Highly specific tcr-mimic antibodies targeting gp100 / hla-a2 and methods of making the same

Abstract

The application provides a simple and efficient TCR-mimic antibody preparation method, and a TCR-mimic antibody for gp100 / HLA-A2 is obtained by screening by using the method. The TCR-mimic antibody provided by the application can recognize a MHC-tumor antigen peptide complex with high specificity and high affinity, and has the potential to be developed as a tumor antibody drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody drugs, in particular, to a high-specificity TCR-mimic antibody targeting gp100 / HLA-A2 and a preparation method thereof. BACKGROUND

[0002] In the field of tumor immunity and therapy, 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 MHC-tumor antigen peptide complex (pMHC) through T cell receptor (TCR), and then activate the immune system to kill tumors.

[0003] The HLA heavy chain (alpha chain) and light chain (beta 2m) of MHC class I molecules form a ternary complex with antigenic peptides in the form of non-covalent bonds. Antigenic peptides are loaded into the groove structure formed by the heavy chain and the light chain, forming a binding interface that interacts with TCR. The natural TCR has low affinity for MHC I, 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 TCR drug development.

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

[0005] In the development of TCR-mimic antibodies, MHC immunogens, MHC protein reagents for screening and in vitro characterization are difficult to obtain. In the literature, pMHC I is usually prepared by recombinant expression of HLA heavy chain and B2M light chain in E. coli, and then three components are complexed in vitro. However, the yield of pMHC I is low, the success rate of complexing is low, and the HLA heavy chain and B2M light chain without antigen peptide are difficult to form stable dimers. In addition, positive TCR-mimic antibodies need to recognize the antigen epitope presented by the groove structure of pMHC, resulting in a low positive rate of TCR-mimic antibodies and difficult screening. SUMMARY

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

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

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

[0009] a heavy chain variable region comprising H-CDR1 of an amino acid sequence as shown in SEQ ID NO: 10, H-CDR2 of an amino acid sequence as shown in SEQ ID NO: 11, and H-CDR3 of 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 a FR region of murine or human origin, and / or the heavy chain variable region comprises a FR region of murine or human origin.

[0012] In another preferred embodiment, the light chain variable region of the antibody or the antigen-binding fragment thereof 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 thereto; and / or

[0013] The heavy chain variable region of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth 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 antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 5, and / or a heavy chain variable region having an amino acid sequence as set forth 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 of murine origin or of human origin.

[0017] In another preferred embodiment, the heavy chain constant region is derived from a mouse heavy chain IgGl.

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

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

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

[0021] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody, a polyclonal antibody, a diabody, a single chain antibody (scFv), a Fab, a Fab', a F(ab')2 antibody.

[0022] In another preferred embodiment, the antibody is a monoclonal antibody comprising a light chain having an amino acid sequence as set forth in SEQ ID NO: 1, and a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 2.

[0023] In another preferred embodiment, the antibody comprises an animal-derived antibody (e.g., a murine-derived antibody of different subtypes), a chimeric antibody (e.g., a human-murine chimeric antibody), a humanized antibody.

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

[0025] (i) a sequence of the TCR-mimic antibody or antigen-binding fragment thereof targeting gp 100 / HLA-A2 as described in the first aspect of the present application; and

[0026] (ii) a signal peptide for facilitating antibody secretion expression and / or a tag sequence for purification, detection.

[0027] In another preferred embodiment, the recombinant antibody has a signal peptide for facilitating antibody secretion expression.

[0028] In another preferred embodiment, the tag sequence is selected from the group consisting of FLAG, Myc, His tag, and the like.

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

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

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

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

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

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

[0035] In another preferred embodiment, the expression vector comprises a eukaryotic cell expression vector, a prokaryotic cell expression vector.

[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 vector, plasmid, transposon, other gene transfer system, or a combination thereof.

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

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

[0040] In another preferred embodiment, the host cell comprises a eukaryotic cell (such as a mammalian cell), a prokaryotic cell.

