Modified immunoglobulin single variable domains

Abstract

Belongs to the field of biomedicine. A modified immunoglobulin single variable domain with reduced pre-existing antibody binding capacity is provided. The immunoglobulin single variable domain is modified by amino acid substitution at a specific site and / or coupling extension, so that the binding of the immunoglobulin single variable domain with a pre-stored antibody is reduced. Uses of the modified immunoglobulin single variable domains are also provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a modified immunoglobulin single variable domain with reduced binding ability to pre-existing antibodies. More specifically, the present invention relates to modifying an immunoglobulin single variable domain by amino acid substitution at a specific site and / or by coupling extension, thereby reducing its binding to pre-existing antibodies. The present invention also relates to the use of the modified immunoglobulin single variable domain. Background of the Invention

[0003] In patients receiving biotherapeutic agents such as antibody drugs or fusion protein drugs, there may be pre-existing antibodies against these biotherapeutic agents. Pre-existing antibodies will have different effects on different therapeutic agents. For example, for cetuximab, pre-existing IgE antibodies against the carbohydrate (galα(1-3)gal) portion can cause severe hypersensitivity reactions; while for panitumumab, pre-existing antibodies will not have an effect on the later stage (The AAPS Journal, Vol.15, No.3, July 2013). Pre-existing antibodies may have an impact on treatment-induced ADA (anti-drug antibodies), safety of treatment, pharmacokinetics (PK), and pharmacodynamics (PD) of biotherapeutic drugs. It is becoming increasingly important to understand the impact of pre-existing antibodies on the risk-benefit profile of biotherapeutics. For specific biological agents, such as antibodies, the art expects to reduce their ability to bind to pre-existing antibodies, thereby reducing the impact of pre-existing antibodies on efficacy.

[0004] Brief description of the invention

[0005] The present invention provides at least the following embodiments:

[0006] Embodiment 1. A modified immunoglobulin single variable domain comprising the following modifications compared to an unmodified parent immunoglobulin single variable domain:

[0007] i) substitution of amino acids at positions 11 and 110, or

[0008] ii) substitution of amino acids at positions 84 and 113,

[0009] Wherein the modified immunoglobulin single variable domain has reduced binding to pre-existing antibodies compared to an unmodified parent immunoglobulin single variable domain.

[0010] Embodiment 2. The modified immunoglobulin single variable domain of embodiment 1, wherein, compared with the unmodified parent immunoglobulin single variable domain, the L at position 11 of the modified immunoglobulin single variable domain is substituted, and the T at position 110 is substituted.

[0011] Embodiment 3. The modified immunoglobulin single variable domain of embodiment 2, wherein the L at position 11 of the modified immunoglobulin single variable domain is replaced by C, and the T at position 110 is replaced by C, compared to the unmodified parent immunoglobulin single variable domain.

[0012] Embodiment 4. The modified immunoglobulin single variable domain of embodiment 1, wherein, compared with the unmodified parent immunoglobulin single variable domain, the A or P at position 84 of the modified immunoglobulin single variable domain is substituted, and the S at position 113 is substituted.

[0013] Embodiment 5. The modified immunoglobulin single variable domain of embodiment 4, wherein the A or P at position 84 of the modified immunoglobulin single variable domain is replaced by C, and the S at position 113 is replaced by C, compared to the unmodified parent immunoglobulin single variable domain.

[0014] Embodiment 6. The modified immunoglobulin single variable domain of any one of embodiments 1-5, wherein said immunoglobulin single variable domain is a VHH.

[0015] Embodiment 7. The modified immunoglobulin single variable domain of any one of embodiments 1-6, wherein the immunoglobulin single variable domain specifically binds to a target antigen selected from HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3, FAP.

[0016] Embodiment 8. The modified immunoglobulin single variable domain of any one of embodiments 1-7, wherein the immunoglobulin single variable domain specifically binds to a HER2 protein, such as a human HER2 protein.

[0017] Embodiment 9. The modified immunoglobulin single variable domain of embodiment 8, wherein the unmodified parent immunoglobulin single variable domain that specifically binds to the HER2 protein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0018] Embodiment 10. The modified immunoglobulin single variable domain of embodiment 8 or 9, wherein the modified immunoglobulin single variable domain that specifically binds to the HER2 protein comprises the amino acid sequence shown in SEQ ID NO: 2 or 5.

[0019] Embodiment 11. The modified immunoglobulin single variable domain of any one of embodiments 1-7, wherein the immunoglobulin single variable domain specifically binds to a CD8a protein, such as a human CD8a protein.

[0020] Embodiment 12. The modified immunoglobulin single variable domain of embodiment 11, wherein the unmodified parent immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO:3.

[0021] Embodiment 13. The modified immunoglobulin single variable domain of embodiment 11 or 12, wherein the modified immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO: 4 or 6.

[0022] Embodiment 14. A conjugate molecule comprising the modified immunoglobulin single variable domain according to any one of embodiments 1 to 13, and at least one chemical or biological moiety coupled thereto.

[0023] Embodiment 15. The conjugate molecule of embodiment 14, wherein the chemical moiety is a chelator, for example a chelator selected from the following: DOTA, DO3A, NODA-GA, DTPA, EDTA, NOTA, NO2A, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHA or their derivatives.

[0024] Embodiment 16. The conjugate molecule of embodiment 15, wherein the chelator chelates with a radionuclide, for example, the radionuclide 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, 99m Tc, 120 I. 123 I. 124I. 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.

[0025] Embodiment 17. The conjugate molecule of embodiment 14, wherein the chemical moiety is a small molecule drug, for example, a small molecule drug selected from the following: Dxd, SN38 (7-ethyl-10-hydroxycamptothecin), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbamide, mechlorethamine, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum (II) (DDP) cisplatin, daunorubicin, doxorubicin, actinomycin D, bleomycin, mithramycin, anthramycin, vincristine, vinblastine.

[0026] Embodiment 18. The conjugate molecule of embodiment 14, wherein the chemical moiety is a water-based polymer such as poly(ethylene glycol) or dextran.

[0027] Embodiment 19. The conjugate molecule of embodiment 14, wherein the biological moiety is selected from the group consisting of a polypeptide, a nucleic acid molecule, a lipid, and a carbohydrate.

[0028] Embodiment 20. The conjugate molecule of embodiment 19, wherein the biological part is selected from a bioluminescent agent such as a fluorescent protein, an enzyme (such as horseradish peroxidase), an immunoglobulin Fc region, abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokine, interleukin-1, interleukin-2, interleukin-6, interleukin-10, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor or IFN.

[0029] Embodiment 21. A pharmaceutical composition comprising the modified immunoglobulin single variable domain of any one of embodiments 1-13 or the conjugated molecule of any one of embodiments 14-20, and a pharmaceutically acceptable carrier.

