High-specificity TCR-mimic antibody targeting gp100 / HLA-A2 and preparation method of high-specificity TCR-mimic antibody

Abstract

The invention provides a simple and efficient preparation method of a TCR-mimic antibody, and the TCR-mimic antibody aiming at gp100 / HLA-A2 is obtained by screening by using the method. The TCR-mimic antibody provided by the invention can recognize an MHC-tumor antigen peptide compound with high specificity and high affinity, and has the potential of being developed as a tumor antibody drug.

Description

Technical Field

[0001] The present invention relates to the field of antibody drugs, and specifically, to a highly specific TCR-mimic antibody targeting gp100 / HLA-A2 and a preparation method thereof. Background Art

[0002] In the field of tumor immunity and treatment, the major histocompatibility complex (MHC) binds and presents intracellular tumor neoantigens for T cell recognition. MHC molecules are divided into class I and class II, which present antigenic peptides to CD8+ T cells and CD4+ T cells respectively. T cells specifically recognize the MHC-tumor antigen peptide complex (pMHC) through the T cell receptor (TCR), and then activate the immune system to kill tumors.

[0003] The HLA heavy chain (α chain) and light chain (β2m) of MHC class I molecules form a ternary complex with the antigen peptide in a non-covalent bond form. The antigen peptide is loaded into the groove structure formed by the heavy chain and the light chain, forming a binding interface that interacts with the TCR. The binding affinity of natural TCR to MHC I is low, with a KD in the μM level. Natural TCR has cross-reactivity and can recognize different MHC I-peptide combinations. Therefore, the affinity and target specificity of TCR are important indicators for the development of TCR drugs.

[0004] HLA-A*02:01 (HLA-A2) is the most common subtype among human MHC class I molecules, presenting tumor antigens such as p53 R175H , NY-ESO-1, WT1, etc. There have been multiple TCR-T cell therapies or soluble TCR protein drugs based on affinity enhancement entering clinical trials. Tebentafusp, a TCR drug with enhanced affinity targeting the melanoma tumor antigen gp100 (YLEPGPVTV) / HLA-A2, was approved by the US FDA for marketing in 2022. On the other hand, pharmaceutical R & D companies are also developing TCR-mimic antibodies, which mimic the ability of TCR to recognize antigen peptides presented by MHC I and have the advantages of high affinity (KD in the nM to pM level) and specific recognition of tumor antigen pMHC.

[0005] In the process of TCR-mimic antibody development, MHC immunogens and MHC protein reagents for screening and in vitro characterization are difficult to obtain. In the literature, pMHCI usually adopts the method of recombinant expression of HLA heavy chain by Escherichia coli, B2M light chain, and in vitro renaturation of the three components with antigen peptides. However, this method has a low yield of pMHCI and a low success rate of renaturation, and the HLA heavy chain and B2M light chain without antigen peptides are difficult to form stable dimers. In addition, positive TCR-mimic antibodies need to recognize the epitopes presented by the pMHC groove structure, resulting in a low positive rate and difficult screening of TCR-mimic antibodies. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing a TCR-mimic antibody, as well as a highly specific TCR-mimic antibody targeting gp100 / HLA-A2 and its application.

[0007] In the first aspect of the present invention, there is provided a TCR-mimic antibody targeting gp100 / HLA-A2 or an antigen-binding fragment thereof, and the antibody or its antigen-binding fragment comprises the following light chain variable region and heavy chain variable region:

[0008] A light chain variable region comprising an L-CDR1 with an amino acid sequence as shown in SEQ ID NO:7, an L-CDR2 with an amino acid sequence as shown in KVS, and an L-CDR3 with an amino acid sequence as shown in SEQ ID NO:8; and

[0009] A heavy chain variable region comprising an H-CDR1 with an amino acid sequence as shown in SEQ ID NO:10, an H-CDR2 with an amino acid sequence as shown in SEQ ID NO:11, and an H-CDR3 with an amino acid sequence as shown in SEQ ID NO:12.

[0010] In another preferred embodiment, the light chain variable region and the heavy chain variable region further comprise FR regions.

[0011] In another preferred embodiment, the light chain variable region comprises murine or human FR regions, and / or the heavy chain variable region comprises murine or human FR regions.

[0012] In another preferred embodiment, the light chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity therewith; and / or

[0013] The heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90% (preferably at least 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0014] In another preferred embodiment, the antibody or its antigen-binding fragment comprises a light chain variable region having the amino acid sequence shown in SEQ ID NO: 5, and / or a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 6.

[0015] In another preferred embodiment, the antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0016] In another preferred embodiment, the heavy chain constant region and / or the light chain constant region is murine or human.

[0017] In another preferred embodiment, the heavy chain constant region is derived from murine heavy chain IgG1.

[0018] In another preferred embodiment, the light chain constant region is derived from murine kappa (κ) chain.

[0019] In another preferred embodiment, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 13.

[0020] In another preferred embodiment, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 9.

[0021] In another preferred embodiment, the antibody or its antigen-binding fragment includes: monoclonal antibody, polyclonal antibody, bispecific antibody, single-chain antibody (scFv), Fab, Fab', F(ab')2 antibody.

[0022] In another preferred embodiment, the antibody is a monoclonal antibody, which comprises a light chain having the amino acid sequence shown in SEQ ID NO: 1, and a heavy chain having the amino acid sequence shown in SEQ ID NO: 2.

[0023] In another preferred embodiment, the antibody includes animal-derived antibodies (such as murine antibodies of different subtypes), chimeric antibodies (such as human-murine chimeric antibodies), humanized antibodies.