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

[0042] (a) the TCR-mimic antibody or antigen-binding fragment thereof targeting gp100 / HLA-A2 according to the first aspect of the present application or the recombinant antibody according to the second aspect of the present application; and

[0043] (b) a conjugating 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 comprises a fluorescent label or a chemiluminescent label.

[0045] In another preferred embodiment, the radionuclide comprises a diagnostic isotope; and / or a therapeutic isotope.

[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 agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an antimetabolite drug, a chemosensitizer, 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 application, there is provided a pharmaceutical composition comprising:

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

[0051] (b) a pharmaceutically acceptable carrier.

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

[0053] In another preferred embodiment, the pharmaceutical composition is for use in the preparation of a medicament for preventing and / or treating a gp100 / HLA-A2 related disease or disorder.

[0054] In another preferred embodiment, the gp100 / HLA-A2 related disease or disorder comprises melanoma.

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

[0056] (1) Detection reagents or kits for detecting gp100 / HLA-A2;

[0057] (2) Medications used to prevent and / or treat gp100 / HLA-A2 related diseases or conditions.

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

[0059] In another preferred embodiment, the detection includes flow cytometry (FACS), cell immunofluorescence assay, and ELISA.

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

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

[0062] In a ninth aspect of the present invention, a method is provided for generating a TCR-mimic antibody or antigen-binding fragment thereof targeting gp100 / HLA-A2 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) Under conditions suitable for producing antibodies or antigen-binding fragments, host cells as described in the fifth aspect of the present invention are cultured to obtain a culture containing the antibodies or antigen-binding fragments.

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

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

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

[0067] (S1) Provide the MHC I-tumor antigen peptide complex (pMHC I) tetramer prepared by in vitro expression in mammalian cells as an immunogen to immunize mice;

[0068] (S2) Spleen cells from immunized mice were fused with logarithmic growth phase myeloma cells SP2 / 0 to prepare hybridoma cells.

[0069] (S3) pMHC I monomer prepared by in vitro mammalian cell expression is used as a screening antigen, which is coated on an ELISA detection plate. The hybridoma cells obtained in step (S2) are screened by indirect ELISA to obtain positive hybridoma polyclonal cell lines.

[0070] (S4) Using MHC I molecules without antigenic peptides prepared by in vitro mammalian cell expression as reverse screening antigens, they are coated on ELISA detection plates. The positive hybridoma polyclonal cell lines obtained in step (S3) are screened by indirect ELISA to exclude positive clones that bind MHC I molecules without antigenic peptides.

[0071] (S5) The positive hybridoma cell lines obtained through steps (S3) and (S4) are subjected to monoclonal culture to obtain monoclonal antibodies that specifically bind to pMHC I.

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

[0073] (a) A single-chain pMHC I was obtained by covalently linking tumor antigen peptide, HLA heavy chain and B2M light chain into a protein molecule and expressing it in mammalian cells in vitro.

[0074] (b) The obtained single-stranded pMHCI was labeled with biotin to obtain biotin-labeled single-stranded pMHC I;

[0075] (c) Biotin-labeled single-stranded pMHCI is assembled with streptavidin SA to form the pMHC I tetramer.

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

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

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

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

[0080] In another preferred embodiment, in step (S1), the 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, and then administer subcutaneous injections of 50-100 μg of immunogen per mouse to female Balb / c mice aged 6-8 weeks.

[0082] (b) Two weeks later, after mixing the same dose with an equal volume of Freund’s complete adjuvant, administer the booster immunization at multiple subcutaneous injection sites.

[0083] (c) After three booster immunizations, the serum titer of mice was tested. The standard for testing was a titer greater than 1:1,000,000.

[0084] (d) Three days before hybridoma cell fusion, administer a booster immunization via intraperitoneal injection of pMHC I tetramer.

[0085] In an eleventh aspect of the present invention, a method for treating gp100 / HLA-A2 related diseases or conditions is provided, comprising administering to a subject in need a TCR-mimic antibody or 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 embodiment, the subjects include humans and non-human mammals.