[0030] Embodiment 22. The pharmaceutical composition of embodiment 21, wherein the pharmaceutical composition is used to treat a disease associated with an antigen specifically targeted by the immunoglobulin single variable domain.

[0031] Embodiment 23. The pharmaceutical composition of embodiment 22, wherein the immunoglobulin single variable domain specifically binds to HER2 protein, and the disease is a HER2-related cancer, for example, the cancer is selected from breast cancer, bladder cancer, lung cancer, head and neck cancer, prostate cancer, liver cancer, gastric cancer, endometrial cancer, kidney cancer, colon cancer, thyroid cancer, ovarian cancer, pancreatic cancer and glioblastoma.

[0032] Embodiment 24. A method for detecting the presence and / or amount of a target antigen in a biological sample, comprising:

[0033] a) contacting the biological sample and the control sample with the modified immunoglobulin single variable domain of any one of embodiments 1-13 or the conjugated molecule of any one of embodiments 14-20 under conditions capable of forming a complex between the modified immunoglobulin single variable domain of any one of embodiments 1-13 or the conjugated molecule of any one of embodiments 14-20 and the target antigen;

[0034] b) detecting the formation of a complex,

[0035] Wherein the difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of the target antigen in the sample.

[0036] Embodiment 25. The method of embodiment 24, wherein the biological sample is an ex vivo sample such as a blood sample.

[0037] Embodiment 26. The method of embodiment 24 or 25, wherein the target antigen is selected from HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3 and FAP.

[0038] Embodiment 27. A detection agent for detecting target antigen-positive cells, comprising the modified immunoglobulin single variable domain of any one of embodiments 1-13 or the conjugated molecule of any one of embodiments 14-20, and optionally a physiologically acceptable carrier.

[0039] Embodiment 28. The detection agent of embodiment 27, wherein the detection agent is used to detect the presence and / or amount of target antigen-positive cells in a tissue in a subject.

[0040] Embodiment 29. The detection agent of embodiment 27 or 28, wherein the tissue is tumor tissue.

[0041] Embodiment 30. The detection agent of any one of Embodiments 27-29, wherein the target antigen is selected from HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3 and FAP.

[0042] Embodiment 31. The detection agent of any one of Embodiments 27-30, wherein the detection agent is a contrast agent.

[0043] Embodiment 32. The detection agent of embodiment 31, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 .L11C-T110C mutation and conjugation reduced the binding of H1 to pre-existing antibodies.

[0046] Figure 2 .P84C-S113C mutation and conjugation reduced the binding of H1 to pre-existing antibodies.

[0047] Figure 3 .L11C-T110C mutation and conjugation reduced the binding of C37H to pre-existing antibodies.

[0048] Figure 4 .A84C-S113C mutation and coupling reduce the coupling of C37H to nanoantibodies.

[0049] Figure 5 .P14C-S113C mutation and conjugation could not reduce the binding of H1 and C37H to pre-existing antibodies.

[0050] Figure 6 .P14C-S112C mutation and conjugation could not reduce the binding of H1 and C37H to pre-existing antibodies.

[0051] Figure 7 .V12C-S112C mutation and conjugation could not reduce the binding of H1 and C37H to pre-existing antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0053] Unless otherwise indicated or defined, all terms used have their usual meaning in the art, which will be understood by those skilled in the art. Reference is made to, for example, standard manuals, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), Volumes 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd edition), Gower Medical Publishing, London, New York (1989), and the general prior art cited herein; in addition, unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art. Reference is also made to, for example, standard manuals, the above-mentioned general prior art and other references cited therein.

[0054] Unless otherwise indicated, the interchangeable terms "antibody" or "immunoglobulin" herein, whether referring to a heavy chain antibody or a conventional 4-chain antibody, are used as general terms to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms of "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0055] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed or transferred to other proteins without losing the rest of the protein and / or the function of the domain.

[0056] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional 4-chain antibody or a chain of a heavy chain antibody), or a polypeptide consisting essentially of such a globular region. An immunoglobulin domain is characterized in that it maintains the immunoglobulin fold characteristic of an antibody molecule.

[0057] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain that is essentially composed of four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to antibodies for antigens by having antigen binding sites.

[0058] The term "immunoglobulin single variable domain" as used herein refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain within the meaning of the present invention is a "domain antibody", such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") of Camelidae as defined below.

[0059] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e. "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VH domain") and the light chain variable domain present in conventional 4-chain antibodies (which is referred to herein as "VL domain"). The VHH domain specifically binds to an epitope without the need for additional antigen-binding domains (this is in contrast to the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.

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

[0061] For example, Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999) Figure 2 As shown in, the amino acid residues used for the VHH domain of Camelidae can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0062] In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen binding molecule or antigen binding protein (e.g., an immunoglobulin single variable domain of the present invention) can bind to. Its specificity can be determined based on the affinity and / or avidity of the antigen binding protein. The affinity, represented 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 stronger the binding strength between the epitope and the antigen binding protein (or, affinity can also be expressed as an association constant (KA), which is 1 / KD). As will be appreciated by those skilled in the art, affinity can be measured in a known manner, depending on the specific antigen of interest. Avidity is a measure of the binding strength between an antigen binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide containing it) and a related antigen. Avidity is related to the following two: the affinity between the antigen binding site on its antigen binding protein, and the number of related binding sites present on the antigen binding protein.

[0063] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or trace component) with which it is normally associated in the source or medium (culture medium) compared to its natural biological source and / or the reaction medium or culture medium from which it was obtained. In particular, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2 times, particularly at least 10 times, more particularly at least 100 times and up to 1000 times or more. An "isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique (e.g., a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).

[0064] As used herein, the term "subject" means a mammal, particularly a primate, especially a human.

[0065] The present inventors surprisingly found that by modifying an immunoglobulin single variable domain by amino acid substitution at specific sites, its binding to pre-existing antibodies can be significantly reduced.

[0066] Therefore, in a first aspect, the present invention provides a modified immunoglobulin single variable domain comprising the following modifications compared to an unmodified parent immunoglobulin single variable domain:

[0067] i) substitution of amino acids at positions 11 and 110, or

[0068] ii) substitution of amino acids at positions 84 and 113,

[0069] Wherein the modified immunoglobulin single variable domain has reduced binding to pre-existing antibodies compared to an unmodified parent immunoglobulin single variable domain.

[0070] Pre-existing antibodies are antibodies that are already present in the subject to which the biotherapeutic is to be administered, particularly those directed against the biotherapeutic. Pre-existing antibodies may be present in a naive subject (i.e., a subject to which the biotherapeutic has never been administered before). The biotherapeutic is, for example, an immunoglobulin single variable domain of the invention.

