93.6
C29 98.4
C30 98.8
C37 99.8
C42 99.9
C45 99.1
C46 99.5
S5 99.7
S6 97.5
S7 99.3
S13 96.3
S18 99.2
S22 97.0
S23 99.3
S29 99.0
S32 99.1
S36 99.7
OKT8 93.0
3.2.抗CD8α单域抗体对人PBMC CD8+T细胞表面抗原的结合
磁珠分离人外周血获得人CD8+T细胞，anti-CD8α PE conjugate(R&D Systems，Cat#FAB1509P)作为阳性对照，分析抗CD8α单域抗体C37-his对CD8+T细胞的结合。
将分离所得细胞浓度调至2.5×10 6个细胞/mL。将150μL细胞的等分试样与3μg 一抗混合，冰上孵育30分钟。细胞洗涤重悬后，加入5μL二抗-PE缀合物，冰上避光孵育30分钟。细胞洗涤两次，重悬，在BD FACSCelestaTM上进行流式细胞分析。对于每个管，收集最少5×10 4个事件。所有分析为单一颜色，在FL1中检测PE。向前散射(FS)和侧散射(SS)数据证明所有细胞群体紧密成群。
流式细胞术用于评价T细胞的CD8α表达(图1)，CD8α单域抗体C37-chis对人PBMC CD8+T细胞表面抗原有较好的结合能力。
3.3.鉴定抗CD8α单域抗体对CD8α结合能力(ForteBio法)
将抗CD8α单域抗体-biontin固定在SA生物传感器上。然后，将浓度为6.25-100nM的CD8α-chis结合到纳米抗体上，随后再进行解离。使用Octet Data Analysis version 9.0评估确定C37、C42、C46、S5四种抗体结合动力学变量，包括Kon、Koff和Kd。
测量的抗CD8α抗体的结合亲和力见表。结果表明C37-his蛋白对CD8α靶标蛋白的亲和力显著，其较高的Ka及较低的Kd值说明该抗体融合蛋白能更快速的结合CD8α抗原并很难解离下来。
表7.抗体对CD8α的结合亲和力
抗体编号 K D(M) K on(1/Ms) K off(1/s)
C37 6.441×10 -10 4.827×10 5 3.109×10 -4
C42 7.272×10 -10 5.782×10 5 4.205×10 -4
C46 1.081×10 -9 2.51×10 5 2.714×10 -4
S5 2.867×10 -9 1.551×10 5 4.446×10 -4
实施例4、 125I标记CD8α单域抗体体外分析
4.1.  125I标记CD8α单域抗体
为了方便表述，将单域抗体C37-cHis命名为SNA006a，将单域抗体C42-cHis命名为SNA006b，将单域抗体C46-cHis命名为SNA006c，将单域抗体S5-cHis命名为SNA006d。采用氯胺T法进行了 125I标记实验。
以0.05mol/L，PH＝7.5的PBS为溶剂配制10mg/mL的氯胺T溶液和偏重亚硫酸钠溶液。将2μL蛋白稀释到50μL PB溶液中，取50μL溶液加入1.5mCi(10μL)的Na 125I混合，混合溶液中加入氯胺T溶液，迅速混匀，室温下反应5min。加入偏重亚硫酸钠溶液50μL混合，终止反应。通过Radio-TLC扫描仪对标记率进行鉴定。经PD-10柱去除未反应的 125I并同时将产物缓冲体系更换为生理盐水。标记结果见图2。
4.2.  125I标记CD8α单域抗体MC38-CD8细胞摄取实验(Cell binding essay)
MC38-CD8细胞(CD8阳性)及MC38细胞(阴性对照)铺板于4个6孔板中，每孔1×10 6细胞，3mL培养基/孔，37℃，5％CO 2过夜培养；细胞取出，4℃孵育30min；取出所有6孔板，每个实验组分为总结合组和非特异结合组，每组三个平行样，故1个六孔板对应1组实验。将其中3孔培养基添加含有25μM未标记蛋白的培养基，作为 非特异性吸附的对照样品。再将培养基全部更换为相应的( 125I标记SNA006a，b，c，d)含有1μci(～5nM) 125I标记CD8单域抗体的培养基；37℃，5％CO 2继续孵育1h；孵育后收集上清，用预冷的含1％BSA的PBS洗涤细胞并入上清；余下的细胞沉淀用含有SDS的PBS重悬后，在γ计数器上分别测定上清和沉淀细胞的放射性计数cpm值，并进行衰变校正。
