Modified immunoglobulin single variable domain

Abstract

The present invention belongs to the field of biological medicine. Provided is a modified immunoglobulin single variable domain having reduced binding capability to preexisting antibodies. Modifying an immunoglobulin single variable domain by means of substitution of an amino acid at a specific site and / or coupling extension reduces the binding thereof to preexisting antibodies. Also provided is the use of the modified immunoglobulin single variable domain.

Description

Modified immunoglobulin single variable domain Technical Field

[0001] The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a modified immunoglobulin single variable domain having reduced binding capacity to pre-existing antibodies. More specifically, the present invention relates to modifying an immunoglobulin single variable domain by amino acid substitution at specific sites and / or by conjugated extension, thereby reducing its binding to pre-existing antibodies. The present invention also relates to uses of such modified immunoglobulin single variable domains.

[0002] Background of the Invention

[0003] Patients receiving biotherapeutics such as antibody drugs or fusion protein drugs may have pre-existing antibodies against these biotherapeutics. Pre-existing antibodies have different effects on different therapeutics. For example, for cetuximab, pre-existing IgE antibodies against the carbohydrate (galα(1-3)gal) portion can cause severe hypersensitivity reactions; whereas for panitumumab, pre-existing antibodies have no effect on later reactions (The AAPS Journal, Vol. 15, No. 3, July 2013). Pre-existing antibodies may affect treatment-induced ADA (anti-drug antibodies), treatment safety, pharmacokinetics (PK), and pharmacodynamics (PD) of biotherapeutics. Understanding the impact of pre-existing antibodies on the risk-benefit profile of biotherapeutics is becoming increasingly important. For specific biological agents, such as antibodies, the field hopes to reduce their ability to bind to pre-existing antibodies, thereby reducing the impact of pre-existing antibodies on efficacy.

[0004] Summary 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 a pre-existing antibody 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, L at position 11 of the modified immunoglobulin single variable domain is substituted, and T at position 110 is substituted.

[0011] Embodiment 3. The modified immunoglobulin single variable domain of embodiment 2, wherein, compared with 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.

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

[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 group consisting of: Dxd, SN38 (7-ethyl-10-hydroxycamptothecin), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, 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 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, a 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 conjugate 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 the 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 is indicative of 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.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

[0051] Figure 7. V12C-S112C mutation and conjugation cannot reduce the binding of H1 and C37H to pre-existing antibodies.

[0052] Detailed Description of the Invention

[0053] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to 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), as well as 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 carried out in a manner known per se, which will be understood by those skilled in the art. Reference is also made, for example, to standard manuals, the above-mentioned general prior art and other references cited therein.

[0054] Unless otherwise indicated, the terms "antibody" or "immunoglobulin" used interchangeably herein, whether referring to heavy chain antibodies or conventional four-chain antibodies, 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 such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") 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 capable of maintaining 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 function of the rest of the protein and / or 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 essentially consists 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," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity to an antibody for an antigen by having an antigen-binding site.

[0058] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain that is capable of specifically binding to an antigenic epitope without being paired with other immunoglobulin variable domains. One 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 an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an "antibody lacking a 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 four-chain antibodies (which is referred to herein as a "VH domain") and the light chain variable domain present in conventional four-chain antibodies (which is referred to herein as a "VL domain"). The VHH domain specifically binds an epitope without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by both the VL and VH domains). 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, as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), 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. Specificity can be determined based on the affinity and / or avidity of an antigen binding protein. Avidity, represented by the dissociation equilibrium constant (KD) of an antigen and an antigen binding protein, is a measure of the binding strength between an epitope and an antigen binding site on an 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 the same) and a related antigen. Avidity is related to both 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 with which it is normally associated in that source or medium (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) 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-fold, particularly 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 (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] Thus, 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 a pre-existing antibody 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 whom the biotherapeutic has never been administered before). The biotherapeutic, for example, is an immunoglobulin single variable domain of the invention.

[0071] In some embodiments, the numbering of the amino acids is the Kabat numbering. Generally speaking, 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, L at position 11 of the modified immunoglobulin single variable domain is substituted with C, and T at position 110 is substituted with 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, 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 with C, and the S at position 113 is substituted with C.

