Anti-TRBC1 nano antibody as well as preparation method and application thereof

Abstract

The invention relates to an anti-TRBC1 nano antibody as well as a preparation method and application thereof. The anti-TRBC1 nano antibody has good affinity. The invention also relates to application of the anti-TRBC1 nano antibody or the antigen binding fragment thereof or the pharmaceutical composition containing the anti-TRBC1 nano antibody or the antigen binding fragment thereof in preparation of drugs for preventing and / or treating TRBC1 related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to an anti-TRBC1 nano antibody and a preparation method and application thereof. Background Art

[0002] Lymphoid malignancies can be broadly divided into those that originate from T cells and those that originate from B cells. T-cell malignancies are a clinically and biologically heterogeneous disease that together account for 10-20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most common histological subtypes are peripheral T-cell lymphoma (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL) and anaplastic large cell lymphoma (ALCL). Of all acute lymphoblastic leukemias (ALL), approximately 20% are of T-cell phenotype.

[0003] Compared to B-cell malignancies, these diseases often present aggressively, with an estimated 5-year survival rate of only 30%. In the case of T-cell lymphomas, they present with a high percentage of patients presenting with disseminated disease, poor The IPI score correlates with the prevalence of extranodal disease. Chemotherapy alone is generally ineffective, with current treatments curing less than 30% of patients.

[0004] Furthermore, while in B-cell malignancies, immunotherapies such as the anti-CD20 monoclonal antibody rituximab can significantly improve outcomes, no equally effective, minimally toxic immunotherapies are currently available for the treatment of T-cell malignancies. A significant difficulty in the development of immunotherapies for T-cell disorders is that there is considerable overlap in the expression of markers for clonal and normal T-cells, with no single antigen being able to clearly identify clonal (malignant) cells.

[0005] The same problem exists when targeting pan-B-cell antigens to treat B-cell malignancies. However, in this case, the concomitant depletion of the B-cell compartment results in relatively mild immunosuppression, which is easily tolerated by most patients. Moreover, in treatments that result in long-term depletion of the normal B-cell compartment, its depletion can be largely eliminated by the administration of mixed immunoglobulins. The situation is completely different when targeting T-cell malignancies. Here, the concomitant depletion of the T-cell compartment results in severe immunosuppression and severe toxicity. Moreover, there is no satisfactory way to mitigate the loss of the T-cell compartment.

[0006] The clinical effects of the therapeutic monoclonal antibody Alemtuzumab partially explain the toxicity. This agent can lyse cells expressing CD52 and has a certain effect on T-cell malignancies. The use of this drug is greatly limited by severe cellular immune defects, mainly due to T-cell exhaustion, and the risk of infection is significantly increased. Therefore, there is a need for an anti-TRBC1 nanobody with good binding affinity to TRBC1 and that can prevent and / or treat diseases associated with TRBC1. Summary of the invention

[0007] In view of this, the present invention proposes an anti-TRBC1 nanobody and a preparation method and application thereof.

[0008] The technical solution of the present invention is achieved as follows: In the first aspect, the present invention provides an anti-TRBC1 nanobody or an antigen-binding fragment thereof, which comprises CDR1, CDR2 and CDR3 contained in the heavy chain variable region (VHH) shown in any one of SEQ ID NO: 1-19.

[0009] The present invention provides a humanized anti-TRBC1 antigen binding domain having a variable heavy chain (VH) and a variable light chain (VL), which include the following complementarity determining regions (CDR 1-3): In certain embodiments, the nanobody or antigen binding fragment thereof, comprising a humanized VH sequence of mouse JOVI-1 CDR, shown as SEQ ID No. 1-2 and 3, may contain 6 or less, 5 or less, 4 or less, 3 or less, 2 or 1 mutations compared to the wild-type human framework region sequence. The humanized VL sequence comprising mouse JOVI-1 CDR, shown as SEQ ID No, 4, 5 and 6, may contain 6 or less, 5 or less, 4 or less, 3 or less, 2 or 1 mutations compared to the wild-type human framework region sequence.