[0024] In a second aspect of the present invention, there is provided a recombinant antibody having:

[0025] (i) the sequence of a TCR-mimic antibody or its antigen-binding fragment targeting gp100 / HLA-A2 as described in the first aspect of the present invention; and

[0026] (ii) Signal peptides that promote the secretory expression of antibodies and / or tag sequences for purification and detection.

[0027] In another preferred embodiment, the recombinant antibody has a signal peptide that promotes the secretory expression of the antibody.

[0028] In another preferred embodiment, the tag sequence is selected from: FLAG, Myc, His tag, etc.

[0029] In another preferred embodiment, the recombinant antibody comprises a light chain with an amino acid sequence as shown in SEQ ID NO: 14, and / or a heavy chain with an amino acid sequence as shown in SEQ ID NO: 14.

[0030] In the third aspect of the present invention, there is provided a polynucleotide molecule encoding a polypeptide selected from the group consisting of:

[0031] (1) The TCR-mimic antibody targeting gp100 / HLA-A2 or its antigen-binding fragment as described in the first aspect of the present invention; or

[0032] (2) The recombinant antibody as described in the second aspect of the present invention.

[0033] In another preferred embodiment, the polynucleotide molecule comprises a nucleotide sequence as shown in SEQ ID NO: 3 and / or 4.

[0034] In the fourth aspect of the present invention, there is provided an expression vector containing the polynucleotide molecule as described in the third aspect of the present invention.

[0035] In another preferred embodiment, the expression vector includes eukaryotic cell expression vectors and prokaryotic cell expression vectors.

[0036] In another preferred embodiment, the expression vector is a mammalian cell expression vector.

[0037] In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0038] In another preferred embodiment, the expression vector is selected from pTT5, pCDNA3.1.

[0039] In the fifth aspect of the present invention, there is provided a host cell containing the expression vector as described in the fourth aspect of the present invention, or having the polynucleotide molecule as described in the third aspect of the present invention integrated into its genome.

[0040] In another preferred embodiment, the host cell includes eukaryotic cells (such as mammalian cells) and prokaryotic cells.

[0041] In a sixth aspect of the present invention, there is provided an antibody conjugate comprising:

[0042] (a) a TCR-mimic antibody targeting gp100 / HLA-A2 as described in the first aspect of the present invention, or an antigen-binding fragment thereof, or a recombinant antibody as described in the second aspect of the present invention; and

[0043] (b) a conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, an enzyme, a cytokine, a radionuclide, a nanoparticle / nanorod.

[0044] In another preferred embodiment, the detectable label includes a fluorescent label or a chemiluminescent label.

[0045] In another preferred embodiment, the radionuclide includes: diagnostic isotopes; and / or therapeutic isotopes.

[0046] In another preferred embodiment, the drug is a cytotoxic drug.

[0047] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding reagent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemotherapy sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

[0048] In another preferred embodiment, the antibody conjugate is an antibody-drug conjugate (ADC).

[0049] In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0050] (a) a TCR-mimic antibody targeting gp100 / HLA-A2 as described in the first aspect of the present invention, or an antigen-binding fragment thereof, a recombinant antibody as described in the second aspect of the present invention, or an antibody conjugate as described in the sixth aspect of the present invention; and

[0051] (b) a pharmaceutically acceptable carrier.

[0052] In another preferred embodiment, the pharmaceutical composition is in an injectable form.

[0053] In another preferred embodiment, the pharmaceutical composition is used for preparing a drug for preventing and / or treating gp100 / HLA-A2 related diseases or disorders.

[0054] In another preferred embodiment, the gp100 / HLA-A2 related diseases or disorders include melanoma.

[0055] In the eighth aspect of the present invention, there is provided the use of a TCR-mimic antibody targeting gp100 / HLA-A2 or an antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant antibody as described in the second aspect of the present invention, or an antibody conjugate as described in the sixth aspect of the present invention for the preparation of:

[0056] (1) A detection reagent or detection kit for detecting gp100 / HLA-A2;

[0057] (2) A drug for preventing and / or treating gp100 / HLA-A2-related diseases or disorders.

[0058] In another preferred example, the gp100 / HLA-A2-related diseases or disorders include melanoma.

[0059] In another preferred example, the detection includes flow cytometry (FACS), cellular immunofluorescence detection, and ELISA detection.

[0060] In another preferred example, the use is non-diagnostic and non-therapeutic.

[0061] In another preferred example, the detection is an in vitro detection.

[0062] In the ninth aspect of the present invention, there is provided a method for generating a TCR-mimic antibody targeting gp100 / HLA-A2 or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a recombinant antibody as described in the second aspect of the present invention, comprising the steps of:

[0063] (s1) Culturing a host cell as described in the fifth aspect of the present invention under conditions suitable for generating an antibody or an antigen-binding fragment to obtain a culture containing the antibody or the antigen-binding fragment;

[0064] (s2) Isolating or recovering the antibody or the antigen-binding fragment from the culture.

[0065] In another preferred example, the method further comprises the step of: (s3) Purifying and / or modifying the antibody or the antigen-binding fragment obtained in step (s2).