[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] 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.

[0080] 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.

[0081] 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:

[0082] 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.

[0083] 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.

[0084] The inventors have also surprisingly discovered 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 or biological moiety. In another aspect, the present invention provides a conjugate molecule comprising the modified immunoglobulin single variable domain of the present invention and at least one chemical or biological moiety coupled thereto.

[0085] 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 chemical moieties or biological moieties to polypeptides are well known to those skilled in the art.

[0086] 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.

[0087] 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.

[0088] 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, decarbazine, 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, and vinblastine.

[0089] In some embodiments, the chemical moiety may also be a water-based polymer such as poly(ethylene glycol) or dextran.

[0090] 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.

[0091] In some embodiments, the biological moiety includes, but is not limited to, a polypeptide, a nucleic acid molecule, a lipid, a carbohydrate, etc. For example, the biological moiety can be a bioluminescent agent such as a fluorescent protein, an enzyme (such as horseradish peroxidase), an immunoglobulin Fc region, etc. Alternatively, the biological moiety can be abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, a 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.

[0092] In some embodiments, the modified immunoglobulin single variable domain or conjugate molecule of the invention has a lower binding affinity and / or avidity for a pre-existing antibody than the unmodified parent immunoglobulin single variable domain. For example, the modified immunoglobulin single variable domain or conjugate molecule binds to a pre-existing antibody with a KD that 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% to about 90%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%) compared to the parent immunoglobulin single variable domain as determined by the method described in the Examples of the present application.

[0093] In some embodiments, the modified immunoglobulin single variable domain or conjugate molecule of the present invention has a comparable binding affinity and / or avidity for the target antigen as compared to the unmodified parent immunoglobulin single variable domain. For example, the affinity of the modified immunoglobulin single variable domain or conjugate molecule to bind 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. Relative affinity can be determined by the methods described in the Examples of this application.

[0094] In another aspect, the present invention relates to nucleic acid molecules encoding the modified immunoglobulin single variable domains of the present invention. The nucleic acids of the present invention may be RNA, DNA, or cDNA. A person skilled in the art can select a nucleic acid molecule encoding the modified immunoglobulin single variable domain of the present invention according to needs or conventional means.

[0095] The nucleic acid of the present invention can also be in the form of a vector, can be present in a vector and / or can be a part of a vector, such as a plasmid, a cosmid or a YAC. The vector can be an expression vector in particular, i.e., a vector for expressing 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. The specific examples of the regulatory elements and other elements useful or necessary for the 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.

[0096] 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.

[0097] In another aspect, the present 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.

[0098] 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).

[0099] 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.

[0100] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0101] However, the present invention can also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0102] 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:

[0103] - culturing the host cell of the invention under conditions allowing expression of the modified immunoglobulin single variable domain of the invention; and

[0104] - recovering the modified immunoglobulin single variable domain expressed by the host cell from the culture; and

[0105] - optionally further purifying and / or modifying the modified immunoglobulin single variable domain of the invention.

[0106] The modified immunoglobulin single variable domains of the present invention can be produced intracellularly in the cells 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.

[0107] 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 isolation and purification techniques suitable for use in methods for producing the modified immunoglobulin single variable domains of the present invention are well known to those skilled in the art.

[0108] 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.

[0109] The present invention provides a pharmaceutical composition comprising a 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.

[0110] 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.

[0111] 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).

[0112] In another aspect, the present invention provides use of the modified immunoglobulin single variable domain or conjugate 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.

[0113] 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.

[0114] 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 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.

[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 detecting the presence and / or amount of a target antigen in a biological sample, comprising:

[0117] 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 allowing formation of a complex between the modified immunoglobulin single variable domain or conjugated molecule of the present invention and the target antigen;

[0118] b) detecting the formation of a complex,

[0119] 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. 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.

[0120] In another aspect, the present invention provides a detection agent for detecting target antigen-positive cells, comprising a modified immunoglobulin single variable domain or conjugated molecule of the present invention, and optionally a physiologically acceptable carrier. In some embodiments, the detection agent is used to detect the presence and / or amount of target antigen-positive cells in a tissue within 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.