[0010] A nanobody or an antigen-binding fragment thereof, comprising the following CDR1, CDR2 and CDR3, the sequences of which are as follows:

[0011]

[0012] The antigen binding domain includes a huanframework region or a huanframework region with one or more mutations. For example, the framework region may contain one or more substitutions compared to the human framework region sequence, and the substitution may be a "reverse mutation" in which one or more amino acids are replaced by equivalent residues in the mouse antibody sequence. The mouse antibody variable heavy chain (VH) sequence is shown below as SEQ ID No.7, and the variable light chain (V) sequence is shown as SEQ ID NO.8.

[0013] Jovi-1 VH

[0014] SEQ ID No.7: EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIOSNERFRGKATLTSDKSSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFFDFWGQGTTLTVSs

[0015] Jovi-1 VL

[0016] SEQ ID No.8: DVVMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKWYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCsoSTHVPYTFGGGTKLEIKR

[0017] In the following two sequences, the CDR sequences are shown in bold and underlined. The VH sequence may include JOVI-1 VH CDRS and human framework H-AF062256. The sequence is shown as SEQ ID No. 9;

[0018] Humanised Jovi-1 H-AF6 framework

[0019] SEQ ID No.9:

[0020] oVOLVoSGAEVKKPGASVKVSCKASGYTFTGYVMHWVROAPGOGLEWMGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGQGTMVTVSs

[0021] The VH sequence may include JOVI-1 VH CDRs and human known H-EF177999, which is shown as SEQ ID No. 10. The CDR sequences are underlined.

[0022] Humanised Jovi-1 H-EF9 framework

[0023] SEQ ID No.10: EVQLVESGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWMGFINPYNDDIOSNERFRGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYNFDGAYRFFDFWGQGTLVTVSs

[0024] The VH sequence may contain JOVI-1 VH CDRS and human known H-KF688165. The sequence is shown as SEQ ID No. 11. The CDR sequences are underlined.

[0025] Humanized Jovi-1 HH-K5 framework

[0026] SEQ ID No.11: QVQLVQSGAEVKKPGASVKVSCKASEYSFTGYVMHWVRQAPGQGLEVVMGFINPYNDDIOSNERFRG RVTMTRDTSISTAYMEVSSLTSDDAAIYYCARGAGYNFDGAYRFFDFWGOGTLVTVSs

[0027] The CDR sequences are underlined. They comprise a heavy chain variable region (VHH) as set forth in any one of SEQ ID NOs: 12-18; or a sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 12-18.

[0028] The VH sequence may comprise one of the sequences shown as SEQ ID Nos. 12 to 18.

[0029] mutation K73

[0030] SEQ ID No.12:

[0031] OVOLVoSGAEVKKPGASVKVSCKASGYTFTGYVMHWVROAPGOGLEWMGFINPYNDDIQSNERFRGRVTMTRDKSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGOGTMVTVSS.

[0032] mutation S71

[0033] SEQ ID No.13:

[0034] OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYVMHWVROAPGOGLEWMGFINPYNDDIOSNERFRGRVTMTSDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGOGTMVTVSS。

[0035] mutations S71,K73

[0036] SEQ ID No.14:

[0037] QVQLVOSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTSDKSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGQGTMVTVSS。

[0038] mutation I48

[0039] SEQ ID No.15:

[0040] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWIGFINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGOGTMVTVSS。

[0041] mutation I48,K73

[0042] SEQ ID No.16:

[0043] OVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWIGFINPYNDDIQSNERFRGRVTMTRDKSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGQGTMVTVSS。

[0044] mutations M20,S71,K73

[0045] SEQ ID No.17:

[0046] QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYVMHWVRQAPGQGLEWMGFINPYNDDIQSNERFRGRVTMTSDKSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGOGTMVTVSS.

[0047] mutations M20,I48

[0048] SEQ ID No.18:

[0049] OVOLVOSGAEVKKPGASVKMSCKASGYTFTGYVMHWVROAPGOGLEWIGFINPYNDDIOSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDGAYRFFDFWGOGTMVTVSS.

[0050] The VL sequence may comprise JOVI-1 VLCDR with human framework 3aaz. The sequence is shown as SEQ ID No.19.

[0051] SEQ ID No.19:

[0052] DIVMTOSPLSLPVTPGEPASISCRSSORLVHSNGNTYLHWYLOKPGOSPRL LI YRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSOSTHVPYTFGQGTKLEIK.

[0053] Any of the sequences shown therein may have one or more amino acid substitutions, deletions or additions compared to the sequence shown therein; preferably, the substitutions are conservative substitutions.

[0054] In certain embodiments, the Nanobody or antigen-binding fragment thereof, wherein the Nanobody or antigen-binding fragment thereof is a camelid antibody, a chimeric antibody or a humanized antibody.

[0055] In a second aspect, the present invention provides a multispecific antibody, which comprises the Nanobody or antigen-binding fragment thereof as described in the first aspect, and other antibodies or antigen-binding fragments thereof or antibody analogs.

[0056] In certain preferred embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0057] The nano antibody described in the first aspect of the present invention or the multispecific antibody described in the second aspect can be prepared by various methods known in the art, for example, obtained by genetic engineering recombinant technology. For example, a DNA molecule encoding the heavy chain variable region gene of the nano antibody of the present invention is obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cell is cultured under specific conditions and expresses the nano antibody or multispecific antibody of the present invention. The antigen-binding fragment of the present invention can be obtained by hydrolyzing the complete nano antibody molecule.

[0058] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the Nanobody or antigen-binding fragment thereof as described in the first aspect or the multispecific antibody as described in the second aspect.

[0059] In a fourth aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising a nucleic acid molecule encoding the separation described in the third aspect. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, and the like.

[0060] In a fifth aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule of the third aspect or the vector of the fourth aspect. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0061] In a sixth aspect, the present invention provides a conjugate comprising the Nanobody or antigen-binding fragment thereof as described in the first aspect, or the multispecific antibody as described in the second aspect, or the nucleic acid molecule as described in the third aspect, or the vector as described in the fourth aspect and a coupling portion.

[0062] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP or biotin, and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection or tracing).

[0063] In certain embodiments, the coupling moiety is selected from a detectable label, such as an enzyme, a radioisotope, a fluorescent dye, a luminescent substance or biotin. The detectable label of the present invention can be any substance detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radioisotopes (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa Fluor). 750), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds), magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastics (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding to avidin modified with the above-mentioned labels (e.g., streptavidin). In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels as described above can be connected to the nanobodies or antigen-binding fragments thereof of the invention by linkers of different lengths to reduce potential steric hindrance.

[0064] In certain embodiments, the coupling portion is selected from nuclides, nucleic acids, cytokines (e.g., IL-6, IL-15, IL-12, IL-1b, IL-23, etc.), immune agonists (e.g., TLR agonists), small molecule toxins, polypeptides, proteins, receptors, ligands with binding activity, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.

[0065] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the Nanobody or its antigen-binding fragment or its prodrug (e.g., probody) according to the first aspect of the present invention, the multispecific antibody according to the second aspect, the isolated nucleic acid molecule according to the third aspect, the vector according to the fourth aspect, the host cell according to the fifth aspect, or the conjugate according to the sixth aspect, and a pharmaceutically acceptable carrier and / or excipient;

[0066] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent; in certain preferred embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as platinum drugs, small molecule inhibitors, etc.; in certain preferred embodiments, the additional pharmaceutically active agent is a drug that enhances immunity, such as interferon, interleukin; in certain preferred embodiments, the additional pharmaceutically active agent is an immunotherapy-related drug, such as an immune checkpoint inhibitor (such as PD-1, CTLA-4 or PD-1 / CTLA-4 inhibitor) or an immune system activator (such as TLR, ONDs, CpG, LPS, SEB, SEA or SEC activator); in certain preferred embodiments, the additional pharmaceutically active agent is a radioactive substance or a colorimetric agent.

[0067] In the eighth aspect, the present invention provides the use of the nanoantibody or its antigen-binding fragment or its prodrug (e.g., probody) described in the first aspect, the multispecific antibody described in the second aspect, the isolated nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, the host cell described in the fifth aspect, the conjugate described in the sixth aspect, or the pharmaceutical composition described in the seventh aspect in the preparation of a medicament for preventing and / or treating and / or adjuvantly treating and / or neoadjuvantly treating a disease associated with TRBC1.

[0068] In certain embodiments, the disease associated with TRBC1 is a tumor; in certain preferred embodiments, the tumor is selected from a solid tumor or a blood tumor.