沉淀计数为结和组的细胞摄取部分(B)，上清和沉淀的放射性计数之和为总放射性(T)，该组B/T减去相应的非特异结合，即为该组10 6细胞摄取的放射免疫活性分数(按％计算)，结果如图3所示。
如图3所示， 125I标记CD8α单域抗体与MC38-CD8细胞有较高的结合百分比，而相应的CD8阴性细胞则没有摄取；且 125I标记CD8α单域抗体与MC38-CD8细胞的结合都能够被未标记的CD8单域抗体封闭；证明了 125I标记CD8单域抗体与MC38-CD8细胞的特异性结合能力。
同时，经计算， 125I-SNA006a在10 6个MC38-CD8细胞上的结合百分比为～50％，且最低结合的 125I-SNA006d也大于10％。表明 125I标记SNA006a、b、c、d均有较高的摄取，并且SNA006-a、b对CD8亲和力要高于SNA006-c、d。
4.3.  125I标记CD8α单域抗体饱和结合分析(Saturation binding essay)
细胞制备：饱和实验在96孔抽滤板中进行。MC38-CD8细胞培养于50mL的培养皿中，实验前，去除培养液，先用0.01M的无菌PBS冲洗两次，使用胰酶消化细胞后，用PBS洗两次，最后用5％BSA制成细胞悬液。细胞按照约10 5个/孔加到96孔抽滤板中。
用于封闭特异性结合的冷抗溶液制备：将10mg/mL的CD8单域抗体溶液(溶于0.01M PBS)使用5％BSA稀释10倍，成1mg/mL的溶液。
受体结合反应：采用放射性配基饱和法，分为总结合和非特异性结合两组。总结合组中设有浓度递增的12个剂量点，非特异性结合组相对也设12个剂量点，每个剂量点设有4组平行样，反应总体积为200μL，相应冷抗过量1000倍。
置于4℃孵育2h。到时间后，抽出上清液，使用4℃预冷的PBS洗6次，每次每孔加入PBS 200μL。最后收集滤膜，在γ计数器上测定每管试样和标准样的放射性计数。最后使用Prism 7.0(GraphPad Software,Inc.)软件按照单点结合模型进行饱和曲线拟合，计算K D和Bmax。
MC38-CD8细胞与 125I-CD8单域抗体的放射性配基结合分析饱和曲线如图4所示。
测量的 125I-CD8单域抗体的结合亲和力结果见表8。 125I标记CD8单域抗体与MC38-CD8细胞的结合都能够被未标记的CD8单域抗体封闭，证明了 125I标记CD8单域抗体与MC38-CD8细胞的特异性结合能力。 125I-SNA006a，其K D值在0.46nM，而 125I-SNA006c为3.8nM， 125I-SNA006d为1.5nM。其中 125I-SNA006a的亲和力明显优于 125I-SNA006-c、d。结果表明CD8单域抗体对CD8靶标蛋白的亲和力显著，其中亲和 力最强的为： 125I-SNA006a。
表8.CD8单域抗体对CD8的结合亲和力
125I-SNA006a K D 4.6X 10 -10M
125I-SNA006c K D 3.8X 10 -9M
125I-SNA006d K D 1.5X 10 -9M
4.4.  125I标记CD8α单域抗体竞争结合实验(Competition binding essay)
细胞制备：饱和实验在96孔抽滤板中进行。MC38-CD8细胞培养于50mL的培养皿中，实验前，去除培养液，先用0.01M的无菌PBS冲洗两次，使用胰酶消化细胞后，用PBS洗两次，最后用5％BSA制成细胞悬液。细胞按照约10 5个/孔加到96孔抽滤板中。
96孔细胞抽滤板使用前以结合缓冲液饱和并晾干。向抽滤板每孔中加入约10 5个细胞，及浓度递增的CD8单域抗体，然后每孔加入约2×10 5计数/min的 125I-CD8单域抗体，以结合缓冲液将反应体积调节到200μL。
4℃反应2h后，以真空泵及抽滤板配套设备将抽滤板中的反应液抽干，向每孔中加入200μL PBS缓冲液(0.01M，pH＝7.4)并抽干，重复洗6次。待抽滤板底部滤膜晾干，收集底部滤膜以γ计数器进行测量，用Prism 7.0(GraphPad Software,Inc.)计算并作图，非线性回归分析计算半数抑制浓度IC50。每个实验点设4个平行样，实验重复两次。本实验给出的结果是平均值加上标准偏差。