[0121] Thus, 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, PET) contrast agent.

[0122] In another aspect, the present invention provides the use of a modified immunoglobulin single variable domain or conjugate 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 within a subject. In some embodiments, the tissue is 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.

[0123] 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's body. In some embodiments, the tissue is tumor tissue. In some embodiments, the method further comprises the 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

[0124] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples.

[0125] Example 1: Screening test for individuals with pre-existing antibody-positive

[0126] Sera were collected from 57 healthy individuals, and individuals with potential positive pre-existing antibodies were screened by ELISA. Nanobody H1, which specifically binds to HER2, and nanobody C37H, which specifically binds to CD8a, were coated on plates at 0.1 μg / well overnight at 4°C. After blocking, 150-fold diluted 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 colorimetric solution was added and the absorbance was read at a wavelength of 450 nm. SoftMax Pro 6.5.1 and Microsoft Excel were used for data processing and analysis. The OD values ​​of all individual serum samples were normalized to the mean OD value of NC (100% PNHS mixed serum) for S / N and Log10 (S / N). Screening Threshold Factor (SCPF) = Mean Response of Log10(S / N) + 1.645 * Stdev Response of Log10(S / N). This yields SCPFs (transferred to S / N) of 1.046 and 1.048 for serum samples against H1 and C37H, respectively. When the OD value of an individual serum sample exceeds the SCPF (transferred to S / N), the individual is considered potentially positive for pre-existing antibodies. Individuals potentially positive for pre-existing antibodies screened using this method are shown in Table 1-1.

[0127] Table 1-1. Potentially positive individuals with pre-existing antibodies

[0128] Example 2: Nanobody L11C-T110C mutation inhibits pre-existing antibody binding

[0129] 2.1. Site mutation

[0130] Nanobodies H1 and C37H were both subjected to the L11C-T110C mutation. Primers were designed for the mutation sites of the mutated antibodies. Using the pre-mutation bacterial solution as a template, the nucleotide sequences of each antibody were amplified by PCR. The nucleotide sequences were then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector. Gene sequencing was performed to confirm the accuracy of the obtained target clone gene sequences, thereby facilitating subsequent experiments. The amino acid sequences before and after the mutation are shown below:

[0131] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0132] H1 (SEQ ID NO: 1):

[0133] H1-L11C-T110C (SEQ ID NO: 2):

[0134] C37H (SEQ ID NO: 3):

[0135] C37H-L11C-T110C(SEQ ID NO:4):

[0136] The mutation sites are underlined.

[0137] Effect of the L11C-T110C mutation on the affinity of nanobodies

[0138] 2.2.1. Affinity of H1-L11C-T110C

[0139] HER2-Fc was coated onto the plate at 0.1 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of H1-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 2-1, compared to the original nanobody H1, the mutant H1-L11C-T110C retained affinity for the antigen target.

[0140] Table 2-1 Affinity of mutant H1-L11C-T110C

[0141] 2.2.2. Affinity of C37H-L11C-T110C

[0142] Plates were coated with CD8a-Fc at 0.5 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of C37-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 2-2, compared to the original nanobody C37H, the mutant C37H-L11C-T110C retained affinity for the antigen target.

[0143] Table 2-2 Affinity of mutant C37H-L11C-T110C

[0144] Effect of the L11C-T110C mutation on the binding of nanobodies to pre-existing antibodies

[0145] Plates were coated with 0.1 μg / well of each of H1, C37H, and the corresponding L11C-T110C mutants overnight at 4°C. After blocking, 150-fold diluted serum from positive individuals was added and incubated at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and incubated at room temperature for 10 minutes. After washing, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1 and Microsoft Excel. The effects of the Nanobody amino acid substitutions and conjugate extensions were evaluated using average % signal inhibition. Higher average % signal inhibition values ​​indicate weaker binding to the pre-existing antibody. Average % signal inhibition = (original Nanobody OD value - mutant OD value) / original Nanobody OD value × 100%. As shown in Table 2-3, the L11C-T110C mutation reduced Nanobody binding to the pre-existing antibody.