[0069] T-cell lymphoma or leukemia expressing TRBC1 can be selected from the following options: peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma, nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0070] In certain embodiments, the Nanobody or antigen-binding fragment thereof, the multispecific antibody, the isolated nucleic acid molecule, the vector, the host cell, the conjugate or the pharmaceutical composition is administered separately, in combination, simultaneously or sequentially with the additional pharmaceutically active agent.

[0071] In certain embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, an immunotherapy-related drug, an immunity-enhancing drug or an immune system activator, a radioactive substance, or a colorimetric agent.

[0072] In the ninth aspect, the present invention provides a method for preventing and / or treating a disease associated with TRBC1, and / or delaying the progression of a disease associated with TRBC1, and / or reducing or inhibiting the recurrence of a disease associated with TRBC1 in a subject, the method comprising administering an effective amount of the pharmaceutical composition described in the seventh aspect to a subject in need thereof.

[0073] In certain embodiments, the disease associated with TRBC1 is a tumor; in certain preferred embodiments, the tumor is selected from a solid tumor or a blood tumor.

[0074] T-cell lymphoma or leukemia expressing TRBC1 can be selected from the following options: peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma, nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0075] In certain embodiments, it further comprises administering to the subject a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, cell therapy, and any combination thereof.

[0076] In a tenth aspect, the present invention provides a method for detecting the presence or level of TRBC1 in a sample, comprising using the Nanobody or antigen-binding fragment thereof described in the first aspect, the multispecific antibody described in the second aspect, the isolated nucleic acid molecule described in the third aspect, or the conjugate described in the sixth aspect of the present invention;

[0077] In certain embodiments, the method is an immunological detection, such as an immunoblot, an enzyme immunoassay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay; in certain embodiments, the conjugate used in the method comprises a Nanobody of the invention, or an antigen-binding fragment thereof, and a detectable label;

[0078] In certain embodiments, the Nanobodies or antigen-binding fragments thereof used in the methods are detectably labeled; in certain embodiments, the Nanobodies or antigen-binding fragments thereof used in the methods are not detectably labeled;

[0079] Thus, the method may also comprise the use of an additional reagent (eg, a second antibody) carrying a detectable label to detect the Nanobody or antigen-binding fragment thereof of the invention.

[0080] In the eleventh aspect, the present invention provides a kit comprising a diagnostic or therapeutic kit of the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the multispecific antibody of the second aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect. In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention carries a detectable label. In certain embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further comprises a detectable label.

[0081] In the twelfth aspect, the present invention provides the use of the nanobody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, and the conjugate described in the sixth aspect in the preparation of a kit for tumor diagnosis or identification.

[0082] T-cell lymphoma or leukemia expressing TRBC1 can be selected from the following options: peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HSTL), extranodal NK / T-cell lymphoma, nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

[0083] The anti-TRBC1 nanobody of the present invention and its preparation method and application have the following beneficial effects compared with the prior art:

[0084] The nanobody or antigen-binding fragment specifically binding to TRBC1 of the present invention has good binding affinity to TRBC1. Therefore, the nanobody or antigen-binding fragment or multispecific antibody specifically binding to TRBC1 of the present invention is suitable for preventing and / or treating diseases or conditions related to TRBC1, and has great clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0086] Figure 1 It is the binding detection of the anti-TRBC1 nanobody and the human TRBC1 protein according to the embodiment of the present invention;

[0087] Figure 2 This is the binding detection of the anti-TRBC1 nanobody and JURKAT cells according to the embodiments of the present invention. DETAILED DESCRIPTION

[0088] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0089] Definition of terms

[0090] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the virology, biochemistry, and immunology laboratory operation steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0091] As used herein, the term "camel-derived antibody" refers to antibodies against an antigen produced by camelids (including camels, alpacas and llamas) after immunization or antigen invasion.

[0092] In this document, unless the context clearly indicates otherwise, when referring to the term "Nanobody", it includes not only complete Nanobodies but also antigen-binding fragments of Nanobodies.

[0093] The donor antibody can be a mouse, rat, rabbit or non-human primate (eg, cynomolgus monkey) antibody having the desired properties (eg, antigen specificity, affinity, reactivity, ability to enhance immune cell activity and / or ability to enhance an immune response).

[0094] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction.