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

[0088] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0089] Figure 1 The results show the specificity of the 42A8 TCRm antibody characterized by ELISA.

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

[0091] Figure 3 The results show the SPR characterization of the binding of the Gp100 positive control drug tebentafusp to gp100(YLEPGPVTA) / HLA-A*02:01. Detailed Implementation

[0092] Through extensive and in-depth research, the inventors have proposed a simpler and more efficient method for preparing TCR-mimic antibodies. This invention utilizes single-chain pMHC expressed by mammalian cells. Specifically, through rational structural design, an antigenic peptide (e.g., gp100), HLA-A2 heavy chain, and B2M light chain are covalently linked into a single protein molecule, maintaining the same antigen-presenting conformation as the natural pMHC ternary complex. Biotin-labeled single-chain pMHC is assembled with streptavidin SA into a tetramer as an immunogen. This immunogen antigenic peptide binds 100% within the pMHC groove structure and exhibits good stability, making it suitable for immunizing animals. Single-chain pMHC expressed by mammalian cells is used as a screening antigen to select for positively binding antibodies. Additionally, during the screening process, empty MHC (peptide-ready MHC, prMHC) expressed by mammalian cells, lacking the antigenic peptide, is used for reverse screening to remove non-specific antibodies that bind to the MHC heavy and light chains but do not recognize the antigen-presenting peptide. Among them, the empty MHC without antigenic peptides can also exist stably through rational structural design, which enables the HLA-A2 heavy chain and B2M light chain dimers to be covalently linked.

[0093] Specifically, this invention screened and obtained a TCR-mimic antibody (hereinafter referred to as TCRm antibody) targeting gp100, clone number 42A8. This antibody exhibits high specificity for gp100(YLEPGPVTA) / HLA-A*02:01, and does not bind to HLA-A*02:01 alone or complexes of HLA-A*02:01 with other peptides. Surface plasmon resonance (SPR) assay showed that the binding affinity (KD) level of the 42A8 TCRm antibody to the GP100pMHC monomer was approximately 0.47 μM. The 42A8 TCRm antibody of this invention, after humanization, has the potential for antibody drug development.

[0094] Based on this, the present invention was completed.

[0095] the term

[0096] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the 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 terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the 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 pertains.

[0098] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. 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 amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0100] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0101] The term "sequence identity" as used 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 sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0102] As used herein, the terms "TCR-mimic antibody" and "TCRm antibody" are used interchangeably. A TCR-mimic antibody is an engineered antibody designed to mimic the function of the T cell receptor (TCR) and specifically recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. Unlike conventional antibodies (which typically target cell surface proteins), TCR-mimic antibodies, by binding to MHC-peptide complexes, can target intracellular antigens (such as tumor-associated antigens or viral antigens), thus expanding the potential of antibody drugs in cancer immunotherapy and infectious diseases. Their design is typically based on structural mimicry of the TCR-MHC-peptide interaction, optimizing the complementarity-determining region (CDR) to improve affinity and specificity. These antibodies have significant value in bispecific antibodies, CAR-T therapy, or direct-targeted therapy, 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 melanoma antigen gp100 by the human MHCI class molecule HLA-A*02:01.

[0104] As used in this article, the terms “GP100pMHC monomer” and “HLA-A*02:01(YLEPGPVTA)” are used interchangeably, both referring 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, both referring to the pMHC tetramer formed by assembling the biotinylated GP100pMHC monomer of the present invention with streptavidin SA.

[0106] Antibody

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

[0108] As used herein, the term "variable" indicates that the variable regions of an antibody differ in a specific sequence, resulting in the affinity and specificity of a particular antibody for a specific antigen. Antibody variable regions include complementarity-determining regions (CDRs) or hypervariable regions, as well as relatively conserved framework regions (FRs). The primary sequence of the heavy and light chain variable regions consists of four FR sequences and three CDR sequences spaced apart (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The sequence and spatial conformation of the heavy and light chain variable regions determine the specific binding of the antibody to the antigenic epitope. Antibody constant regions do not directly participate in antibody-antigen binding, but they affect the performance of antibody capture and detection.