[0066] In the tenth aspect of the present invention, there is provided a method for preparing a TCR-mimic antibody, the method comprising the following steps:

[0067] (S1) Providing a tetramer of MHCI-tumor antigen peptide complex (pMHC I) prepared by in vitro mammalian cell expression as an immunizing antigen to immunize mice;

[0068] (S2) Fusing spleen cells of the immunized mice with myeloma cells SP2 / 0 in the logarithmic growth phase to prepare hybridoma cells;

[0069] (S3) Provide the pMHC I monomer prepared by in vitro mammalian cell expression as a screening antigen, coat it on an ELISA detection plate, and screen the hybridoma cells obtained in step (S2) by indirect ELISA to obtain a positive hybridoma polyclonal cell line;

[0070] (S4) Use the MHC I molecule without antigen peptide prepared by in vitro mammalian cell expression as a reverse screening antigen, coat it on an ELISA detection plate, and screen the positive hybridoma polyclonal cell line obtained in step (S3) by indirect ELISA to exclude the positive clones that bind to the MHC I molecule without antigen peptide;

[0071] (S5) Monoclonal culture the positive hybridoma cell line screened in steps (S3) and (S4) to obtain a monoclonal antibody that specifically binds to pMHC I.

[0072] In another preferred example, the MHCI-tumor antigen peptide complex (pMHCI) tetramer is prepared by the following method:

[0073] (a) Covalently link the tumor antigen peptide, HLA heavy chain, and B2M light chain into a protein molecule and express it in in vitro mammalian cells to obtain single-chain pMHC I;

[0074] (b) Label the obtained single-chain pMHCI with biotin to obtain biotin-labeled single-chain pMHC I;

[0075] (c) Assemble the biotin-labeled single-chain pMHCI with streptavidin SA to form the pMHC I tetramer.

[0076] In another preferred example, the pMHCI monomer is the single-chain pMHC I obtained by covalently linking the tumor antigen peptide, HLA heavy chain, and B2M light chain into a protein molecule and expressing it in in vitro mammalian cells.

[0077] In another preferred example, the MHC I is HLA-A*02:01 (HLA-A2).

[0078] In another preferred example, the tumor antigen peptide is selected from gp100 (YLEPGPVTV), p53 R175H , NY-ESO-1, WT1.

[0079] In another preferred example, the pMHC I is gp100 (YLEPGPVTA) / HLA-A*02:01.

[0080] In another preferred example, in step (S1), mice are immunized according to the following steps:

[0081] (a) Mix 50 - 100 μg of pMHCI tetramer with an equal volume of Freund's complete adjuvant, emulsify it, and then immunize female Balb / c mice aged 6 - 8 weeks by subcutaneous multi-point injection at a dose of 50 - 100 μg of immunizing antigen per mouse;

[0082] (b) After an interval of two weeks, mix it with an equal volume of Freund's complete adjuvant at the same dose and boost immunize by subcutaneous multi-point injection;

[0083] (c) After boosting immunization three times, detect the serum titer of the mice, and the detection standard is that the titer is greater than 1:1000000;

[0084] (d) Three days before hybridoma cell fusion, boost immunize once intraperitoneally with pMHC I tetramer.

[0085] In the eleventh aspect of the present invention, a method for treating gp100 / HLA-A2 related diseases or disorders is provided, including administering to a subject in need a TCR-mimic antibody or an antigen-binding fragment thereof targeting gp100 / HLA-A2 as described in the first aspect of the present invention, an antibody conjugate as described in the sixth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention.

[0086] In another preferred example, the subject includes humans and non-human mammals.

[0087] In another preferred example, the gp100 / HLA-A2 related diseases or disorders include melanoma.

[0088] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Shows the results of ELISA characterizing the specificity of the 42A8 TCRm antibody.

[0090] Figure 2 Shows the results of surface plasmon resonance (SPR) characterizing the binding of the 42A8 TCRm antibody to gp100(YLEPGPVTA) / HLA-A*02:01.

[0091] Figure 3 Shows the results of SPR characterizing the binding of the Gp100 positive control drug tebentafusp to gp100(YLEPGPVTA) / HLA-A*02:01. Detailed implementation manners

[0092] Through extensive and in-depth research, the inventor of the present invention has for the first time proposed a simpler and more efficient method for preparing TCR-mimic antibodies. The present invention uses single-chain pMHC expressed by mammalian cells, that is, through rational structural design, an antigen peptide (such as gp100), HLA-A2 heavy chain and B2M light chain are covalently linked into a protein molecule, and the spatial structure maintains the same antigen-presenting conformation as the natural pMHC ternary complex. The biotin-labeled single-chain pMHC and streptavidin SA are assembled into a tetramer as an immunogen. The antigen peptide in this immunogen binds 100% in the pMHC groove structure and has good stability, which is suitable for immunizing animals. The single-chain pMHC expressed by mammalian cells is used as a screening antigen to screen for positive-binding antibodies. In addition, during the screening process, an empty MHC (peptide ready MHC, prMHC) without an antigen peptide expressed by mammalian cells is used to counter-screen and remove non-specific antibodies that bind to the MHC heavy chain and light chain but do not recognize the antigen-presenting peptide. Among them, the empty MHC without an antigen peptide can also stably exist by covalently linking the HLA-A2 heavy chain and B2M light chain dimer through rational structural design.

[0093] Specifically, the present invention has screened and obtained a TCR-mimic antibody (hereinafter referred to as TCRm antibody) against gp100, with the clone number 42A8. This antibody has high specificity for gp100 (YLEPGPVTA) / HLA-A*02:01 and does not bind to single HLA-A*02:01 and the complex of HLA-A*02:01 and other peptides. The binding affinity (KD) level of the 42A8 TCRm antibody and GP100pMHC monomer was detected by surface plasmon resonance technology (SPR) to be approximately 0.47 uM. After humanization of the 42A8 TCRm antibody of the present invention, it has the potential for antibody drug development.