[0095] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of a protein or polypeptide comprising an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0096] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0097] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a tumor (eg, a tumor expressing TRBC1), or is at risk of suffering from the above-mentioned disease.

[0098] Herein, "combination" includes therapies that can be administered separately, such as separately formulated for separate administration (e.g., can be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co-formulation"). In certain embodiments, the anti-TRBC1 nanobody or its antigen-binding fragment of the present invention can be administered sequentially. In other embodiments, the anti-TRBC1 nanobody or its antigen-binding fragment can be administered simultaneously. The anti-TRBBC1 nanobody or its antigen-binding fragment of the present invention can be used in combination with at least one other (active) agent in any manner.

[0099] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compendium of Molecular Biology Experiments, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturers. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0100] Example 1. Alpaca immunization

[0101] Anti-TRBC1 nanoantibodies were screened by immunizing alpacas. Two alpacas were immunized. The immune antigen was hFc-tagged human TRBC1 recombinant protein. The first immunization was subcutaneously immunized with Freund's complete adjuvant (CFA) mixed with huTRBC1-hFc, and the remaining booster immunizations were subcutaneously immunized three times with Freund's incomplete adjuvant (IFA) mixed with huTRBC1-hFc.

[0102] Example 2. Establishment of phage antibody library

[0103] After the alpaca was immunized four times, 50 ml of peripheral blood was collected and alpaca PBMC was isolated according to the instructions for lymphocyte separation solution (Haoyang Biology, HY2015). Then, total RNA was extracted with RNAiso Plus (Takara, 9109) reagent, and 5 μg of RNA was co-transcribed to obtain cDNA, referring to the instructions for PrimeScriptTMII1st Strand cDNA Synthesis Kit (Takara, 6210B). The obtained cDNA was used as a template to amplify the heavy chain variable region (VHH) fragment, which was then connected to the vector after enzyme digestion and electroporated into TG1 competent cells (NBbiolab, P008). The bacterial storage capacity of one alpaca was 2.14×109 (pfu), and the bacterial storage capacity of the other alpaca was 7.8×109 (pfu).

[0104] Example 3. Screening of anti-TRBC1 nanobodies

[0105] The two alpaca libraries constructed were both selected by cell and protein selection. After three or four rounds of cell selection and two rounds of protein selection, the plasmid of the selected bacterial library was extracted, the VHH fragment was amplified by PCR, connected to the expression vector, and then the clone was selected for culture and induced VHH secretion expression. The coated antigens were human TRBC1-his (huTRBC1-his: Acro, TRBC1-H5223), monkey TRBC1-his (cynoTRBC1-his: Acro, TRBC1-C52H6), and mouse TRBC1-his (muTRBC1-his: Acro, TRBC1-M52H3). The antigen was diluted to 2 μg / ml with antigen coating solution (carbonate buffer at pH 9.6), and then added to the ELISA plate at 50 μl / well and coated overnight at 2-8°C; the next day, the plates were washed three times with PBST, and 2% BSA was added to block at 37°C for 2 h; 100 μl of induced VHH supernatant was added to each well and incubated at 37°C for 1 h; the plates were washed three times with PBST, and HRP-labeled anti-DYKDDDDK antibody (Proteintech, HRP-66008) was added at 50 μl / well and incubated at 37°C for 1 h; the plates were washed three times with PBST, and TMB was added for color development and reading.

[0106] Five anti-TRBC1 candidate nanobodies with good crossover between humans, monkeys and mice were obtained, and they were named Jovi-1chimera HC, H-AF062256, H-EF177999-graft, and H-KF688165-graft. Then, DNA sequencing was performed on the four anti-TRBC1 nanobodies, and sequence analysis was performed using CLC software to obtain clones of single sequences and store them in glycerol. The sequencing results were analyzed and the CDR regions were defined. The variable regions and CDR sequences of the obtained anti-TRBC1 nanobodies are shown in Table 1:

[0107] Table 1 Amino acid sequences of variable regions and CDRs of anti-TRBC1 nanobodies

[0108] TABLE 1

[0109]

[0110] Example 4. Induced expression and purification of anti-TRBC1 nanobody

[0111] The clones containing candidate nanobodies stored in glycerol were activated in 2YT medium (containing Amp) at 37°C and 250rpm overnight, and inoculated into fresh 2YT medium at 1:100 the next day at 37°C and 250rpm until OD600 = 0.6, and induction medium TB (Shanghai Biotech, B540123-0500), potassium ion buffer and IPTG were added, and induced at 30°C and 250rpm overnight. The supernatant was collected by centrifugation of the induced bacterial solution, and the anti-TRBC1 nanobodies were purified by gravity column loaded with Ni (NTA type) affinity chromatography medium.