[0109] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified into one of the κ and λ classes based on the amino acid sequence of their constant region. According to the amino acid sequence of their heavy chain constant region, immunoglobulins can be divided into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM, as well as antibody subtypes (isotypes), such as mouse IgG including IgG1, IgG2a, and IgG2b subtypes. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0110] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a largely homogeneous population, meaning that the individual antibodies contained in that population are identical. Monoclonal antibodies target a single antigenic determinant (epitope) with high specificity. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a largely homogeneous population of antibodies, which should not be interpreted as requiring any special methods to produce the antibody.

[0111] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the monoclonal antibody targeting gp100 / HLA-A2, monoclonal antibodies having the variable region chain of the monoclonal antibody 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 a variable region (complementarity-determining region, CDR), provided that the variable region is the same as or has at least 90% homology with the variable regions of the light chain and heavy chain of the present invention, preferably at least 95% homology.

[0112] As known to those skilled in the art, antibody conjugates and fusion expression products include: conjugates formed by binding fluorescent or luminescent markers, radiolabels, enzymes capable of producing detectable products, gold nanoparticles / nanoran and other detectable molecules to the antibody targeting gp100 / HLA-A2 or its antigen-binding fragment; or conjugates formed by binding therapeutic molecules such as therapeutic drugs, toxins, and radionuclides 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 maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds on their chain regions; and (iii) scFv fragments consisting of the VH and VL domains of a single arm of the antibody.

[0114] This invention includes not only complete monoclonal antibodies, but also antibody fragments with binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0115] The term "epitope" or "antigenic determinant" refers to the site on an antigen where immunoglobulins or antibodies specifically bind. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0116] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

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

[0118] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0119] In this invention, antibodies include murine antibodies prepared using 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 this invention refers to a monoclonal antibody against gp100 / HLA-A2 prepared according to knowledge and skills in the art.

[0120] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0121] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "light chain variable region" and "VL" are used interchangeably.

[0122] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242.

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

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

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

[0126] The terms "fragment," "derivative," and "analyte" of an antibody refer to a polypeptide that substantially retains the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved 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 substituent groups in one or more amino acid residues; or (iii) polypeptides formed by coupling a mature polypeptide with another compound (e.g., a compound used for chemiluminescence, such as acridinium ester); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, secretion sequence, or tag protein sequence or other fusion protein sequence used to purify or detect this polypeptide). These fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0127] The antibody of this invention refers to a polypeptide containing the aforementioned CDR region and possessing gp100(YLEPGPVTV) / HLA-A2 binding activity. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. This term also includes active fragments and active derivatives of the antibody of this invention.

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

[0129] The present invention also includes fragments of the antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, 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 of the antibody of the present invention.

[0130] Polynucleotide molecules, carriers and host cells

[0131] This invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized 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 identical to the coding region sequence shown in SEQ ID NO:3-4 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a sequence having the same amino acid sequence as the polypeptide of this invention, but differing from the coding region sequence shown in SEQ ID NO:3-4.

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

[0133] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include polynucleotides that include additional coding and / or non-coding sequences. The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Furthermore, heavy or light chains can be fused with proteins or tag sequences (such as fluorescent proteins, flag tags) to form fusion proteins.

[0134] Once the antibody sequence is obtained, recombinant antibodies can be prepared using bioengineering methods. Typically, the gene encoding the antibody sequence is cloned into a vector, the expression vector is then transferred into cells for expression, the cells or expression supernatant are harvested, and the recombinant antibody is obtained through purification. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0135] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or other expression vectors) known in the art. Furthermore, mutations can be introduced into the antibody sequence of this invention through chemical synthesis.

[0136] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

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

[0138] Transforming host cells with recombinant DNA to express antibodies is a routine technique well-known to those skilled in the art. The recombinantly expressed antibodies can be isolated and purified using routine techniques well-known to those skilled in the art, which will not be elaborated upon here.