[0071] In some embodiments, the numbering of the amino acids is the Kabat numbering. In general, it is well known to those skilled in the art that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.

[0072] In some embodiments, compared to the unmodified parent immunoglobulin single variable domain, L at position 11 is substituted and T at position 110 is substituted in the modified immunoglobulin single variable domain.

[0073] In some embodiments, compared to the unmodified parent immunoglobulin single variable domain, the L at position 11 of the modified immunoglobulin single variable domain is replaced by C, and the T at position 110 is replaced by C.

[0074] In some embodiments, compared to the unmodified parent immunoglobulin single variable domain, the A or P at position 84 of the modified immunoglobulin single variable domain is substituted, and the S at position 113 is substituted.

[0075] In some embodiments, the A or P at position 84 of the modified immunoglobulin single variable domain is substituted with C, and the S at position 113 is substituted with C, compared to the unmodified parent immunoglobulin single variable domain.

[0076] In some embodiments, the immunoglobulin single variable domain is a VHH.

[0077] In some embodiments, the immunoglobulin single variable domain specifically binds to a target antigen. In some embodiments, the target antigen includes but is not limited to HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3, FAP.

[0078] In some embodiments, the immunoglobulin single variable domain specifically binds to a HER2 protein, such as a human HER2 protein. An exemplary human HER2 protein comprises the amino acid sequence shown in SEQ ID NO: 13:

[0079] MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCY QDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAED GTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNP HQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT

[0080] In some embodiments, the unmodified parent immunoglobulin single variable domain that specifically binds to the HER2 protein comprises the amino acid sequence shown in SEQ ID NO:1.

[0081] In some embodiments, the modified immunoglobulin single variable domain that specifically binds to a HER2 protein comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the modified immunoglobulin single variable domain that specifically binds to a HER2 protein comprises the amino acid sequence shown in SEQ ID NO: 5.

[0082] In some embodiments, the immunoglobulin single variable domain specifically binds to a CD8a protein, such as a human CD8a protein. An exemplary human CD8a protein comprises the amino acid sequence shown in SEQ ID NO: 14:

[0083] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAG

[0084] In some embodiments, the unmodified parent immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO:3.

[0085] In some embodiments, the modified immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the modified immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO: 6.

[0086] The inventors have also surprisingly found that the binding of the modified immunoglobulin single variable domain to pre-existing antibodies can be further reduced by chemically coupling at least one chemical part or biological part. On the other hand, the present invention provides a conjugate molecule comprising the modified immunoglobulin single variable domain of the present invention and at least one chemical part or biological part coupled thereto.

[0087] In some embodiments, the modified immunoglobulin single variable domain is covalently coupled to the chemical moiety or biological moiety. In some embodiments, the modified immunoglobulin single variable domain is chemically coupled to the chemical moiety or biological moiety directly or through a linker. Methods for coupling a chemical moiety or biological moiety to a polypeptide are well known to those skilled in the art.

[0088] In some embodiments, the chemical moiety can be a chelating agent. For example, suitable chelating agents include, but are not limited to, DOTA, DO3A, NODA-GA, DTPA, EDTA, NOTA, NO2A, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHA, or derivatives thereof.

[0089] In some embodiments, the chelating agent chelates with a radionuclide. The radionuclide may be a radionuclide with an energy between 20-4000 KeV. The radionuclide may include but is 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, 99m 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.

[0090] In some embodiments, the chemical moiety can also be a small molecule drug. For example, suitable small molecule drugs include but are not limited to Dxd, SN38 (7-ethyl-10-hydroxycamptothecin), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbamide, mechlorethamine, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, daunorubicin, doxorubicin, actinomycin D, bleomycin, mithramycin, anthramycin, vincristine, vinblastine.

[0091] In some embodiments, the chemical moiety may also be a water-based polymer such as poly(ethylene glycol) or dextran.

[0092] In some preferred embodiments, the chemical moiety is DOTA.In some preferred embodiments, the chemical moiety is Dxd.In some preferred embodiments, the chemical moiety is NODA-GA.

[0093] In some embodiments, the biological part includes, but is not limited to, polypeptides, nucleic acid molecules, lipids, carbohydrates, etc. For example, the biological part can be a bioluminescent agent such as a fluorescent protein, an enzyme (such as horseradish peroxidase), an immunoglobulin Fc region, etc. Alternatively, the biological part can be abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokine, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), interleukin-10 ("IL-10"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or IFN, etc.

[0094] In some embodiments, the modified immunoglobulin single variable domain or conjugate molecule of the present invention has a lower binding affinity and / or avidity to a pre-existing antibody than an unmodified parent immunoglobulin single variable domain. For example, the KD of the modified immunoglobulin single variable domain or conjugate molecule binding to a pre-existing antibody is 150% or more (e.g., 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650% or more) of the KD of the parent immunoglobulin single variable domain to the pre-existing antibody. Alternatively, the modified immunoglobulin single variable domain or conjugate molecule has an average % signal inhibition of at least 10% or more (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more, e.g., about 10%-about 90%, about 10%-about 50%, about 10%-about 40%, about 10%-about 30%) compared to the parent immunoglobulin single variable domain as determined by the method described in the Examples of the present application.

[0095] In some embodiments, the modified immunoglobulin single variable domain or conjugate molecule of the present invention has a binding affinity and / or avidity comparable to that of the unmodified parent immunoglobulin single variable domain for the target antigen. For example, the affinity of the modified immunoglobulin single variable domain or conjugate molecule binding to the target antigen is at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 150% or more of the affinity of the parent immunoglobulin single variable domain to the target antigen. The relative affinity can be determined by the method described in the examples of the present application.

[0096] In another aspect, the present invention relates to nucleic acid molecules encoding the modified immunoglobulin single variable domains of the present invention. The nucleic acid of the present invention may be RNA, DNA or cDNA. Those skilled in the art may select nucleic acid molecules encoding the modified immunoglobulin single variable domains of the present invention as needed or by conventional means.

[0097] The nucleic acid of the present invention may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, a cosmid or a YAC. The vector may be an expression vector in particular, i.e., a vector that can provide for expression of the modified immunoglobulin single variable domain in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector generally comprises at least one nucleic acid of the present invention, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host. Specific examples of regulatory elements and other elements useful or necessary for expression of the modified immunoglobulin single variable domain of the present invention, such as promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0098] The nucleic acids of the invention can be prepared or obtained by known means (e.g. by automated DNA synthesis and / or recombinant DNA technology) based on the information on the amino acid sequences of the polypeptides of the invention given herein, and / or can be isolated from suitable natural sources.