[0112] Example 5. Affinity detection of anti-TRBC1 nanobody

[0113] 5.1 ELISA to determine the affinity of anti-TRBC1 nanobodies

[0114] The ELISA plate was coated with huTRBC1 at 4°C overnight. After blocking with 2% BSA blocking solution for 2 hours, VHH antibodies of different dilutions were added to each well (nanoantibodies 1F10, 2F11 and 3E1 from 150nM, 4F4 and 4H5 from 1000nM; 3-fold dilutions for 8 concentrations) and incubated for 1 hour; HRP-labeled anti-DYKDDDDK antibody (Sigma, A8592) was added, and after color development with TMB solution, it was terminated with 2M hydrochloric acid and the absorbance at 450nm was read. EC50 was counted using GraphpadPrism software.

[0115] Table 2 Binding detection of anti-TRBC1 nanobody and human TRBC1 protein

[0116]

[0117] 5.2 Determination of the cell-level affinity of anti-TRBC1 nanobodies

[0118] 50 μl (2×10^5 cells) of Jurkat cells highly expressing human TRBC1 were added to each well of a 96-well V-shaped plate, and 50 μl of gradient diluted nanoantibodies (nanoantibodies starting from 150 nM, 3-fold dilutions to 8 concentrations) were added to each well and incubated on ice for 1 hour. Unbound nanoantibodies were washed away, and anti-DYKDDDDK antibodies (Biolegend, 637318) labeled with AlexaFluro488 diluted according to the reagent instructions were added. After incubation on ice for 1 hour, the cells were washed 4 times, and 100 μl of PBS was added to each well to resuspend the cells, and the cells were detected by flow cytometry. EC50 was calculated using GraphpadPrism software.

[0119] The results of affinity at the cell level are shown in Table 3 . The anti-TRBC1 nanoantibodies in Table 3 can bind to Jurkat cells that highly express human TRBC1.

[0120] Table 3 Binding of anti-TRBC1 nanobodies to Jurkat cells

[0121]

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-TRBC1 nanobody, characterized in that include: Variable heavy chain VH, variable light chain VL and complementary determining regions VH CDR 1-3 and VL CDR 1-3, the amino acid sequence of VH CDR 1-3 is shown in SEQ ID NO.1-3, the amino acid sequence of VLCDR 1-3 is shown in SEQ ID NO.4-6; the amino acid sequence of the variable heavy chain VH is shown in SEQ ID NO.7, and the amino acid sequence of the variable light chain VL is shown in SEQ ID NO.

8.

2. The anti-TRBC1 nanobody according to claim 1, characterized in that The nanobody is a camelid antibody, a chimeric antibody or a humanized antibody.

3. A multispecific antibody, characterized in that: The nanobody comprising the nanobody according to any one of claims 1 to 2, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

4. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody according to any one of claims 1 to 2 or the multispecific antibody according to claim 3.

5. A carrier, characterized in that Comprising the nucleic acid molecule of claim 3.

6. A host cell, characterized in that Comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

7. The method for preparing an anti-TRBC1 nanobody according to claim 1, comprising culturing the host cell according to claim 6 under conditions that allow protein expression and collecting the nanobody or multispecific antibody from the cultured host cell culture.

8. A pharmaceutical composition, characterized in that Comprising the nanobody according to any one of claims 1 to 2, or the multispecific antibody according to claim 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6, and a pharmaceutically acceptable carrier and / or excipient.

9. A kit, characterized in that: Comprising the nanobody according to any one of claims 1 to 2, or the multispecific antibody according to claim 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6, or the pharmaceutical composition according to claim 7.

10. Use of the nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, or the multispecific antibody according to claim 3, or the nucleic acid molecule according to claim 4, or the vector according to claim 5, or the host cell according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a drug for preventing and / or treating a disease associated with TRBC1.

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