如图5所示， 125I-SNA006a与MC38-CD8细胞CD8受体的结合也能被SNA006a“冷”蛋白以浓度依赖的方式进行抑制，经Prism 7.0(GraphPad Software,Inc.)软件拟合得到的IC50值为～3.0nM。本文所述的竞争性结合测定表明了SNA006a对CD8抗原结合的高亲和力与高特异性。
实施例5、 125I标记CD8α单域抗体及其SPECT/CT成像
5.1.用于研究 125I标记CD8α单域抗体的动物模型。
用6～8周龄的雌性裸小鼠进行体内研究。在SPF环境中饲养裸鼠，可随意获取食物和水，标准12小时白天-夜晚照明循环。对于异种移植，在小鼠右前腿皮下植入100μl的细胞(MC38-CD8或MC38)/PBS。细胞接种密度约为5～6×10 6个细胞/小鼠。在异氟烷麻醉下实施植入。在这些条件下，在多于90％的注射的动物中，在1～2周后得到可用的肿瘤(100-300mm 3)(MC38-CD8或MC38)。
实验结束后，收取肿瘤组织，进行CD8免疫组化染色，分析CD8的表达水平，并与PET/CT检测的肿瘤整体CD8表达水平进行对比。
5.2.  125I标记CD8α单域抗体体内SPECT成像
按照5.1的方法接种MC38-CD8+/-肿瘤模型，将小鼠异氟烷麻醉后置于SPECT/CT 床，～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。分别在1h、2h、3h进行SPECT扫描，分析多个时间点的肿瘤及组织器官的放射性吸收情况。结果如图6所示。
注射 125I-CD8单域抗体后120min内，MC38-CD8移植瘤均清晰可见。从图6可以看到，右侧MC38-CD8+肿瘤与对侧阴性对照肿瘤MC38-CD8-相比具有良好的对比度， 125I-CD8单域抗体在肾脏中具有显著的浓聚，这表明其主要通过肾代谢。由于以上所述的碘标记方法均不可避免的产生脱碘现象，故SPECT结果会发生甲状腺及胃部的放射性浓聚。且 125I能量较弱，应用于SPECT显影会造成一定的组织自吸收。所以 125I标记的CD8单域抗体应用于SPECT显象目的是为了筛选出最佳的候选抗体，以应用于后续的 68Ga标记显像剂。结果表明 125I标记SNA006a、SNA006c、SNA006d均在肿瘤部位有较高的摄取，而SNA006a的显影效果明显优于SNA006c、SNA006d。SPECT的体内筛选实与相应的体外实验结果相吻合，该结果进一步证明了SNA006a在前述所有单域抗体中最具CD8示踪潜力。
实施例6、NOTA-CD8α单域抗体前体合成及 68Ga-NOTA-CD8α单域抗体放射性标记
6.1.NOTA-CD8α单域抗体前体合成
将双功能螯合剂p-SCN-NOTA偶联CD8单域抗体，偶联产物通过PD-10柱纯化并更换缓冲液至生理盐水，紫外分光光度计测280nm的吸光度，通过ESI-Q-TOF-MS分析偶联效率。采用体外细胞试验(肿瘤细胞摄取及亲和力检测)考察偶联产物对肿瘤细胞的靶向性，通过ELISA分析其体外与CD8α蛋白的亲和能力。
用PD-10柱(GE)将CD8α-cHis单域抗体(单域抗体包括C37、C46、S5)的缓冲液更换为一定pH的NaHCO 3缓冲液，p-SCN-Bn-NOTA(Macrocyclics，货号B-605)溶解于DMSO中，浓度为25mg/mL，将p-SCN-Bn-NOTA溶液以单域抗体上赖氨酸摩尔数的2倍加入到CD8α单域抗体中，室温反应一定时间，缀合产物使用PD-10柱(GE)纯化并超滤浓缩。
用ELISA测定上述标记后CD8α-NOTA的活性，具体方法如下：CD8α-Fc融合蛋白5μg/孔包被平板，4℃放置过夜。3％的BSA 37℃封闭后，加入梯度稀释的样品(以未偶联的CD8α单域抗体为标准品)，37℃下作用1小时。之后加入抗-his-HRP(购自Abcam公司)，室温反应1小时。之后加入显色液，450nm波长读取吸收值。ELISA的活性总结如表9。
表9.CD8α-cHis-NOTA的ELISA活性结果汇总
单域抗体编号 C37 C46 S5