[0146] Table 2-3 Effect of L11C-T110C mutation on the binding of nanobodies to pre-existing antibodies

[0147] Example 3: Nanobody A / P84C-S113C mutation inhibits pre-existing antibody binding

[0148] 3.1. Site mutation

[0149] Nanobodies H1 and C37H were both subjected to A / P84C-S113C mutations (the two pairs of mutation sites are shown in Figure 1). Corresponding primers were designed for the mutation sites of the mutated antibodies. Using the pre-mutation bacterial solution as a template, the nucleotide sequence of each antibody was amplified by PCR and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector. Gene sequencing was performed to confirm the correctness of the obtained target clone gene sequence, thereby advancing subsequent experiments. The amino acid sequence after mutation is shown below:

[0150] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0151] H1-P84C-S113C (SEQ ID NO: 5):

[0152] C37H-A84C-S113C (SEQ ID NO:6):

[0153] The mutation sites are underlined.

[0154] Effect of the A / P84C-S113C mutation on the affinity of nanobodies

[0155] 3.2.1. Affinity of H1-P84C-S113C

[0156] HER2-Fc was coated onto the plate at 0.1 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of H1-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 3-1, the mutant H1-P84C-S113C retained affinity for the antigen target compared to the original nanobody.

[0157] Table 3-1 Affinity of mutant H1-P84C-S113C

[0158] 3.2.2. Affinity of C37H-A84C-S113C

[0159] Plates were coated with CD8a-Fc at 0.5 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of C37-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 3-2, the mutant C37H-A84C-S113C retained affinity for the antigen target compared to the original nanobody.

[0160] Table 3-2 Affinity of mutant C37H-A84C-S113C

[0161] Effect of the A / P84C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0162] Plates were coated with 0.1 μg / well of each of H1, C37H, and the corresponding A / P84C-S113C mutants overnight at 4°C. After blocking, 150-fold diluted serum from positive individuals was added and incubated at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and incubated at room temperature for 10 minutes. After washing, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1 and Microsoft Excel. The effects of the Nanobody amino acid substitutions and conjugate extensions were evaluated using average % signal inhibition. Higher average % signal inhibition values ​​indicate weaker binding to the pre-existing antibody. Average % signal inhibition = (original Nanobody OD value - mutant OD value) / original Nanobody OD value × 100%. As shown in Table 3-3, the A / P84C-S113C mutation reduced Nanobody binding to the pre-existing antibody.

[0163] Table 3-3 Effect of A / P84C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0164] Example 4: Nanobody P14C-S113C mutation cannot inhibit pre-existing antibody binding

[0165] 4.1. Site mutation

[0166] Nanobodies H1 and C37H were both subjected to the P14C-S113C mutation (the two pairs of mutation sites are shown in Figure 1). Corresponding primers were designed for the mutation sites of the mutated antibodies. Using the pre-mutation bacterial solution as a template, the nucleotide sequence of each antibody was amplified by PCR and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector. Gene sequencing was performed to confirm the correctness of the obtained target clone gene sequence, thereby advancing subsequent experiments. The amino acid sequence after mutation is shown below:

[0167] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0168] H1-P14C-S113C (SEQ ID NO: 7):

[0169] C37H-P14C-S113C (SEQ ID NO:8):

[0170] The mutation sites are underlined.

[0171] Effect of the P14C-S113C mutation on the affinity of nanobodies

[0172] 4.2.1. Affinity of H1-P14C-S113C

[0173] HER2-Fc was coated onto the plate at 0.1 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of H1-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 4-1, the mutant H1-P14C-S113C retained affinity for the antigen target compared to the original nanobody.

[0174] Table 4-1 Affinity of mutant H1-P14C-S113C

[0175] 4.2.2. Affinity of C37H-P14C-S113C

[0176] Plates were coated with CD8a-Fc at 0.5 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of C37-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 4-2, the mutant C37H-P14C-S113C retained affinity for the antigen target compared to the original nanobody.