[0099] In another aspect, the invention relates to a host cell expressing or capable of expressing one or more modified immunoglobulin single variable domains of the invention and / or containing a nucleic acid or vector of the invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

[0100] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0101] Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0102] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0103] However, the present invention may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0104] The present invention also provides a method for producing the modified immunoglobulin single variable domain of the present invention, the method generally comprising the following steps:

[0105] - culturing the host cell of the invention under conditions allowing expression of the modified immunoglobulin single variable domain of the invention; and

[0106] - recovering the modified immunoglobulin single variable domain expressed by said host cell from the culture; and

[0107] - Optionally further purifying and / or modifying the modified immunoglobulin single variable domain of the invention.

[0108] The modified immunoglobulin single variable domains of the present invention can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0109] Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. are known in the art. Similarly, protein separation and purification techniques suitable for use in the methods of making the modified immunoglobulin single variable domains of the present invention are well known to those skilled in the art.

[0110] However, the modified immunoglobulin single variable domains of the present invention can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0111] The present invention provides a pharmaceutical composition comprising the modified immunoglobulin single variable domain or conjugated molecule of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used to treat a disease associated with an antigen specifically targeted by the immunoglobulin single variable domain.

[0112] In some embodiments, the immunoglobulin single variable domain specifically binds to the HER2 protein, and the disease is a HER2-related disease, such as a HER2-related cancer. The cancer includes, but is not limited to, breast cancer, bladder cancer, lung cancer, head and neck cancer, prostate cancer, liver cancer, gastric cancer, endometrial cancer, kidney cancer, colon cancer, thyroid cancer, ovarian cancer, pancreatic cancer, and glioblastoma. Preferably, the cancer is a cancer in which the tumor tissue overexpresses HER2. More preferably, the cancer is breast cancer.

[0113] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0114] In another aspect, the present invention provides use of the modified immunoglobulin single variable domain or conjugated molecule of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease associated with an antigen specifically targeted by the immunoglobulin single variable domain.

[0115] In some embodiments, the immunoglobulin single variable domain specifically binds to the HER2 protein, and the disease is a HER2-related disease, such as a HER2-related cancer. The cancer includes, but is not limited to, breast cancer, bladder cancer, lung cancer, head and neck cancer, prostate cancer, liver cancer, gastric cancer, endometrial cancer, kidney cancer, colon cancer, thyroid cancer, ovarian cancer, pancreatic cancer, and glioblastoma. Preferably, the cancer is a cancer in which the tumor tissue overexpresses HER2. More preferably, the cancer is breast cancer.

[0116] In another aspect, the present invention provides a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a modified immunoglobulin single variable domain or a conjugated molecule of the present invention or a pharmaceutical composition of the present invention, wherein the disease is a disease associated with an antigen specifically targeted by the immunoglobulin single variable domain.

[0117] In some embodiments, the immunoglobulin single variable domain specifically binds to the HER2 protein, and the disease is a HER2-related disease, such as a HER2-related cancer. The cancer includes, but is not limited to, breast cancer, bladder cancer, lung cancer, head and neck cancer, prostate cancer, liver cancer, gastric cancer, endometrial cancer, kidney cancer, colon cancer, thyroid cancer, ovarian cancer, pancreatic cancer, and glioblastoma. Preferably, the cancer is a cancer in which the tumor tissue overexpresses HER2. More preferably, the cancer is breast cancer.

[0118] In another aspect, the present invention provides a method for detecting the presence and / or amount of a target antigen in a biological sample, comprising:

[0119] a) contacting the biological sample and the control sample with the modified immunoglobulin single variable domain or conjugated molecule of the present invention under conditions in which a complex can be formed between the modified immunoglobulin single variable domain or conjugated molecule of the present invention and the target antigen;

[0120] b) detecting the formation of a complex,

[0121] The difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of the target antigen in the sample. In some embodiments, the biological sample is an ex vivo sample such as a blood sample. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD8a.

[0122] On the other hand, the present invention provides a detection agent for detecting target antigen positive cells, which comprises a modified immunoglobulin single variable domain or conjugated molecule of the present invention, and an optional physiologically acceptable carrier. In some embodiments, the detection agent is used to detect the presence and / or amount of target antigen positive cells in tissues in a subject. In some embodiments, the tissue is tumor tissue. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD8a.

[0123] Therefore, in some embodiments, the detection agent is a contrast agent. In some embodiments, the contrast agent is an ECT (Emission Computed Tomography) contrast agent, such as a SPECT (Single-Photon Emission Computed Tomography) contrast agent or a PET (Positron Emission Tomography, Positron Emission Tomography, PET) contrast agent.

[0124] On the other hand, the present invention provides the use of the modified immunoglobulin single variable domain or conjugated molecule of the present invention in the preparation of a detection agent for detecting target antigen-positive cells. In some embodiments, the detection agent is used to detect the presence and / or amount of target antigen-positive cells in a tissue in a subject. In some embodiments, the tissue is a tumor tissue. In some embodiments, the detection agent is a contrast agent. In some embodiments, the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD8a.

[0125] On the other hand, the present invention provides a method for detecting target antigen-positive cells in a subject, comprising administering to the subject a modified immunoglobulin single variable domain or conjugated molecule of the present invention or a detection agent of the present invention. In some embodiments, the method is used to detect the presence and / or amount of target antigen-positive cells in a tissue in the subject. In some embodiments, the tissue is a tumor tissue. In some embodiments, the method further comprises a step of imaging the subject, such as ECT imaging. In some embodiments, the ECT imaging is SPECT imaging. In some embodiments, 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 can be used. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is CD8a. Example

[0126] The present invention will be further described below by way of examples, but the present invention is not limited to the described examples.

[0127] Example 1: Screening test for individuals with pre-existing antibody positive

[0128] Serum was collected from 57 healthy individuals, and individuals with potential positive pre-existing antibodies were screened by ELISA. Nanoantibody H1 that specifically binds to HER2 and nanoantibody C37H that specifically binds to CD8a were coated with 0.1 μg / well at 4°C overnight, and 150-fold diluted individual serum was added after blocking, and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added, and the absorbance value was read at a wavelength of 450nm. SoftMaxPro 6.5.1 and Microsoft excel were used for data processing and analysis, and the OD values ​​of all individual serum samples were normalized by S / N and Log10 (S / N) with the mean OD value of NC (100% PNHS mixed serum). Screening threshold factor (SCPF) = Mean Response of Log10 (S / N) + 1.645 * Stdev Response of Log10 (S / N), from which the SCPF (transferred to S / N) of serum samples for H1 and C37H were 1.046 and 1.048 respectively. When the OD value of individual serum sample > SCPF (transferred to S / N), the individual is judged to be potentially positive for pre-existing antibodies. The potentially positive individuals for pre-existing antibodies screened by this method are shown in Table 1-1.