[0139] Pharmaceutical Composition

[0140] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-described antibody or its active fragment, or its fusion protein, or its immunoconjugate, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

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

[0142] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or its conjugate) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the antibodies or immunoconjugates of the present invention can also be used with other therapeutic agents.

[0143] In one embodiment of the invention, when using the pharmaceutical composition, a safe and effective amount of the antibody or immunoconjugate of the invention is administered to a mammal, wherein the safe and effective amount is typically 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 between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0144] application

[0145] The antibody of this invention has broad biological and clinical application value, encompassing multiple fields such as the diagnosis and treatment of tumors like melanoma, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of melanoma. The TCR-mimic antibody of this invention mimics the ability of TCRs to recognize MHC I-presented gp100 antigen peptides, specifically recognizing gp100(YLEPGPVTV) / HLA-A2 with high affinity, thus enabling its use in targeted tumor therapy.

[0146] Methods for preparing TCR-mimic antibodies

[0147] This invention also provides a method for preparing TCR-mimic antibodies, which can be used to screen for specific antibodies against antigenic peptides presented by MHCI. The method includes the following steps:

[0148] (S1) A single-chain pMHC I was obtained by covalently linking the tumor antigen peptide, HLA heavy chain and B2M light chain into a protein molecule and expressing it in mammalian cells in vitro. Then, the single-chain pMHC I was biotinylated and assembled with streptavidin SA to form a MHC I-tumor antigen peptide complex (pMHC I) tetramer. The pMHC I tetramer was used as an immunogen to immunize mice.

[0149] (S2) Spleen cells from immunized mice were fused with logarithmic growth phase myeloma cells SP2 / 0 to prepare hybridoma cells.

[0150] (S3) pMHC I monomer prepared by in vitro mammalian cell expression is used as a screening antigen, which is coated on an ELISA detection plate. The hybridoma cells obtained in step (S2) are screened by indirect ELISA to obtain positive hybridoma polyclonal cell lines.

[0151] (S4) Using MHC I molecules without antigenic peptides prepared by in vitro mammalian cell expression as reverse screening antigens, they are coated on ELISA detection plates. The positive hybridoma polyclonal cell lines obtained in step (S3) are screened by indirect ELISA to exclude positive clones that bind MHC I molecules without antigenic peptides.

[0152] (S5) The positive hybridoma cell lines obtained through steps (S3) and (S4) are subjected to monoclonal culture to obtain monoclonal antibodies that specifically bind to pMHC I.

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

[0154] (S1) The tumor antigen peptide gp100 (YLEPGPVTA), HLA-A2 heavy chain and B2M light chain were covalently linked into a protein molecule, which was expressed in mammalian cells in vitro to obtain GP100pMHC monomer. Then, the GP100pMHC monomer was biotinylated and labeled. The biotinylated GP100pMHC monomer was assembled with streptavidin SA to form GP100pMHC tetramer. The GP100pMHC tetramer was used as an immunogen to immunize mice.

[0155] (S2) Spleen cells from immunized mice were fused with logarithmic growth phase myeloma cells SP2 / 0 to prepare hybridoma cells.

[0156] (S3) Provide GP100pMHC 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 positive hybridoma polyclonal cell lines.

[0157] (S4) Using HLA-A*02:01 molecules without antigenic peptides prepared by in vitro mammalian cell expression as reverse screening antigens, they are coated onto ELISA detection plates. Positive hybridoma polyclonal cell lines obtained in step (S3) are screened by indirect ELISA to exclude positive clones that bind to HLA-A*02:01 molecules without antigenic peptides.

[0158] (S5) The positive hybridoma cell lines obtained through steps (S3) and (S4) are subjected to monoclonal culture to obtain a monoclonal antibody that specifically binds to gp100(YLEPGPVTA) / HLA-A*02:01.

[0159] The main advantages of this invention include:

[0160] (1) This 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 tetramer is prepared by in vitro lactation expression and used as an antigen to immunize mice, which greatly increases the abundance of peptides and improves the success rate of obtaining specific TCRm antibodies.