[0094] On this basis, the present invention has been completed.

[0095] Terms

[0096] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0098] As used herein, when used in reference to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0099] The three-letter and single-letter codes for amino acids used in this invention are as described in J.biol.chem, 243, p3558 (1968).

[0100] As used herein, the term "optionally" or "optionally" means that the subsequent described event or situation can occur but is not required to occur. For example, "optionally comprising 1-3 variable regions of the antibody heavy chain" means that the variable regions of the antibody heavy chain of a specific sequence can be present but are not required to be present, and can be 1, 2, or 3.

[0101] The "sequence identity" described in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and the sequences having identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0102] As used herein, the terms "TCR-mimic antibody" and "TCRm antibody" can be used interchangeably. A TCR-mimic antibody is an engineered antibody designed by mimicking the function of the T cell receptor (TCR), which can specifically recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. Different from traditional antibodies (which usually target cell surface proteins), TCR-mimic antibodies can target antigens of intracellular origin (such as tumor-associated antigens or viral antigens) by binding to MHC-peptide complexes, thus expanding the application potential of antibody drugs in cancer immunotherapy and infectious diseases. Its design is usually based on the structural mimicry of the interaction between TCR and MHC-peptide, and the complementarity-determining regions (CDRs) are optimized to improve affinity and specificity. Such antibodies have important value in bispecific antibodies, CAR-T therapies, or direct targeting therapies, but also face challenges such as immunogenicity, off-target effects, and MHC polymorphism.

[0103] As used herein, the terms "gp100 / HLA-A2", "gp100(YLEPGPVTA) / HLA-A*02:01", and "gp100(YLEPGPVTV) / HLA-A2" are used interchangeably and all refer to the MHC-tumor antigen peptide complex formed by the presentation of the melanoma antigen gp100 by the human MHC class I molecule HLA-A*02:01.

[0104] As used herein, the terms "GP100pMHC monomer" and "HLA-A*02:01(YLEPGPVTA)" are used interchangeably and all refer to the pMHC monomer obtained by covalently linking the tumor antigen peptide gp100(YLEPGPVTA), the HLA-A2 heavy chain, and the B2M light chain into a protein molecule and expressing it in mammalian cells in vitro.

[0105] As used herein, the terms "GP100pMHC tetramer" and "GP100pMHC tetramer" are used interchangeably and all refer to the pMHC tetramer formed by biotinylating the GP100pMHC monomer of the present invention and then assembling it with streptavidin SA.

[0106] Antibody

[0107] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetramer formed by two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is the variable region (VH), which is connected to the heavy chain constant region. The N-terminus of each light chain is the variable region (VL), which is connected to the light chain constant region.

[0108] As used herein, the term "variable" means that the variable regions in antibodies are different in specific sequences, forming the affinity and specificity of a specific antibody for binding to a specific antigen. The antibody variable region includes complementarity-determining regions (CDRs) or hypervariable regions and relatively conserved framework regions (FRs). The primary sequences of the heavy and light chain variable regions are composed of 4 FR sequences and 3 CDR sequences arranged alternately (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The sequences and spatial conformational structures of the heavy and light chain variable regions determine the specific binding of the antibody to the antigen epitope. The antibody constant region does not directly participate in the binding of the antibody to the antigen, but affects the performance of capturing and detecting the antibody.

[0109] The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of κ and λ based on the amino acid sequence of its constant region. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant regions, mainly including 5 classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, as well as antibody subtypes (isotypes), such as mouse IgG includes IgG1, IgG2a, and IgG2b subtypes. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well-known to those skilled in the art.

[0110] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population, that is, the individual antibodies contained in the population are identical. Monoclonal antibodies specifically target a single antigenic determinant (epitope). The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a substantially homogeneous antibody population, and this should not be construed as requiring any special method for producing the antibody.

[0111] The present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the monoclonal antibodies targeting gp100 / HLA-A2, monoclonal antibodies having the variable region chains of the monoclonal antibodies targeting gp100 / HLA-A2, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing variable regions (complementary determining regions, CDRs), provided that the variable regions are the same as or have at least 90% homology, preferably at least 95% homology, to the variable regions of the light chain and heavy chain of the present invention.

[0112] As is known to those skilled in the art, antibody conjugates and fusion expression products include: conjugates formed by binding fluorescent or luminescent markers, radioactive markers, enzymes capable of producing detectable products, gold nanoparticles / nanorods, and other molecules for detection to the antibody targeting gp100 / HLA-A2 or its antigen-binding fragment; or conjugates formed by binding therapeutic drugs, toxins, radionuclides, and other therapeutic molecules to the antibody targeting gp100 / HLA-A2 or its antigen-binding fragment.

[0113] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of full-length antibodies can be used for the antigen-binding function of antibodies. Examples of binding fragments included within the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab’)2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) scFv fragments consisting of the VH and VL domains of a single arm of an antibody.

[0114] The present invention includes not only intact monoclonal antibodies, but also antibody fragments with binding activity, such as Fab or (Fab’)2 fragments; antibody heavy chains; antibody light chains or scFvs.

[0115] The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.

[0116] The terms "specifically bind", "selectively bind", "selectively binds", and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen.

[0117] As used herein, the term "antigenic determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present invention.

[0118] The present invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives, and analogs of the antibodies.