CD8α-cHis-NaHCO 3相对活性(％) 76 76 98
CD8α-cHis-NOTA相对活性(％) 77 20 105
6.2.  68Ga-NOTA-CD8α单域抗体放射性标记
用0.1M的无菌HCl淋洗Eckert&Ziegler IGG100锗68/镓68(Ge 68/Ga 68)发生器制备 68Ga淋洗液，添加等体积0.2M醋酸钠溶液，并加入1/4体积含有上述NOTA-CD8抗体pH为5.3的0.1M醋酸钠缓冲液，反应体系的pH在4.5～4.7之间，室温反应10分钟。经PD-10柱去除未反应的离子镓并同时将产物缓冲体系更换为生理盐水，并用0.22um滤膜过滤。产物通过分析PH、Radio-TLC、Radio-HPLC、放射活度、放射化学纯度等进行质量控制。另：也可用0.05M的无菌HCl淋洗ITG锗68/镓68(Ge-68/Ga-68)发生器制备 68Ga淋洗液，添加1/2体积0.2M醋酸钠溶液，其他反应及质量控制条件均相同。
6.3.薄层色谱ITLC分析
ITLC-SG预切至1cm x 12cm条和用铅笔标记条各端彼此距离1cm。在展开槽中倾倒展开剂，覆盖槽，和令其平衡。在离ITLC条底部1cm铅笔线处加一滴 68Ga-CD8单域抗体注射液。将ITLC条放置在展开槽中和令其展开从加样点距离10cm(即至顶部铅笔线标志)。用辐射测量的ITLC扫描仪扫描ITLC，通过在层析图上对峰积分计算放射化学纯度(RCP)，分析结果如图7所示，表明正电子核素 68Ga成功标记单域抗体CD8。
实施例7、 68Ga-NOTA-CD8α单域抗体体内分布
7.1.用于研究 68Ga-NOTA-CD8α单域抗体的动物模型。
用6～8周龄的雌性裸小鼠进行体内研究。在SPF环境中饲养裸鼠，可随意获取食物和水，标准12小时白天-夜晚照明循环。对于异种移植，在小鼠右前腿皮下植入100μl的细胞(MC38-CD8或MC38)/PBS。细胞接种密度约为5～6×10 6个细胞/小鼠。在异氟烷麻醉下实施植入。在这些条件下，在多于90％的注射的动物中，在1～2周后得到可用的肿瘤(100-300mm 3)(MC38-CD8或MC38)。
实验结束后，收取肿瘤组织，进行CD8免疫组化染色，分析CD8的表达水平，并与PET/CT检测的肿瘤整体CD8表达水平进行对比。
7.2. 68Ga-NOTA-CD8α单域抗体体内分布
按照7.1的方法接种MC38-CD8+/-肿瘤模型，将～10μg放射性标记的单域抗体(～100μCi/10μg)尾静脉注射给予小鼠。在1、1.5小时采集数据，每个时间点使3只小鼠安乐死。对血液、肾、肝、脾、肺、心、肠、胃、肌肉、皮肤、脑、骨及CD8+/-肿瘤等目的组织解剖，并且在γ计数器上计数以收集数据。以注射剂量为总注射剂量。对于每一种器官，基于该总注射剂量确定％注射剂量(％ID)，并且将器官称重，用于确定每克的％注射剂量(％ID/g)。
图8显示了在携带MC38-CD8肿瘤的完好雄性小鼠中 68Ga-NOTA-CD8α单域抗体体内摄取的生物分布数据。其为在具有皮下肿瘤的完好雄性小鼠中(n＝3) 68Ga-NOTA-CD8摄取的离体生物分布数据。在静脉给药后1、1.5小时采集数据。 数据以平均％ID/g±标准偏差(S.D.)表示。所述比值的误差以标准偏差的几何平均数计算。如图8所示的数据，其中误差条代表该组的标准偏差。
如图9显示对于这些实验的肿瘤组织和血液摄取比例以及肿瘤组织和对侧正常肌肉摄取比例的结果。CD8单域抗体在表达靶标的MC38-CD8肿瘤中显示良好的肿瘤摄取，峰值肿瘤/肌肉比高，显示出 68Ga-NOTA-SNA006a单域抗体良好的肿瘤特异性。此标记物 68Ga-NOTA-SNA006a可能成为一种良好的肿瘤显像剂。
实施例8、 68Ga-NOTA-CD8α单域抗体体内PET成像
按照6.1的方法接种MC38-CD8+/-肿瘤模型。采用Micro-PET/CT(IRIS PET/CT,inviscan,Strasbourg,France)扫描，测量病灶％ID/g。在120分钟进行连续小动物PET/CT扫描，分析多个时间点的肿瘤、肌肉及肾脏的放射性吸收情况，如图10所示。采用基于蒙特卡洛模型的三维有序子集期望最大化算法(Monte-Carlo based 3D OSEM)进行图像重建。在肿瘤、肌肉、肝等器官用感兴趣区(ROI)法计算放射性活度(MBq/mL)，所得值除以注射剂量获得各组织对PET示踪剂摄取值(％ID/g)(假定组织密度为1g/ml)。计算结果分别如图10所示。