[0129] Table 1-1. Potentially positive individuals with pre-existing antibodies

[0130] Nanobodies Pre-stored antibody potential positive Donor No. H1 24、30、32、33、36、38、43、50 C37H 24、30、32、38、43、50

[0131] Example 2: Nanobody L11C-T110C mutation inhibits pre-existing antibody binding

[0132] 2.1. Site mutation

[0133] Nanobodies H1 and C37H were both subjected to L11C-T110C mutation. Corresponding primers were designed for the mutation sites of the mutated antibodies, and the nucleotide sequences of each antibody were amplified by PCR using the pre-mutation bacterial solution as a template, and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector, and the gene sequence was determined to determine the correctness of the obtained target clone gene sequence, thereby advancing subsequent experiments. The amino acid sequences before and after the mutation are shown below:

[0134] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0135] H1 (SEQ ID NO: 1):

[0136] QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVSS

[0137] H1-L11C-T110C (SEQ ID NO: 2):

[0138] QLQLVESGGG C VQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQV C VSS

[0139] C37H (SEQ ID NO: 3):

[0140] QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVSS

[0141] C37H-L11C-T110C(SEQ ID NO:4):

[0142] QVQLVESGGG C VQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLV C VSS

[0143] The mutation sites are underlined.

[0144] 2.2. Effect of L11C-T110C mutation on the affinity of nanobodies

[0145] 2.2.1. Affinity of H1-L11C-T110C

[0146] HER2-Fc was coated on the plate at 0.1 μg / well at 4°C overnight. After blocking, samples diluted with H1-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, colorimetric solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 2-1, compared with the original nanoantibody H1, the mutant H1-L11C-T110C retained its affinity for the antigen target.

[0147] Table 2-1 Affinity of mutant H1-L11C-T110C

[0148] Antibody Name EC50(ng / mL) Relative activity (%) H1 76.7 100.0 H1-L11C-T110C 65.4 117.3

[0149] 2.2.2. Affinity of C37H-L11C-T110C

[0150] CD8a-Fc was coated on the plate at 0.5 μg / well at 4°C overnight. After blocking, samples diluted with C37-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, colorimetric solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 2-2, compared with the original nanoantibody C37H, the mutant C37H-L11C-T110C retained its affinity for the antigen target.

[0151] Table 2-2 Affinity of mutant C37H-L11C-T110C

[0152] Antibody Name EC50(ng / mL) Relative activity (%) C37H 5.1 100.0 C37H-L11C-T110C 3.1 162.6

[0153] 2.3. Effect of L11C-T110C mutation on the binding of nanobodies to pre-existing antibodies

[0154] H1, C37H and the corresponding L11C-T110C mutants were coated with 0.1 μg / well at 4°C overnight, and 150-fold diluted positive individual serum was added after blocking, and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 450nm. The software SoftMax Pro 6.5.1 and Microsoft excel were used for data processing and graphical analysis, and the average % signal inhibition was used to evaluate the effects of nano antibody amino acid substitution and coupling extension. The higher the average % signal inhibition value, the weaker the binding to the pre-existing antibody. Average % signal inhibition = (original nano antibody OD value-mutant OD value) / original nano antibody OD value × 100%. As shown in Table 2-3, the L11C-T110C mutation can reduce the binding of nano antibodies to pre-existing antibodies.

[0155] Table 2 - Effect of 3L11C-T110C mutation on the binding of nanobodies to pre-existing antibodies

[0156] Parental clone Sequence modification Average % signal inhibition (average of positive individuals) H1 H1-L11C-T110C 20 C37H C37H-L11C-T110C 24

[0157] Example 3: Nanobody A / P84C-S113C mutation inhibits pre-existing antibody binding

[0158] 3.1. Site mutation

[0159] Nanobody H1 and C37H were both subjected to A / P84C-S113C mutation (the two pairs of mutation sites were as follows Figure 1 As shown). Corresponding primers were designed for the mutation sites of the mutated antibodies, and the nucleotide sequences of each antibody were amplified by PCR using the pre-mutation bacterial solution as a template, and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector, respectively. The gene sequence was determined to determine the correctness of the obtained target clone gene sequence, so as to promote subsequent experiments. The amino acid sequence after mutation is shown below:

[0160] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0161] H1-P84C-S113C (SEQ ID NO: 5):

[0162] QLQLVESGGGLVQPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLK C EDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVS C

[0163] C37H-A84C-S113C (SEQ ID NO:6):

[0164] QVQLVESGGGLVQPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDG NTYYADSVKGRFTISRDNSKNHVYLQMNSLR C EDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVS C

[0165] The mutation sites are underlined.

[0166] Effect of the A / P84C-S113C mutation on the affinity of nanobodies

[0167] 3.2.1. Affinity of H1-P84C-S113C

[0168] HER2-Fc was coated on the plate at 0.1 μg / well at 4°C overnight. After blocking, samples diluted with H1-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 3-1, compared with the original nanoantibody, the mutant H1-P84C-S113C retained its affinity for the antigen target.

[0169] Table 3-1 Affinity of mutant H1-P84C-S113C

[0170] Antibody Name EC50(ng / mL) Relative activity (%) H1 29.7 100.0 H1-P84C-S113C 32.1 92.5

[0171] 3.2.2. Affinity of C37H-A84C-S113C

[0172] CD8a-Fc was coated on the plate at 0.5 μg / well at 4°C overnight. After blocking, samples diluted with C37-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 3-2, compared with the original nanoantibody, the mutant C37H-A84C-S113C retained its affinity for the antigen target.

[0173] Table 3-2 Affinity of mutant C37H-A84C-S113C

[0174] Antibody Name EC50(ng / mL) Relative activity (%) C37H 7.7 100.0 C37H-A84C-S113C 8.2 93.3

[0175] Effect of 3.3A / P84C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0176] H1, C37H and the corresponding A / P84C-S113C mutants were coated with 0.1 μg / well at 4°C overnight, and 150-fold diluted positive individual serum was added after blocking, and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 450nm. The software SoftMax Pro 6.5.1 and Microsoft excel were used for data processing and graphical analysis, and the average % signal inhibition was used to evaluate the effects of amino acid substitution and coupling extension of nano antibodies. The higher the average % signal inhibition value, the weaker the binding to the pre-existing antibody. Average % signal inhibition = (original nano antibody OD value - mutant OD value) / original nano antibody OD value × 100%. As shown in Table 3-3, the A / P84C-S113C mutation can reduce the binding of nano antibodies to pre-existing antibodies.