[0119] In the present invention, antibodies include murine antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art. The term "murine antibody" in the present invention refers to a monoclonal antibody against gp100 / HLA-A2 prepared according to knowledge and skills in the art.

[0120] In the present invention, an antibody can be monospecific, bispecific, trispecific, or more multispecific.

[0121] As used herein, the term "heavy chain variable region" is used interchangeably with "VH". The term "light chain variable region" is used interchangeably with "VL".

[0122] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that mainly contribute to antigen binding. One of the most commonly used definitions of the six CDRs was provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

[0123] In one aspect of the present invention, a TCR-mimic antibody targeting gp100 / HLA-A2 or an antigen-binding fragment thereof is provided. A TCR-mimic antibody targeting gp100 / HLA-A2, named 42A8 TCRm antibody, was obtained through mouse immunological screening in the present invention.

[0124] The function of the antibody of the present invention is determined by the variable region sequences of the light and heavy chains of the antibody and the antibody structure conformation, and can specifically bind to the melanoma tumor antigen gp100 (YLEPGPVTV) / HLA-A2. Using the variable region gene or complementary determining region (CDR) gene of this antibody, different forms of genetically engineered antibodies can be modified and produced in any expression system using prokaryotic and eukaryotic cells.

[0125] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and all refer to an antibody that specifically binds to gp100 (YLEPGPVTV) / HLA-A2, such as a protein or polypeptide having a heavy chain (amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4) and a light chain (amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3).

[0126] "Fragments", "derivatives", and "analogs" of an antibody refer to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by coupling a mature polypeptide with another compound (such as a compound for chemiluminescence, such as acridinium ester), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a tag protein sequence for purifying or detecting this polypeptide or other fusion protein sequences). These fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0127] The antibody of the present invention refers to a polypeptide having gp100(YLEPGPVTV) / HLA-A2 binding activity and including the above CDR regions. This term also includes variant forms of the polypeptide containing the above CDR regions and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0128] The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained using the hybridoma cell lines against the present invention.

[0129] The present invention also includes fragments of the antibody of the present invention. Generally, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0130] Polynucleotide molecules, vectors and host cells

[0131] The present invention also provides a polynucleotide molecule encoding the above antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be the same as or a degenerate variant of the coding region sequences shown in SEQ ID NO:3-4. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention but different from the coding region sequences shown in SEQ ID NO:3-4.

[0132] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0133] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding this polypeptide, or may also include a polynucleotide further comprising additional coding and / or non-coding sequences. The full-length nucleotide sequence of the antibody of the present invention or its fragment can generally be obtained by PCR amplification, recombination or artificial synthesis. In addition, the heavy chain or light chain can also be fused with a protein or a tag sequence (such as a fluorescent protein, Flag tag) to form a fusion protein.

[0134] Once the antibody sequence is obtained, recombinant antibody preparation can be carried out by bioengineering methods. Usually, the gene encoding the antibody sequence is cloned into a vector, and then the expression vector is transferred into cells for expression. The cells or the expression supernatant are harvested, and the recombinant antibody is purified. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0135] Currently, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing plasmids (or other expression vectors) known in the art. In addition, mutations can also be introduced into the antibody sequence of the present invention by chemical synthesis.

[0136] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.

[0137] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0138] Transforming host cells with recombinant DNA and recombinantly expressing antibodies are conventional techniques well-known to those skilled in the art. The recombinantly expressed antibodies can be separated and purified by conventional techniques well-known to those skilled in the art, which will not be elaborated here.

[0139] Pharmaceutical composition

[0140] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition which contains the above-mentioned antibody or its active fragment, or its fusion protein, or its immunoconjugate, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value may vary according to the nature of the substances to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0141] The active ingredient in the pharmaceutical composition of the present invention can specifically bind to the melanoma tumor antigen gp100 (YLEPGPVTV) / HLA-A2, and thus can be used to treat related diseases, such as melanoma. In addition, other therapeutic agents can also be used simultaneously.

[0142] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or its conjugate) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The pharmaceutical preparation should be matched with the administration route. The pharmaceutical composition of the present invention can be made into an injectable form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectables and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. In addition, the antibody or immunoconjugate of the present invention can also be used together with other therapeutic agents.

[0143] In one embodiment of the present invention, when using the pharmaceutical composition, a safe and effective amount of the antibody or immunoconjugate of the present invention is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dose should also consider factors such as the administration route and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0144] Application

[0145] The antibodies of the present invention have extensive biological and clinical application values, and their applications involve multiple fields such as the diagnosis and treatment of diseases such as tumors (e.g., melanoma), basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of melanoma. The TCR-mimic antibodies of the present invention mimic the ability of TCR to recognize the gp100 antigen peptide presented by MHC I, and specifically recognize gp100(YLEPGPVTV) / HLA-A2 with high affinity, so they can be used for tumor targeted therapy.