[0162] (3) In the method of the present invention, GP100pMHC(HLA-A*02:01(YLEPGPVTA)) monomer prepared by in vitro lactation expression is used as a screening antigen, which can directly screen out the real specific TCRm antibody that only binds to HLA-A*02:01(YLEPGPVTA).

[0163] (4) The present invention uses empty MHC of different genotypes (e.g., HLA-A*03:01 and HLA-A*11:01) and HLA-A*02:01 and complexes of HLA-A*02:01 with other antigenic peptides, and characterizes the specificity of the obtained TCR-mimic antibody targeting GP100 / HLA-A*02:01 by SPR and ELISA.

[0164] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Experiments in the embodiments or test examples of the present invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material / commodity manufacturer; reagents whose specific source is not specified are commercially available conventional reagents.

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

[0166] The GP100pMHC monomers (Human HLA-A*02:01&B2M&GP100(YLEPGPVTA) Monomer Protein, catalog number: MHC-HM402, Kactus Biosystems), GP100pMHC tetramer (Human HLA-A*02:01&B2M&GP100(YLEPGPVTA) Tetramer Protein, catalog number: MHC-HM402T, Kactus Biosystems), and HLA*02:01 (Human Peptide Ready HLA-A*02:01&B2M Monomer Protein, catalog number: MHC-HM43R, Kactus Biosystems) used in this embodiment were prepared by expression in mammalian cells (HEK293 cells).

[0167] 1.1 Mouse Immunization

[0168] 80 μg of GP100pMHC tetramer was mixed with an equal volume of Freund's complete adjuvant (CFA), emulsified, and administered subcutaneously at multiple sites to 6-8 week old female Balb / c mice at a dose of 80 μg / mouse. Two weeks later, booster immunizations were administered subcutaneously at multiple sites with the same dose of CFA adjuvant. Serum titers were measured after three booster immunizations. Specifically, using immunogen-coated plates, the collected serum was serially diluted and analyzed by ELISA. For serum titers greater than 1:1,000,000, a booster immunization with GP100pMHC tetramer was administered intraperitoneally three days before hybridoma cell fusion.

[0169] 1.2 Screening of hybridoma cell lines

[0170] Three days after booster immunization of mice, spleen cells were fused with logarithmic growth phase myeloma cells SP2 / 0 using PEG1500 to prepare hybridoma cells. Ten days later, the cells were plated with 0.5 μg / ml of GP100pMHC monomer and screened for positive hybridoma polyclonal cell lines with an OD value greater than 2.5 using indirect ELISA. Further, HLA*02:01 without antigenic peptides was plated at 0.5 μg / ml, and positive clones bound to HLA*02:01 without antigenic peptides were excluded by indirect ELISA screening, ultimately obtaining cell line 42A8.

[0171] 1.3 Obtaining Monoclonal Antibodies

[0172] The selected 42A8 cell line was limited to monoclonal dilution and then expanded and cryopreserved. Simultaneously, hybridoma monoclonal cell lines were amplified and purified to obtain monoclonal antibodies. The obtained monoclonal antibodies were sequenced, and the results are as follows:

[0173] Light chain amino acid sequence (SEQ ID NO:1):

[0174]

[0175]

[0176] The normally typed portion represents the light chain variable region of the antibody (SEQ ID NO:5), and the underlined portions represent the CDRs of the light chain variable region in sequence: 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 portion represents the light chain constant region (SEQ ID NO:9).

[0177] Heavy chain amino acid sequence (SEQ ID NO:2):

[0178]

[0179] The text in normal font represents the heavy chain variable region of the antibody (SEQ ID NO:6), and the underlined parts are the CDRs of the heavy chain variable region: 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 text in italics and bold represents the heavy chain constant region (SEQ ID NO:13).