[0177] Table 3-3 Effect of the A / P84C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0178]

[0179]

[0180] Example 4: Nanobody P14C-S113C mutation cannot inhibit pre-existing antibody binding

[0181] 4.1. Site mutation

[0182] Nanobody H1 and C37H were both subjected to P14C-S113C mutation (the two pairs of mutated sites are as follows Figure 1 As shown). Corresponding primers were designed for the mutation sites of the mutated antibodies, and the nucleotide sequences of each antibody were amplified by PCR using the pre-mutation bacterial solution as a template, and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector, respectively. The gene sequence was determined to determine the correctness of the obtained target clone gene sequence, so as to promote subsequent experiments. The amino acid sequence after mutation is shown below:

[0183] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0184] H1-P14C-S113C (SEQ ID NO: 7):

[0185] QLQLVESGGGLVQ C GGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTVS C

[0186] C37H-P14C-S113C (SEQ ID NO:8):

[0187] QVQLVESGGGLVQ C GGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTVS C

[0188] The mutation sites are underlined.

[0189] Effect of P14C-S113C mutation on the affinity of nanobodies

[0190] 4.2.1. Affinity of H1-P14C-S113C

[0191] HER2-Fc was coated on the plate at 0.1 μg / well at 4°C overnight. After blocking, samples diluted with H1-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 4-1, compared with the original nanoantibody, the mutant H1-P14C-S113C retained its affinity for the antigen target.

[0192] Table 4-1 Affinity of mutant H1-P14C-S113C

[0193] Antibody Name EC50(ng / mL) Relative activity (%) H1 411.4 100.0 H1-P14C-S113C 435.7 94.4

[0194] 4.2.2. Affinity of C37H-P14C-S113C

[0195] CD8a-Fc was coated on the plate at 0.5 μg / well at 4°C overnight. After blocking, samples diluted with C37-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, colorimetric solution was added and the absorbance was read at 450 nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 4-2, compared with the original nanoantibody, the mutant C37H-P14C-S113C retained its affinity for the antigen target.

[0196] Table 4-2 Affinity of mutant C37H-P14C-S113C

[0197] Antibody Name EC50(ng / mL) Relative activity (%) C37H 30.8 100.0 C37H-P14C-S113C 60.7 50.8

[0198] Effect of P14C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0199] H1, C37H and the corresponding P14C-S113C mutants were coated with 0.1 μg / well at 4°C overnight, and 150-fold diluted positive individual serum was added after blocking, and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 450nm. The software SoftMax Pro 6.5.1 and Microsoft excel were used for data processing and graphical analysis, and the average % signal inhibition was used to evaluate the effects of amino acid substitution and coupling extension of nano antibodies. The higher the average % signal inhibition value, the weaker the binding to the pre-existing antibody. Average % signal inhibition = (original nano antibody OD value - mutant OD value) / original nano antibody OD value × 100%. As shown in Table 4-3, the P14C-S113C mutation cannot reduce the binding of nano antibodies to pre-existing antibodies.

[0200] Table 4-3 Effect of P14C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0201] Parental clone Sequence modification Average % signal inhibition (average of positive individuals) H1 H1-P14C-S113C -30 C37H C37H-P14C-S113C -3

[0202] Example 5: Nanobody P14C-S112C mutation cannot inhibit pre-existing antibody binding

[0203] 5.1. Site mutation

[0204] Nanobody H1 and C37H were both subjected to P14C-S112C mutation (the two pairs of mutated sites are as follows Figure 1As shown). Corresponding primers were designed for the mutation sites of the mutated antibodies, and the nucleotide sequences of each antibody were amplified by PCR using the pre-mutation bacterial solution as a template, and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector, respectively. The gene sequence was determined to determine the correctness of the obtained target clone gene sequence, so as to promote subsequent experiments. The amino acid sequence after mutation is shown below:

[0205] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0206] H1-P14C-S112C (SEQ ID NO: 9):

[0207] QLQLVESGGGLVQ C GGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTV C S

[0208] C37H-P14C-S112C (SEQ ID NO:10):

[0209] QVQLVESGGGLVQ C GGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTV C S

[0210] The mutation sites are underlined.

[0211] Effect of P14C-S112C mutation on the affinity of nanobodies

[0212] 5.2.1. Affinity of H1-P14C-S112C

[0213] HER2-Fc was coated on the plate at 0.1 μg / well at 4°C overnight. After blocking, samples diluted with H1-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 5-1, compared with the original nanoantibody, the mutant H1-P14C-S112C retained its affinity for the antigen target.

[0214] Table 5-1 Affinity of mutant H1-P14C-S112C

[0215] Antibody Name EC50(ng / mL) Relative activity (%) H1 194.5 100.0 H1-P14C-S112C 176.3 110.3

[0216] 5.2.2. Affinity of C37H-P14C-S112C

[0217] CD8a-Fc was coated on the plate at 0.5 μg / well at 4°C overnight. After blocking, samples diluted with C37-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 5-2, compared with the original nanoantibody, the mutant C37H-P14C-S112C retained its affinity for the antigen target.

[0218] Table 5-2 Affinity of mutant C37H-P14C-S112C

[0219] Antibody Name EC50(ng / mL) Relative activity (%) C37H 15.7 100.0 C37H-P14C-S112C 14.2 110.9

[0220] Effect of P14C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0221] H1, C37H and the corresponding P14C-S112C mutants were coated with 0.1 μg / well at 4°C overnight, and 150-fold diluted positive individual serum was added after blocking, and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 450nm. The software SoftMax Pro 6.5.1 and Microsoft excel were used for data processing and graphical analysis, and the average % signal inhibition was used to evaluate the effects of amino acid substitution and coupling extension of nano antibodies. The higher the average % signal inhibition value, the weaker the binding to the pre-existing antibody. Average % signal inhibition = (original nano antibody OD value - mutant OD value) / original nano antibody OD value × 100%. As shown in Table 5-3, the P14C-S112C mutation cannot reduce the binding of nano antibodies to pre-existing antibodies.

[0222] Table 5-3 Effect of P14C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0223] Parental clone Sequence modification Average % signal inhibition (average of positive individuals) H1 H1-P14C-S112C -21 C37H C37H-P14C-S112C -8

[0224] Example 6: Nanobody V12C-S112C mutation cannot inhibit pre-existing antibody binding

[0225] 6.1. Site mutation

[0226] Nanobody H1 and C37H were subjected to V12C-S112C mutation (the two pairs of mutation sites are as follows Figure 1 As shown). Corresponding primers were designed for the mutation sites of the mutated antibodies, and the nucleotide sequences of each antibody were amplified by PCR using the pre-mutation bacterial solution as a template, and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector, respectively. The gene sequence was determined to determine the correctness of the obtained target clone gene sequence, so as to promote subsequent experiments. The amino acid sequence after mutation is shown below:

[0227] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0228] H1-P14C-S112C (SEQ ID NO:11):

[0229] QLQLVESGGGL C QPGGSLRLSCAASSSIFSVNNMGWYRQAAGEQRELVASISRLGTTNYKDSVKGRVTISRDDAKSTVYLQMNSLKPEDTAVYYCNTDPPWGDDPFERSASWGQGTQVTV C S

[0230] C37H-P14C-S112C (SEQ ID NO:12):

[0231] QVQLVESGGGL C QPGGSLRLSCAASGLTFSDYAIGWFRQAPGKEREGISCIRIYDGNTYYADSVKGRFTISRDNSKNHVYLQMNSLRAEDTAVYYCAAGSYYSCSVYPAYDLDYWGKGTLVTV C S

[0232] The mutation sites are underlined.