[0146] Method for preparing TCR-mimic antibody

[0147] The present invention also provides a method for preparing TCR-mimic antibody, which can be used to screen and obtain specific antibodies against antigen peptides presented by MHCI. The method comprises the following steps:

[0148] (S1) Covalently link a tumor antigen peptide, an HLA heavy chain, and a B2M light chain into a protein molecule, express a single-chain pMHC I in vitro mammalian cells, then biotinylate the single-chain pMHC I, and assemble the biotinylated single-chain pMHC I with streptavidin SA to form a MHC I-tumor antigen peptide complex (pMHC I) tetramer; immunize mice with this pMHC I tetramer as an immunizing antigen;

[0149] (S2) Take the spleen cells of the immunized mice and fuse them with myeloma cells SP2 / 0 in the logarithmic growth phase to prepare hybridoma cells;

[0150] (S3) Provide a pMHC I monomer prepared by in vitro mammalian cell expression as a screening antigen, coat it on an ELISA detection plate, and screen the hybridoma cells obtained in step (S2) by indirect ELISA to obtain a positive hybridoma polyclonal cell line;

[0151] (S4) Use an MHC I molecule without antigen peptide prepared by in vitro mammalian cell expression as a reverse screening antigen, coat it on an ELISA detection plate, and screen the positive hybridoma polyclonal cell line obtained in step (S3) by indirect ELISA to exclude positive clones that bind to the MHC I molecule without antigen peptide;

[0152] (S5) Monoclonally culture the positive hybridoma cell line screened through steps (S3) and (S4) to obtain a monoclonal antibody that specifically binds to pMHC I.

[0153] In a specific embodiment of the present invention, a method for preparing a TCR-mimic antibody against the antigen peptide gp100(YLEPGPVTA) / HLA-A*02:01 presented by MHC I is provided, comprising the following steps:

[0154] (S1) Covalently link the tumor antigen peptide gp100(YLEPGPVTA), HLA-A2 heavy chain, and B2M light chain into a protein molecule, express it in vitro in mammalian cells to obtain GP100pMHC monomers, then biotinylate the GP100pMHC monomers, and assemble the biotin-labeled GP100pMHC monomers with streptavidin SA to form GP100pMHC tetramers; immunize mice with the GP100pMHC tetramers as immunogens;

[0155] (S2) Take the spleen cells of immunized mice and fuse them with myeloma cells SP2 / 0 in the logarithmic growth phase to prepare hybridoma cells;

[0156] (S3) Provide GP100pMHC monomers prepared by in vitro mammalian cell expression as screening antigens, coat them on an ELISA detection plate, and screen the hybridoma cells obtained in step (S2) by indirect ELISA to obtain positive hybridoma polyclonal cell lines;

[0157] (S4) Use HLA-A*02:01 molecules without antigen peptides prepared by in vitro mammalian cell expression as reverse screening antigens, coat them on an ELISA detection plate, and screen the positive hybridoma polyclonal cell lines obtained in step (S3) by indirect ELISA to exclude positive clones that bind HLA-A*02:01 molecules without antigen peptides;

[0158] (S5) Monoclonally culture the positive hybridoma cell lines screened in steps (S3) and (S4) to obtain monoclonal antibodies that specifically bind gp100(YLEPGPVTA) / HLA-A*02:01.

[0159] The main advantages of the present invention include:

[0160] (1) The present invention provides a simpler and more efficient method for preparing TCR-mimic antibodies, which can be used to prepare TCR-mimic antibodies that specifically recognize peptide antigens presented by major histocompatibility complex molecules.

[0161] (2) In the method of the present invention, GP100pMHC tetramers prepared by in vitro mammalian expression are used as antigens to immunize mice, greatly increasing the abundance of peptides and improving the success rate of obtaining specific TCRm antibodies.

[0162] (3) In the method of the present invention, the GP100 pMHC (HLA-A*02:01 (YLEPGPVTA)) monomer prepared by in vitro mammalian expression is used as a screening antigen, and specific TCRm antibodies that truly bind only to HLA-A*02:01 (YLEPGPVTA) can be directly screened out.

[0163] (4) The present invention uses empty MHCs of different genotypes (such as HLA-A*03:01 and HLA-A*11:01), HLA-A*02:01, and the complex of HLA-A*02:01 and other antigen peptides to characterize the specificity of the obtained TCR-mimic antibodies targeting GP100 / HLA-A*02:01 by SPR and ELISA.

[0164] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Experiments without specific conditions noted in the embodiments or test examples of the present invention are usually carried out under conventional conditions or according to the conditions recommended by the raw material / commodity manufacturer; reagents without specific sources noted are conventional reagents purchased from the market.

[0165] Example 1 Development of TCR-mimic antibody cell line targeting gp100(YLEPGPVTA) / HLA-A*02:01

[0166] The GP100 pMHC monomer (Human HLA-A*02:01&B2M&GP100(YLEPGPVTA)Monomer Protein, product number: MHC-HM402, Kactus Biosystems), GP100 pMHC tetramer (Human HLA-A*02:01&B2M&GP100(YLEPGPVTA)Tetramer Protein, product number: MHC-HM402T, Kactus Biosystems), and HLA*02:01 without antigen peptide (Human Peptide Ready HLA-A*02:01&B2M Monomer Protein, product number: MHC-HM43R, Kactus Biosystems) used in this example were prepared by expression in mammalian cells (HEK293 cells).

[0167] 1.1 Mouse immunization

[0168] Mix 80 μg of GP100 pMHC tetramer with an equal volume of complete Freund's adjuvant (CFA), emulsify it, and immunize female Balb / c mice aged 6 - 8 weeks subcutaneously at multiple sites at a dose of 80 μg per mouse. Strengthen the immunization by subcutaneous multi-site injection at the same dose mixed with CFA adjuvant every two weeks. After three boosts, detect the serum titer. Specifically, coat the plate with the immunogen, serially dilute the collected serum, and perform ELISA detection. If the serum titer is greater than 1:1000000, give an intraperitoneal boost immunization with GP100 pMHC tetramer three days before hybridoma cell fusion.