[0180] 1.4 Recombinant Antibody Expression and Preparation

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

[0182] 42A8 TCRm antibody light chain nucleotide sequence (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] GP100pMHC 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 diluted to 5 μg / ml with PBS and coated onto ELISA plates for 2 hours. The plates were then blocked with 3% BSA in PBST for 1 hour. 42A8 TCRm antibody was diluted to 130 nM with blocking buffer and used as the first well, followed by 3-fold serial dilutions. The last well contained 100 μL of blocking buffer as a negative control. After incubation at 37°C for 1 hour, the plates were washed twice. 100 μL of diluted goat anti-mouse HRP secondary antibody was added to each well, and the plates were incubated at 37°C for 1 hour. After washing twice, the plates were developed with TMB for 10 minutes, and the reaction was stopped with 0.5 M sulfuric acid. The OD450 value was read using a microplate reader.

[0192] The results showed that the 42A8TCRm antibody only bound to the GP100pMHC monomer and did not bind to HLA-A*02:01 conjugated with other peptides or to homotype HLA-A without antigenic peptides, demonstrating high specificity. Figure 1 ).

[0193] Example 3: Surface plasmon resonance (SPR) characterization of the binding of 42A8 TCRm antibody to GP100pMHC monomer.

[0194] The binding of the 42A8 TCRm antibody to the GP100pMHC monomer was characterized by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva) instrument.

[0195] SPR running buffer was 1×HBS-EP+ buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7.4). Using an SA chip, biotinylated GP100pMHC and biotinylated HLA-A* (02:01) without antigenic peptide were diluted to 20ug / ml with buffer, flowed through the chip at a flow rate of 10ul / min for 20s, and captured onto the chip by binding to streptavidin on the chip. The analyte 42A8 TCRm antibody and the positive control GP100 drug tebentafusp were bound to biotinylated GP100pMHC (KT501-K) and biotinylated HLA*02:01 (KR501-7M) without antigen peptide, respectively, captured on the chip in two channels. The antibody flowed through the antigen chip surface at five concentration gradients: 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM, at a flow rate of 30 μL / min. The binding time was 120 s, and the dissociation time was 300 s. The antigen-antibody binding kinetic parameters were obtained as follows:Figure 2 and Figure 3 As shown.

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

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

Claims

1. A TCR-mimic antibody or antigen binding fragment thereof targeting gp100 / HLA-A2, characterized in that, The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region of: a light chain variable region comprising an L-CDR1 of an amino acid sequence as set forth in SEQ ID NO: 7, an L-CDR2 of an amino acid sequence as set forth in KVS, and an L-CDR3 of an amino acid sequence as set forth in SEQ ID NO: 8; and a heavy chain variable region comprising an H-CDR1 of an amino acid sequence as set forth in SEQ ID NO: 10, an H-CDR2 of an amino acid sequence as set forth in SEQ ID NO: 11, and an H-CDR3 of an amino acid sequence as set forth in SEQ ID NO: 12; the gp100 / HLA-A2 is YLEPGPVTA / HLA-A*02:

01.

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

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

4. The recombinant antibody of claim 3, wherein The recombinant antibody comprises a light chain of an amino acid sequence as set forth in SEQ ID NO: 14, and / or a heavy chain of an amino acid sequence as set forth in SEQ ID NO:

15.

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

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

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

8. An antibody conjugate, characterized in that, The antibody conjugate consists of: (a) the TCR-mimic antibody or antigen-binding fragment thereof targeting gp100 / HLA-A2 as claimed in claim 1 or 2, or the recombinant antibody as claimed in claim 3; and (b) a conjugating moiety, which is a detectable label.

9. The antibody conjugate of claim 8, wherein, The conjugating moiety is selected from the group consisting of an enzyme capable of producing a detectable product, a radionuclide, a nanoparticle, or a nanorod.

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

11. Use of the TCR-mimic antibody or antigen-binding fragment thereof targeting gp100 / HLA-A2 of claim 1 or 2, the recombinant antibody of claim 3, or the antibody conjugate of claim 8 for the manufacture of a detection reagent or a detection kit for the detection of gp100 / HLA-A2, said gp100 / HLA-A2 being YLEPGPVTA / HLA-A*02:01.

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