[0233] 6.2. Effect of V12C-S112C mutation on the affinity of nanobodies

[0234] 6.2.1. Affinity of H1-V12C-S112C

[0235] HER2-Fc was coated on the plate at 0.1 μg / well at 4°C overnight. After blocking, samples diluted with H1-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 6-1, compared with the original nanoantibody, the mutant H1-V12C-S112C retained its affinity for the antigen target.

[0236] Table 6-1 Affinity of mutant H1-P14C-S112C

[0237] Antibody Name EC50(ng / mL) Relative activity (%) H1 412.1 100.0 H1-V12C-S112C 327.7 125.8

[0238] 6.2.2. Affinity of C37H-V12C-S112C

[0239] CD8a-Fc was coated on the plate at 0.5 μg / well at 4°C overnight. After blocking, samples diluted with C37-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 6-2, compared with the original nanoantibody, the mutant C37H-V12C-S112C retained its affinity for the antigen target.

[0240] Table 6-2 Affinity of mutant C37H-V12C-S112C

[0241] Antibody Name EC50(ng / mL) Relative activity (%) C37H 25.8 100.0 C37H-V12C-S112C 13.6 189.8

[0242] 6.3. Effect of V12C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0243] H1, C37H and the corresponding P14C-S112C mutants were coated with 0.1 μg / well at 4°C overnight, and 150-fold diluted positive individual serum was added after blocking, and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 450nm. The software SoftMax Pro 6.5.1 and Microsoft excel were used for data processing and graphical analysis, and the average % signal inhibition was used to evaluate the effects of amino acid substitution and coupling extension of nano antibodies. The higher the average % signal inhibition value, the weaker the binding to the pre-existing antibody. Average % signal inhibition = (original nano antibody OD value - mutant OD value) / original nano antibody OD value × 100%. As shown in Table 6-3, the V12C-S112C mutation cannot reduce the binding of nano antibodies to pre-existing antibodies.

[0244] Table 6-3 Effect of V12C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0245] Parental clone Sequence modification Average % signal inhibition (average of positive individuals) H1 H1-P14C-S112C -42 C37H C37H-P14C-S112C -17

[0246] Example 7: Coupling extension of L11C-T110C, A / P84C-S113C, P14C-S113C, P14C-S112C, V12C-S112C mutants

[0247] 7.1. Coupling

[0248] H1-L11C-T110C and C37H-A84C-S113C were mixed with TCEP at a molar ratio of 1:5 and reduced at 37°C for 2 hours. After reduction, the protein was mixed with DOTA-Mal at a molar ratio of 1:10 and coupled at 37°C for 2 hours. After the reaction was completed, the ultrafiltration tube was centrifuged and purified, and the solution was changed to 0.25M ammonium acetate, centrifuged at 4200rpm, 15°C for 15 minutes, and centrifuged 5 times.

[0249] H1-P84C-S113C was mixed with TCEP at a molar ratio of 1:5 and reduced at 37°C for 2h. After reduction, the protein was mixed with GGFG-DXD at a molar ratio of 1:10, and DMSO was added to make the added DMSO volume 10% of the total reaction volume, and coupled at 37°C for 2h. After the reaction was completed, the ultrafiltration tube was centrifuged and purified to change the solution to 0.05M sodium acetate, centrifuged at 4200rpm, 15°C for 15min, and centrifuged 5 times.

[0250] C37H-L11C-T110C, H1-P14C-S113C, C37H-P14C-S113C, H1-P14C-S112C, C37H-P14C-S112C, H1-V12C-S112C, and C37H-V12C-S112C were mixed with TCEP at a molar ratio of 1:5 and reduced at 37°C for 2 hours. After reduction, the protein was mixed with Mal-NODA-GA at a molar ratio of 1:10 and coupled at 37°C for 2 hours. After the reaction was completed, the ultrafiltration tube was centrifuged and purified, and the solution was changed to 0.05M sodium acetate, centrifuged at 4200rpm, 15°C for 15 minutes, and centrifuged 5 times.

[0251] 7.2. Affinity of L11C-T110C, A / P84C-S113C, P14C-S113C, P14C-S112C, and V12C-S112C mutants for antigenic targets after conjugation

[0252] 7.2.1. Affinity of H1 mutants after conjugation

[0253] HER2-Fc was coated on the plate at 0.1 μg / well at 4°C overnight. After blocking, samples diluted with H1-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, colorimetric solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 7-1, compared with the original nanoantibody, the H1 mutant retained its affinity for the antigen target after coupling.

[0254] Table 7-1H1 mutant affinity after coupling

[0255]

[0256] 7.2.2. Affinity of the C37H mutant after conjugation

[0257] CD8a-Fc was coated on the plate at 0.5 μg / well at 4°C overnight. After blocking, samples diluted with C37-chis were added and reacted at room temperature for 1 hour. After washing, anti-his tag (HRP) was added and reacted at room temperature for 1 hour. After washing, color development solution was added and the absorbance was read at 450nm. SoftMax Pro 6.5.1 was used for data processing and plotting analysis. As shown in Table 7-2, compared with the original nanoantibody, the C37H mutant retained its affinity for the antigen target after coupling.

[0258] Table 7-2 Affinity of C37H mutant and conjugated extension

[0259]

[0260] 7.3. Binding ability of nanobody mutants to pre-existing antibodies after coupling

[0261] After H1, C37H and mutant coupling, the proteins were coated with 0.1 μg / well at 4°C overnight. After blocking, 150-fold diluted positive individual serum was added and reacted at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and reacted at room temperature for 10 minutes. After washing, the color developing solution was added and the absorbance value was read at a wavelength of 450nm. The software SoftMax Pro 6.5.1 and Microsoft excel were used for data processing and graphical analysis. The average % signal inhibition was used to evaluate the effect of amino acid substitution and coupling extension of nano antibodies. The higher the average % signal inhibition value, the weaker the binding to the pre-existing antibody. Average % signal inhibition = (original nano antibody OD value-mutant OD value) / original nano antibody OD value × 100%. As shown in Table 7-3, after L11C-T110C and A / P84C-S113C mutants were coupled and extended with GGFG-DXD, DOTA or NODA-GA, the binding ability of nano antibodies to pre-existing antibodies was further reduced. After NODA-GA coupling of the P14C-S113C, P14C-S112C, and V12C-S112C mutants, the binding ability of the nanobody to the pre-existing antibody was not reduced.