[0169] 1.2 Screening of hybridoma cell lines

[0170] Three days after the mice are boosted, take the spleen cells of the mice and fuse them with the myeloma cells SP2 / 0 in the logarithmic growth phase through PEG1500 to prepare hybridoma cells. Ten days later, coat the plate with 0.5 μg / ml of GP100 pMHC monomer, and screen positive hybridoma polyclonal cell lines with an OD value greater than 2.5 by indirect ELISA. Further, coat the plate with HLA*02:01 without antigen peptide at 0.5 μg / ml, and screen by indirect ELISA to exclude positive clones that bind to HLA*02:01 without antigen peptide, and finally obtain the cell line with the clone number 42A8.

[0171] 1.3 Obtaining monoclonal antibodies

[0172] Dilute the obtained 42A8 cell line to monoclonal state by limiting dilution, then expand the culture and cryopreserve it. At the same time, amplify and culture the hybridoma monoclonal cell line, and purify to obtain monoclonal antibodies. Sequence the obtained monoclonal antibodies, and the results are as follows:

[0173] Amino acid sequence of the light chain (SEQ ID NO:1):

[0174]

[0175]

[0176] Among them, the normal font part is the variable region of the light chain of the antibody (SEQ ID NO:5), the underlined parts are the CDRs of the light chain variable region in turn: L-CDR1 (SEQ ID NO:7), L-CDR2 (KVS), and L-CDR3 (SEQ ID NO:8), divided according to the IMGT rules; the italicized and bold part is the constant region of the light chain (SEQ ID NO:9).

[0177] Amino acid sequence of the heavy chain (SEQ ID NO:2):

[0178]

[0179] Among them, the normal font part is the variable region of the heavy chain of the antibody (SEQ ID NO: 6), and the underlined parts are the CDRs of the variable region of the heavy chain in sequence: H-CDR1 (SEQ ID NO: 10), H-CDR2 (SEQ ID NO: 11), and H-CDR3 (SEQ ID NO: 12), divided according to the IMGT rules; the italicized and bold part is the constant region of the heavy chain (SEQ ID NO: 13).

[0180] 1.4 Preparation of recombinant antibody expression

[0181] The gene sequences of the light and heavy chains of the antibody encoding the signal peptide, variable region and constant region (SEQ ID NO: 3 and 4) were synthesized and respectively constructed into mammalian cell expression vectors. The expression vectors can be any commercially available mammalian cell expression vectors such as pTT5 and pCDNA3.1. The recombinant plasmid was transfected into Expi293 mammalian cells to secrete and express the 42A8 TCRm antibody. The cell expression supernatant was purified by protein A affinity to obtain the 42A8 TCRm antibody (the amino acid sequences of the light and heavy chains are shown in SEQ ID NO: 14 and 15), which was used for antibody characterization.

[0182] Nucleotide sequence of the light chain of the 42A8 TCRm antibody (SEQ ID NO: 3):

[0183] ATGAAACTGCCTGTGAGACTCCTCGTGTTGATGTTTTGGATACCTGCAAGCAGCTCCGATGTCGTTATGACACAGACACCACTGTCACTCCCCGTGTCACTCGGAGACCAGGCCTCTATATCATGCCGGTCCTCACAGTCACTCGTCCATTCCAATGGCAACACGTATCTGCACTGGTATCTGCAGAAGCCAGGCCAATCCCCCAACCTGCTTATATCCAAGGTGAGCAATCGCTTCAGCGGGGTGCCAGACAGGTTTAGTGGGTCTGGGTCCGGAACGGACTTTACCCTGAAAATTAGCCGAGTTGAGGCTGAGGACCTCGGAGTGTACTTTTGTAGCCAGTCAACACACGTGCCGTTCACTTTCGGCTCAGGTACGAAACTGGAAATCAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC

[0184] 42A8 TCRm heavy chain nucleotide sequence (SEQ ID NO:4):

[0185]

[0186] Amino acid sequence of the light chain of the 42A8 TCRm antibody (SEQ ID NO: 14):

[0187] MKLPVRLLVLMFWIPASSS DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPNLLISKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0188] Amino acid sequence of the heavy chain of the 42A8 TCRm antibody (SEQ ID NO: 15):

[0189] MGWSQITLFLVAAITCASS QIQLQQSGAELASPGASVTLSCKASGYTFTDHIMNWVKKRPGQGLEWIGRIYPVSGETNYNQKFMGKATFSVDRSSSTVYMVLNSLTSEDPAVYYCGRSYGNYGSYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0190] Example 2 ELISA characterization of the specificity of the 42A8 TCRm antibody

[0191] The GP100 pMHC monomer, HLA-A*02:01 (ALYDKTKRIFL) monomer, HLA-A*11:01 monomer without antigenic peptide, HLA-A*03:01 monomer and HLA-A*02:01 monomer were separately diluted to 5 μg / ml with PBS and coated on an ELISA plate for 2 hours, and then blocked with PBST containing 3% BSA for 1 hour. The 42A8 TCRm antibody was diluted to 130 nM with the blocking solution and used as the first well, and then serially diluted 3-fold. The last well was used as a negative control with the blocking solution. 100 μl was added to each well, and after incubation at 37 °C for 1 hour, the plate was washed twice. Then, 100 μl of the diluted goat anti-mouse HRP secondary antibody was added to each well, and after incubation at 37 °C for 1 hour, the plate was washed twice. TMB was used for color development for 10 minutes, and the reaction was terminated with 0.5 M sulfuric acid. The OD450 value was read using an ELISA reader.