如图10结果显示，注射上述PET示踪剂 68Ga-NOTA-SNA006a后直到120min，MC38-CD8移植瘤均清晰可见，而CD8阴性表达的MC38移植瘤在注射后未见有摄取。因此，MC38-CD8肿瘤与对侧阴性对照肿瘤相比具有良好的区分度。根据PET扫描结果，分析多个时间点的肿瘤、肌肉及肾脏的放射性吸收情况。生物分布ROI结果使用活动时间曲线分析，如图11所示。
结果表明 68Ga-NOTA-SNA006a、 68Ga-NOTA-SNA006c、 68Ga-NOTA-SNA006d均在肿瘤部位有较高的摄取，而 68Ga-NOTA-SNA006a的显影效果明显优于c、d。PET的体内筛选实与相应的体外实验结果相吻合，该结果进一步证明了 68Ga-NOTA-SNA006a在前述所有单域抗体中最具CD8示踪潜力。
实施例9、 68Ga-NOTA-SNA006a单域抗体体内稳定性实验
取正常小鼠，自尾静脉注射 68Ga-NOTA-SNA006a 74MBq(1mCi/100μL PBS)。注射后0.5h和1h后，采集尿液样品，将所有样品加一定量的50％乙腈水溶液溶解后，8000转/min离心15min，取上清过0.22μm滤膜后，滤液用Radio-TLC进行分析。
Radio-TLC结果如图12显示，未见明显降解或 68Ga脱落现象，说明在此条件下，放射性标记抗体结构稳定，体内稳定应较好。

Claims (45)

  1. 一种CD8结合多肽，其包含至少一个能够特异性结合CD8α的免疫球蛋白单一可变结构域，所述至少一个免疫球蛋白单一可变结构域包含SEQ ID NO:5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77、81中任一个中的CDR1、CDR2和CDR3。
  2. 权利要求1的CD8结合多肽，其中所述至少一个免疫球蛋白单一可变结构域包含选自以下的CDR1、CDR2和CDR3：
    (1)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2，SEQ ID NO:4所示的CDR3；
    (2)SEQ ID NO:6所示的CDR1，SEQ ID NO:7所示的CDR2，SEQ ID NO:8所示的CDR3；
    (3)SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2，SEQ ID NO:12所示的CDR3；
    (4)SEQ ID NO:14所示的CDR1，SEQ ID NO:15所示的CDR2，SEQ ID NO:16所示的CDR3；
    (5)SEQ ID NO:18所示的CDR1，SEQ ID NO:19所示的CDR2，SEQ ID NO:20所示的CDR3；
    (6)SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2，SEQ ID NO:24所示的CDR3；
    (7)SEQ ID NO:26所示的CDR1，SEQ ID NO:27所示的CDR2，SEQ ID NO:28所示的CDR3；
    (8)SEQ ID NO:30所示的CDR1，SEQ ID NO:31所示的CDR2，SEQ ID NO:32所示的CDR3；
    (9)SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2，SEQ ID NO:37所示的CDR3；
    (10)SEQ ID NO:38所示的CDR1，SEQ ID NO:39所示的CDR2，SEQ ID NO:40所示的CDR3；
    (11)SEQ ID NO:42所示的CDR1，SEQ ID NO:43所示的CDR2，SEQ ID NO:44所示的CDR3；