[0262] Table 7-3 Binding ability of nanobody mutants to pre-existing antibodies after coupling and extension

[0263]

Claims

1. A modified immunoglobulin single variable domain comprising the following modifications compared to an unmodified parent immunoglobulin single variable domain: i) substitution of amino acids at positions 11 and 110, or ii) substitution of amino acids at positions 84 and 113, Wherein the modified immunoglobulin single variable domain has reduced binding to pre-existing antibodies compared to an unmodified parent immunoglobulin single variable domain.

2. The modified immunoglobulin single variable domain of claim 1, wherein compared to the unmodified parent immunoglobulin single variable domain, the L at position 11 of the modified immunoglobulin single variable domain is substituted, and the T at position 110 is substituted.

3. The modified immunoglobulin single variable domain of claim 2, wherein compared to the unmodified parent immunoglobulin single variable domain, the L at position 11 of the modified immunoglobulin single variable domain is replaced by C, and the T at position 110 is replaced by C.

4. The modified immunoglobulin single variable domain of claim 1, wherein the A or P at position 84 of the modified immunoglobulin single variable domain is substituted, and the S at position 113 is substituted, compared to the unmodified parent immunoglobulin single variable domain.

5. The modified immunoglobulin single variable domain of claim 4, wherein the A or P at position 84 of the modified immunoglobulin single variable domain is replaced by C, and the S at position 113 is replaced by C, compared to the unmodified parent immunoglobulin single variable domain.

6. The modified immunoglobulin single variable domain of any one of claims 1 to 5, wherein the immunoglobulin single variable domain is a VHH.

7. The modified immunoglobulin single variable domain of any one of claims 1 to 6, wherein the immunoglobulin single variable domain specifically binds to a target antigen selected from HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3 and FAP.

8. The modified immunoglobulin single variable domain of any one of claims 1 to 7, wherein the immunoglobulin single variable domain specifically binds to a HER2 protein, such as a human HER2 protein.

9. The modified immunoglobulin single variable domain of claim 8, wherein the unmodified parent immunoglobulin single variable domain that specifically binds to the HER2 protein comprises the amino acid sequence shown in SEQ ID NO:

1.

10. The modified immunoglobulin single variable domain of claim 8 or 9, wherein the modified immunoglobulin single variable domain that specifically binds to the HER2 protein comprises the amino acid sequence shown in SEQ ID NO: 2 or 5.

11. The modified immunoglobulin single variable domain of any one of claims 1 to 7, wherein the immunoglobulin single variable domain specifically binds to a CD8a protein, such as a human CD8a protein.

12. The modified immunoglobulin single variable domain of claim 11, wherein the unmodified parent immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO:

3.

13. The modified immunoglobulin single variable domain of claim 11 or 12, wherein the modified immunoglobulin single variable domain that specifically binds to the CD8a protein comprises the amino acid sequence shown in SEQ ID NO: 4 or 6.

14. A conjugate molecule comprising the modified immunoglobulin single variable domain according to any one of claims 1 to 13, and at least one chemical part or biological part coupled thereto.

15. The conjugate molecule of claim 14, wherein the chemical moiety is a chelator, for example a chelator selected from the group consisting of DOTA, DO3A, NODA-GA, DTPA, EDTA, NOTA, NO2A, TRAP, TETA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, HEHA or derivatives thereof.

16. The conjugate molecule of claim 15, wherein the chelator chelates with a radionuclide, for example, the radionuclide 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, 99m 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.

17. The conjugate molecule of claim 14, wherein the chemical moiety is a small molecule drug, for example, a small molecule drug selected from the group consisting of Dxd, SN38 (7-ethyl-10-hydroxycamptothecin), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbamide, mechlorethamine, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum (II) (DDP) cisplatin, daunorubicin, doxorubicin, actinomycin D, bleomycin, mithramycin, anthramycin, vincristine, vinblastine.

18. The conjugate molecule of claim 14, wherein the chemical moiety is a water-based polymer such as poly(ethylene glycol) or dextran.

19. The conjugate molecule of claim 14, wherein the biological moiety is selected from the group consisting of a polypeptide, a nucleic acid molecule, a lipid, and a carbohydrate.

20. The conjugate molecule of claim 19, wherein the biological moiety is selected from a bioluminescent agent such as a fluorescent protein, an enzyme (such as horseradish peroxidase), an immunoglobulin Fc region, abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokine, interleukin-1, interleukin-2, interleukin-6, interleukin-10, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor or IFN.

21. A pharmaceutical composition comprising the modified immunoglobulin single variable domain according to any one of claims 1 to 13 or the conjugated molecule according to any one of claims 14 to 20, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is used to treat a disease associated with an antigen specifically targeted by the immunoglobulin single variable domain.

23. The pharmaceutical composition of claim 22, wherein the immunoglobulin single variable domain specifically binds to HER2 protein, and the disease is a HER2-related cancer, for example, the cancer is selected from breast cancer, bladder cancer, lung cancer, head and neck cancer, prostate cancer, liver cancer, gastric cancer, endometrial cancer, kidney cancer, colon cancer, thyroid cancer, ovarian cancer, pancreatic cancer and glioblastoma.

24. A method for detecting the presence and / or amount of a target antigen in a biological sample, comprising: a) contacting the biological sample and the control sample with the modified immunoglobulin single variable domain of any one of claims 1 to 13 or the conjugated molecule of any one of claims 14 to 20 under conditions that a complex can be formed between the modified immunoglobulin single variable domain of any one of claims 1 to 13 or the conjugated molecule of any one of claims 14 to 20 and the target antigen; b) detecting the formation of a complex, Wherein the difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of the target antigen in the sample.

25. The method of claim 24, wherein the biological sample is an ex vivo sample such as a blood sample.

26. The method of claim 24 or 25, wherein the target antigen is selected from HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3 and FAP.

27. A detection agent for detecting target antigen-positive cells, comprising the modified immunoglobulin single variable domain of any one of claims 1 to 13 or the conjugated molecule of any one of claims 14 to 20, and optionally a physiologically acceptable carrier.

28. The detection agent of claim 27, wherein the detection agent is used to detect the presence and / or amount of target antigen-positive cells in a tissue in a subject.

29. The detection agent of claim 27 or 28, wherein the tissue is tumor tissue.

30. The detection agent of any one of claims 27 to 29, wherein the target antigen is selected from HER2, CD8a, PD-L1, Claudin18.2, Trop2, EGFR, GPC3, B7H3 and FAP.

31. The detection agent of any one of claims 27-30, wherein the detection agent is a contrast agent.

32. The detection agent of claim 31, wherein the contrast agent is an ECT contrast agent, such as a SPECT contrast agent or a PET contrast agent.

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