[0177] Table 4-2 Affinity of mutant C37H-P14C-S113C

[0178] Effect of the P14C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0179] Plates were coated with 0.1 μg / well of each of H1, C37H, and the corresponding P14C-S113C mutants overnight at 4°C. After blocking, 150-fold diluted serum from positive individuals was added and incubated at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and incubated at room temperature for 10 minutes. After washing, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1 and Microsoft Excel. The effects of the Nanobody amino acid substitutions and conjugate extensions were evaluated using average % signal inhibition. Higher average % signal inhibition values ​​indicate weaker binding to the pre-existing antibody. Average % signal inhibition = (original Nanobody OD value - mutant OD value) / original Nanobody OD value × 100%. As shown in Table 4-3, the P14C-S113C mutation did not reduce Nanobody binding to the pre-existing antibody.

[0180] Table 4-3 Effect of P14C-S113C mutation on the binding of nanobodies to pre-existing antibodies

[0181] Example 5: Nanobody P14C-S112C mutation cannot inhibit pre-existing antibody binding

[0182] 5.1. Site mutation

[0183] Nanobodies H1 and C37H were both subjected to the P14C-S112C mutation (the two pairs of mutation sites are shown in Figure 1). Corresponding primers were designed for the mutation sites of the mutated antibodies. Using the pre-mutation bacterial solution as a template, the nucleotide sequence of each antibody was amplified by PCR and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector. Gene sequencing was performed to confirm the correctness of the obtained target clone gene sequence, thereby advancing subsequent experiments. The amino acid sequence after mutation is shown below:

[0184] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0185] H1-P14C-S112C (SEQ ID NO: 9):

[0186] C37H-P14C-S112C (SEQ ID NO:10):

[0187] The mutation sites are underlined.

[0188] Effect of the P14C-S112C mutation on the affinity of nanobodies

[0189] 5.2.1. Affinity of H1-P14C-S112C

[0190] HER2-Fc was coated onto the plate at 0.1 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of H1-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 5-1, the mutant H1-P14C-S112C retained affinity for the antigen target compared to the original nanobody.

[0191] Table 5-1 Affinity of mutant H1-P14C-S112C

[0192] 5.2.2. Affinity of C37H-P14C-S112C

[0193] Plates were coated with CD8a-Fc at 0.5 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of C37-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 5-2, the mutant C37H-P14C-S112C retained affinity for the antigen target compared to the original nanobody.

[0194] Table 5-2 Affinity of mutant C37H-P14C-S112C

[0195] Effect of the P14C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0196] Plates were coated with 0.1 μg / well of each of H1, C37H, and the corresponding P14C-S112C mutants overnight at 4°C. After blocking, 150-fold diluted serum from positive individuals was added and incubated at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and incubated at room temperature for 10 minutes. After washing, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1 and Microsoft Excel. The effects of the Nanobody amino acid substitutions and conjugate extensions were evaluated using average % signal inhibition. Higher average % signal inhibition values ​​indicate weaker binding to the pre-existing antibody. Average % signal inhibition = (original Nanobody OD value - mutant OD value) / original Nanobody OD value × 100%. As shown in Table 5-3, the P14C-S112C mutation did not reduce binding of the Nanobody to the pre-existing antibody.

[0197] Table 5-3 Effect of P14C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0198] Example 6: Nanobody V12C-S112C mutation cannot inhibit pre-existing antibody binding

[0199] 6.1. Site mutation

[0200] Nanobodies H1 and C37H were both subjected to V12C-S112C mutations (the two pairs of mutation sites are shown in Figure 1). Corresponding primers were designed for the mutation sites of the mutated antibodies. Using the pre-mutation bacterial solution as a template, the nucleotide sequences of each antibody were amplified by PCR and then cloned into the PSNA008 [pCDNA4 (Invitrogen, Cat V86220)] vector. Gene sequencing was performed to confirm the correctness of the obtained target clone gene sequence, thereby advancing subsequent experiments. The amino acid sequence after mutation is shown below:

[0201] The naming of the mutated antibody is based on the specific position of the mutation site in its antibody sequence.