    (12)SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2，SEQ ID NO:48所示的CDR3；
    (13)SEQ ID NO:50所示的CDR1，SEQ ID NO:51所示的CDR2，SEQ ID NO:52所示的CDR3；
    (14)SEQ ID NO:54所示的CDR1，SEQ ID NO:55所示的CDR2，SEQ ID NO:56所示的CDR3；
    (15)SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2，SEQ ID NO:60所示的CDR3；
    (16)SEQ ID NO:62所示的CDR1，SEQ ID NO:63所示的CDR2，SEQ ID NO:64所示的CDR3；
    (17)SEQ ID NO:66所示的CDR1，SEQ ID NO:67所示的CDR2，SEQ ID NO:68所示的CDR3；
    (18)SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2，SEQ ID NO:72所示的CDR3；
    (19)SEQ ID NO:74所示的CDR1，SEQ ID NO:75所示的CDR2，SEQ ID NO:76所示的CDR3；
    (20)SEQ ID NO:78所示的CDR1，SEQ ID NO:79所示的CDR2，SEQ ID NO:80所示的CDR3。
  3. 权利要求1或2的CD8结合多肽，其中所述免疫球蛋白单一可变结构域包含与SEQ ID NO：5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77和81之一的氨基酸序列具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％序列相同性的氨基酸序列。
  4. 权利要求1-3中任一项的CD8结合多肽，其中所述免疫球蛋白单一可变结构域包含SEQ ID NO：5、9、13、17、21、25、29、33、37、41、45、49、53、57、61、65、69、73、77和81之一的氨基酸序列。
  5. 权利要求1-4中任一项的CD8结合多肽，其中所述免疫球蛋白单一可变结构域是VHH。
  6. 核酸分子，其编码权利要求1-5中任一项的CD8结合多肽。
  7. 表达载体，其包含与表达调控元件可操作地连接的权利要求6的核酸分子。
  8. 宿主细胞，其包含权利要求6的核酸分子或以权利要求7的表达载体转化，并能够表达所述CD8结合多肽。
  9. 产生权利要求1-5中任一项的CD8结合多肽的方法，包括：
    a)在允许所述CD8结合多肽表达的条件下培养权利要求8的宿主细胞；
    b)从得自步骤a)的培养物回收由所述宿主细胞表达的CD8结合多肽；及
    c)任选进一步纯化和/或修饰得自步骤b)的CD8结合多肽。
  10. 一种缀合分子，其包含权利要求1-5任一项的CD8结合多肽，以及与所述CD8结合多肽缀合的至少一种可检测标记。
  11. 权利要求10的缀合分子，其中所述可检测标记选自放射性核素、荧光剂、化学发光剂、生物发光剂、顺磁离子和酶。
  12. 权利要求11的缀合分子，其中所述可检测标记选自 110In、 111In、 177Lu、 18F、 52Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 68Ge、 86Y、 90Y、 89Zr、 94mTc、 120I、 123I、 124I、 125I、 131I、 154-158Gd、 32P、 11C、 13N、 15O、 186Re、 188Re、 51Mn、 52mMn、 55Co、 72As、 75Br、 76Br、 82mRb、 83Sr或其它γ-、β-、或正电子发射体，例如，所述可检测标记为 68Ga或 125I。
  13. 权利要求10-12中任一项的缀合分子，其中所述CD8结合多肽通过螯合剂与所述可检测标记缀合。