[0192] The results showed that the 42A8 TCRm antibody only bound to the GP100 pMHC monomer, did not bind to HLA-A*02:01 complexed with other peptides and HLA-A isotype without antigenic peptide, and had high specificity ( Figure 1 ).

[0193] Example 3 Characterization of the binding of the 42A8 TCRm antibody to the GP100 pMHC monomer by surface plasmon resonance (SPR)

[0194] The binding of the 42A8 TCRm antibody to the GP100 pMHC monomer was characterized by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva) instrument.

[0195] The SPR running buffer was 1×HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). Using an SA chip, biotinylated GP100 pMHC and biotinylated HLA-A*(02:01) without antigenic peptide were diluted to 20 μg / ml with the buffer, and the flow rate was 10 μl / min. They were passed over the chip for 20 s and captured on the chip by binding to streptavidin on the chip. The analyte to be tested, the 42A8 TCRm antibody, and the positive control GP100 drug tebentafusp were separately bound to biotinylated GP100 pMHC (KT501-K) and biotinylated HLA*02:01 without antigenic peptide (KR501-7M) captured on the chip in two channels. The antibody flowed over the surface of the antigen chip at five concentration gradients of 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM, with a flow rate of 30 μL / min, a binding time of 120 s, and a dissociation time of 300 s, to obtain the antigen-antibody binding kinetic parameters, as shown respectively inFigure 2 and Figure 3 as shown

[0196] The results showed that the 42A8 TCRm antibody ( Figure 2 ) had similar binding properties to the Gp100 positive control drug tebentafusp ( Figure 3 ).

[0197] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A TCR-mimic antibody targeting gp100 / HLA-A2 or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises the following light chain variable region and heavy chain variable region: A light chain variable region comprising an L-CDR1 having an amino acid sequence as shown in SEQ ID NO:7, an L-CDR2 having an amino acid sequence as shown in KVS, and an L-CDR3 having an amino acid sequence as shown in SEQ ID NO:8; and A heavy chain variable region comprising an H-CDR1 having an amino acid sequence as shown in SEQ ID NO:10, an H-CDR2 having an amino acid sequence as shown in SEQ ID NO:11, and an H-CDR3 having an amino acid sequence as shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The light chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence having at least 90% sequence identity therewith; and / or The heavy chain variable region of the antibody or its antigen-binding fragment comprises the amino acid sequence as shown in SEQ ID NO:6, or an amino acid sequence having at least 90% sequence identity therewith.

3. A recombinant antibody, which has: (i) The sequence of the TCR-mimic antibody targeting gp100 / HLA-A2 or its antigen-binding fragment as claimed in claim 1 or 2; and (ii) A signal peptide for promoting antibody secretion and expression and / or a tag sequence for purification and detection.

4. A polynucleotide molecule encoding a polypeptide selected from the group consisting of: (1) The TCR-mimic antibody targeting gp100 / HLA-A2 or its antigen-binding fragment as claimed in claim 1 or 2; or (2) The recombinant antibody as claimed in claim 3.

5. An expression vector containing the polynucleotide molecule as claimed in claim 4.

6. A host cell containing the expression vector as claimed in claim 5, or having the polynucleotide molecule as claimed in claim 4 integrated into its genome.

7. An antibody conjugate, characterized in that, The antibody conjugate comprises: (a) The TCR-mimic antibody targeting gp100 / HLA-A2 or its antigen-binding fragment as claimed in claim 1 or 2 or the recombinant antibody as claimed in claim 3; and (b) A conjugate moiety selected from the group consisting of: a detectable label, a drug, a toxin, an enzyme, a cytokine, a radionuclide, a nanoparticle / nanorod.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) The TCR-mimic antibody targeting gp100 / HLA-A2 or its antigen-binding fragment as claimed in claim 1 or 2, the recombinant antibody as claimed in claim 3, or the antibody conjugate as claimed in claim 7; and (b) A pharmaceutically acceptable carrier.

9. Use of the TCR-mimic antibody targeting gp100 / HLA-A2 or its antigen-binding fragment as claimed in claim 1 or 2, the recombinant antibody as claimed in claim 3, or the antibody conjugate as claimed in claim 7 for the preparation of: (1) A detection reagent or detection kit for detecting gp100 / HLA-A2; (2) A drug for preventing and / or treating gp100 / HLA-A2-related diseases or disorders.

10. A method for preparing a TCR-mimic antibody, characterized in that, The method comprises the following steps: (S1) Immunize mice with a tetramer of MHC I-tumor antigen peptide complex (pMHC I) prepared by in vitro mammalian cell expression as an immunizing antigen; (S2) Fuse spleen cells from the immunized mice with myeloma cells SP2 / 0 in the logarithmic growth phase to prepare hybridoma cells; (S3) Provide a pMHC I monomer prepared by in vitro mammalian cell expression as a screening antigen, coat it on an ELISA detection plate, and screen the hybridoma cells obtained in step (S2) by indirect ELISA to obtain a positive hybridoma polyclonal cell line; (S4) Use an MHC I molecule without antigen peptide prepared by in vitro mammalian cell expression as a reverse screening antigen, coat it on an ELISA detection plate, and screen the positive hybridoma polyclonal cell line obtained in step (S3) by indirect ELISA to exclude positive clones that bind to the MHC I molecule without antigen peptide; (S5) Monoclonally culture the positive hybridoma cell line obtained by screening in steps (S3) and (S4) to obtain a monoclonal antibody that specifically binds to pMHC I.

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