[0202] H1-P14C-S112C (SEQ ID NO:11):

[0203] C37H-P14C-S112C (SEQ ID NO:12):

[0204] The mutation sites are underlined.

[0205] 6.2. Effect of the V12C-S112C mutation on the affinity of nanobodies

[0206] 6.2.1. Affinity of H1-V12C-S112C

[0207] HER2-Fc was coated onto the plate at 0.1 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of H1-chis buffer 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, a colorimetric solution was added, and the absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 6-1, the mutant H1-V12C-S112C retained affinity for the antigen target compared to the original nanobody.

[0208] Table 6-1 Affinity of mutant H1-P14C-S112C

[0209] 6.2.2. Affinity of C37H-V12C-S112C

[0210] Plates were coated with CD8a-Fc at 0.5 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of C37-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 6-2, the mutant C37H-V12C-S112C retained affinity for the antigen target compared to the original nanobody.

[0211] Table 6-2 Affinity of mutant C37H-V12C-S112C

[0212] Effect of the V12C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0213] Plates were coated with 0.1 μg / well of each of H1, C37H, and the corresponding P14C-S112C mutants overnight at 4°C. After blocking, 150-fold diluted serum from positive individuals was added and incubated at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and incubated at room temperature for 10 minutes. After washing, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1 and Microsoft Excel. The effects of the Nanobody amino acid substitutions and conjugate extensions were evaluated using average % signal inhibition. Higher average % signal inhibition values ​​indicate weaker binding to the pre-existing antibody. Average % signal inhibition = (original Nanobody OD value - mutant OD value) / original Nanobody OD value × 100%. As shown in Table 6-3, the V12C-S112C mutation did not reduce Nanobody binding to the pre-existing antibody.

[0214] Table 6-3 Effect of V12C-S112C mutation on the binding of nanobodies to pre-existing antibodies

[0215] Example 7: Coupling extension of L11C-T110C, A / P84C-S113C, P14C-S113C, P14C-S112C, and V12C-S112C mutants

[0216] 7.1. Coupling

[0217] 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. Following reduction, the proteins were mixed with DOTA-Mal at a molar ratio of 1:10 and coupled at 37°C for 2 hours. After completion of the reaction, the solution was purified by ultrafiltration and centrifugation in 0.25 M ammonium acetate at 4200 rpm, 15°C for 15 minutes, and centrifuged five times.

[0218] H1-P84C-S113C was mixed with TCEP at a molar ratio of 1:5 and reduced at 37°C for 2 hours. Following reduction, the protein was mixed with GGFG-DXD at a molar ratio of 1:10. DMSO was added to a volume of 10% of the total reaction volume, and the reaction was coupled at 37°C for 2 hours. After completion of the reaction, the solution was purified by ultrafiltration and centrifuged five times with 0.05M sodium acetate at 4200 rpm and 15°C for 15 minutes.

[0219] 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. Following reduction, the proteins were mixed with Mal-NODA-GA at a molar ratio of 1:10 and coupled at 37°C for 2 hours. After completion of the reaction, the solution was purified by ultrafiltration by centrifugation, exchanged with 0.05 M sodium acetate, and centrifuged five times at 4200 rpm at 15°C for 15 minutes.

[0220] 7.2. Affinity of the L11C-T110C, A / P84C-S113C, P14C-S113C, P14C-S112C, and V12C-S112C Mutants for Antigen Targets after Conjugation

[0221] 7.2.1. Affinity of H1 mutants after conjugation

[0222] HER2-Fc was coated onto the plate at 0.1 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of H1-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 7-1, the conjugated H1 mutant retained affinity for the antigen target compared to the original nanobody.

[0223] Table 7-1 Affinity of H1 mutants after coupling

[0224] 7.2.2. Affinity of the C37H mutant after conjugation

[0225] Plates were coated with CD8a-Fc at 0.5 μg / well overnight at 4°C. After blocking, samples were added in a serial dilution of C37-chis buffer 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, a colorimetric solution was added, and absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1. As shown in Table 7-2, the C37H mutant retained affinity for the antigen target after conjugation compared to the original nanobody.