  14. 权利要求13的缀合分子，其中所述螯合剂选自DTPA、EDTA、NOTA、DOTA、TRAP、TETA、NETA、CB-TE2A、Cyclen、Cyclam、Bispidine、TACN、ATSM、SarAr、AmBaSar、MAG 3、MAG 2、HYNIC、DADT、EC、NS 3、H2dedpa、HBED、DFO、PEPA或者HEHA及其衍生物。
  15. 权利要求14的缀合分子，其中所述可检测标记是 68Ga且所述螯合剂为NOTA。
  16. 一种检测生物学样品中CD8的存在和/或量的方法，包括：
    a)在权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子与CD8之间能够形成复合物的条件下，使所述生物学样品和对照样品接触本发明的CD8结合多肽或本发明的缀合分子；
    b)检测复合物的形成，
    其中所述生物学样品与对照样品之间复合物形成的差异指示样品中CD8的存在和/或量。
  17. 一种用于检测CD8阳性细胞的检测剂，其包含权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子，以及任选的生理学上可接受的载体。
  18. 权利要求17的检测剂，其是造影剂。
  19. 权利要求18的检测剂，其中所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
  20. 权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子在制备用于检测CD8阳性细胞的检测剂中的用途。
  21. 权利要求20的检测剂，其是造影剂。
  22. 权利要求21的检测剂，其中所述造影剂是ECT造影剂，例如SPECT造影剂或PET造影剂。
  23. 一种用于检测组织中CD8阳性细胞的存在和/或量的方法，包括
    a)使所述组织与权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂接触；和
    b)确定组织中CD8阳性细胞的存在和/或量。
  24. 权利要求23的方法，其中所述组织选自血液组织、淋巴组织、和肿瘤组织。
  25. 权利要求23或24的方法，其中所述CD8阳性细胞是CD8阳性T细胞。
  26. 权利要求23-25中任一项的方法，其中通过对所述组织成像确定组织中CD8阳性细胞的存在和/或量。
  27. 权利要求23-25中任一项的方法，其中通过流式细胞术确定组织中CD8阳性细胞的存在和/或量。
  28. 一种用于检测对象体内的组织中的CD8阳性细胞的存在和/或量的方法，包括 给所述对象施用权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂接触。
  29. 权利要求28的方法，其中所述组织是肿瘤组织。
  30. 权利要求28或29的方法，其中所述CD8阳性细胞是CD8阳性T细胞。
  31. 权利要求28-30中任一项的方法，其中所述方法还包括对所述对象进行成像例如ECT成像的步骤，例如所述ECT成像是SPECT成像或PET成像。
  32. 一种用于确定患有肿瘤的对象是否适于抗肿瘤疗法的方法，所述方法包含
    1)给所述对象施用权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂，和
    2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
    其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则将所述对象鉴别为适于抗肿瘤疗法。
  33. 一种用于预测患有肿瘤的对象对抗肿瘤疗法的反应的方法，所述方法包含
    1)向所述对象施用权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂，和
    2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
    其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则个体可能对抗肿瘤疗法起反应。
  34. 一种治疗对象中的肿瘤的方法，所述方法包括：
    1)向所述对象施用权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂，和
    2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤是否包含CD8阳性细胞，
    其中如果检测到所述肿瘤中CD8阳性细胞的存在，例如所述对象的肿瘤由CD8阳性细胞浸润，则给所述对象施用抗肿瘤疗法。
  35. 一种用于监测抗肿瘤疗法在对象中的功效的方法，所述方法包含
    1)给患有肿瘤且使用抗肿瘤疗法治疗的对象施用权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂；
    2)对所述对象进行成像例如ECT成像，以确定所述对象的肿瘤中CD8阳性细胞的量。
  36. 权利要求32-35中任一项的方法，其中所述抗肿瘤疗法是免疫检查点抑制剂疗法。
  37. 权利要求32-36中任一项的方法，其中抗肿瘤疗法选自施用PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、TIM3抑制剂、BTLA抑制剂、TIGIT抑制剂、CD47抑制剂、 GITR抑制剂、LAG3抑制剂、另一T细胞共抑制剂或配体的拮抗剂、吲哚胺-2,3-双加氧酶(IDO)抑制剂、血管内皮生长因子(VEGF)拮抗剂、Ang2抑制剂、转化生长因子β(TGFβ)抑制剂、表皮生长因子受体(EGFR)抑制剂、CD20抑制剂、针对肿瘤特异性抗原的抗体、疫苗、增加抗原呈递的佐剂、双特异性抗体、细胞毒素、化疗剂、环磷酰胺、放疗、IL-6R抑制剂、IL-4R抑制剂、IL-10抑制剂、细胞因子以及抗体-药物缀合物(ADC)。
  38. 权利要求32-37中任一项的方法，其中所述肿瘤是实体肿瘤。
  39. 权利要求38的方法，其中所述实体肿瘤选自结肠直肠癌、卵巢癌、前列腺癌、乳腺癌、脑癌、宫颈癌、膀胱癌、肛门癌、子宫癌、结肠癌、肝癌、胰腺癌、肺癌、子宫内膜癌、骨癌、睾丸癌、皮肤癌、肾癌、胃癌、食管癌、头颈癌、唾液腺癌以及骨髓瘤。
  40. 一种用于分离和/或纯化CD8阳性细胞的方法，所述方法包括：
    (a)提供疑似包含CD8阳性细胞的细胞群；
    (b)鉴定所述细胞群的亚群，所述亚群的细胞结合权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子；和
    (c)分离所述亚群。
  41. 一种用于分离CD8阳性细胞的方法，所述方法包括：
    (a)提供疑似包含CD8阳性细胞的细胞群；
    (b)使所述细胞群与权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子接触，从而允许CD8阳性细胞结合至权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子；
    (c)去除未结合至权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子的细胞；和
    (c)回收结合至权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子的CD8阳性细胞。
  42. 权利要求40或41的方法，其中所述CD8阳性细胞是CD8阳性T细胞。
  43. 权利要求40-42中任一项的方法，其中所述包含CD8阳性细胞的细胞群是人外周血单个核细胞(PBMC)。
  44. 权利要求40-44中任一项的方法，其中权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子固定化于固体表面上，例如固定化于凝胶或磁珠表面。
  45. 一种试剂盒，其包含权利要求1-5任一项的CD8结合多肽或权利要求10-15任一项的缀合分子或权利要求17-19任一项的检测剂。
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