[0226] Table 7-2 Affinity of C37H mutant and after coupling extension

[0227] 7.3. Binding Ability of Nanobody Mutants to Pre-Existing Antibodies after Conjugation

[0228] The H1, C37H, and mutant conjugated proteins were coated on plates at 0.1 μg / well overnight at 4°C. After blocking, a 150-fold dilution of positive individual serum was added and the reaction was carried out at room temperature for 1 hour. After washing, anti-human IgG (HRP) was added and the reaction was carried out at room temperature for 10 minutes. After washing, a colorimetric solution was added, and the absorbance was read at 450 nm. Data processing and graphical analysis were performed using SoftMax Pro 6.5.1 and Microsoft Excel. The effects of the Nanobody amino acid substitutions and conjugation extensions were evaluated using the average % signal inhibition. Higher average % signal inhibition values ​​indicate weaker binding to the pre-existing antibody. Average % signal inhibition = (original Nanobody OD value - mutant OD value) / original Nanobody OD value × 100%. As shown in Table 7-3, the binding ability of the Nanobody to the pre-existing antibody was further reduced after the L11C-T110C and A / P84C-S113C mutants were conjugated and extended with GGFG-DXD, DOTA, or NODA-GA. 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.

[0229] Table 7-3 Binding ability of nanobody mutants to pre-existing antibodies after conjugation and extension

Claims

1. A modified single variable domain of an immunoglobulin, which contains the following modifications compared to the unmodified parental single variable domain of the immunoglobulin: i) Substitutions of the amino acids at positions 11 and 110, or ii) Substitutions of the amino acids at positions 84 and 113, wherein the modified single variable domain of the immunoglobulin has a reduced binding to pre-existing antibodies compared to the unmodified parental single variable domain of the immunoglobulin.

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

3. The modified single variable domain of the immunoglobulin according to claim 2, wherein compared to the unmodified parental single variable domain of the immunoglobulin, the L at position 11 of the modified single variable domain of the immunoglobulin is substituted with C, and the T at position 110 is substituted with C.

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

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

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

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

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

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

1.

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

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

12. The modified immunoglobulin single variable domain of claim 11, wherein the unmodified parental 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 1-13, and at least one chemical moiety or biological moiety coupled thereto.

15. The conjugate molecule of claim 14, wherein the chemical moiety is a chelator, such as 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 derivatives thereof.

16. The conjugate molecule of claim 15, wherein the chelating agent 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 γ-, β-, 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 following: Dxd, SN38 (7-ethyl-10-hydroxycamptothecin), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, chlorambucil, melphalan, carmustine, lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, cis-dichlorodiamine platinum (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-soluble polymer such as poly(ethylene glycol) or dextran.

19. The conjugate molecule of claim 14, wherein the biological moiety is selected from polypeptides, nucleic acid molecules, lipids, carbohydrates.

20. The conjugate molecule of claim 19, wherein the biological moiety is selected from bioluminescent agents such as fluorescent proteins, enzymes (such as horseradish peroxidase), immunoglobulin Fc regions, abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, lymphokines, 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 1-13 or the conjugate molecule according to any one of claims 14-20, and a pharmaceutically acceptable carrier.

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

23. The pharmaceutical composition of claim 22, wherein the single variable domain of the immunoglobulin specifically binds to the HER2 protein, and the disease is HER2-related cancer, such as 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 a control sample with the modified single variable domain of the immunoglobulin according to any one of claims 1-13 or the conjugate molecule according to any one of claims 14-20 under conditions capable of forming a complex between the modified single variable domain of the immunoglobulin according to any one of claims 1-13 or the conjugate molecule according to any one of claims 14-20 and the target antigen; b) detecting the formation of the complex, wherein the difference in the formation of the complex 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 single variable domain of the immunoglobulin according to any one of claims 1-13 or the conjugate molecule according to any one of claims 14-20, and optionally a physiologically acceptable carrier.

28. The detection agent of claim 27, wherein the detection agent is for detecting the presence and / or amount of target antigen-positive cells in tissues in a subject.

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

30. The detection agent of any one of claims 27-29, 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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