WO2025044998A1 - Single-domain antibody targeting prame polypeptide and use thereof - Google Patents

Abstract

A single-domain antibody targeting a PRAME polypeptide. The single-domain antibody targeting the PRAME polypeptide can bind to the PRAME polypeptide with high affinity and has relatively good specificity, thereby laying a new material foundation for the development of an anti-tumor drug targeting the PRAME polypeptide. Further disclosed are a bispecific antibody, a chimeric antigen receptor, a chimeric antigen receptor-T cell, an ADC, etc., prepared by means of using the single-domain antibody.

Description

Single domain antibody targeting PRAME polypeptide and its use Technical Field

The present invention relates to the field of biotechnology. More specifically, the present invention relates to a single domain antibody targeting a PRAME polypeptide and uses thereof.

Background Art

PRAME (PReferentially expressed Antigen in MElanoma) is an intracellular protein encoded by the PRAME gene. PRAME is expressed at high levels in many tumors, such as melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc., and is rarely expressed in normal human tissues. These characteristics make PRAME an ideal target for tumor targeted therapy (Am J Surg Pathol. 2018Nov; 42(11):1456-1465.). After being processed by the intracellular antigen presentation system, the PRAME protein has a special polypeptide sequence: SLLQHLIGL, which can be presented to the cell surface by the major histocompatibility antigen HLA-A02 molecule. The HLA-A02 complex bound to the PRAME polypeptide can be used as a cell membrane target to develop T cell receptor (T cell receptor, TCR) or TCR-like antibody-related therapies.

Single-domain antibodies (sdAb) are a special type of antibody with a smaller molecular weight. Single-domain antibodies are composed of only two identical heavy chains. Compared with the molecular weight of 150-160kDa of traditional double-chain antibodies, the molecular weight of single-domain antibodies is about 110KD. Single-domain antibodies generally have high specificity, high affinity, low immunogenicity, and good permeability. The antigen binding region of a single-domain antibody is composed of only one chain, and the variable region (VHH) of a single-domain antibody is only 12-15kDa. Therefore, it is very simple to structurally modify single-domain antibodies, and there will be no problem of mismatching of the light and heavy chains of traditional double-linked antibodies, nor will there be a decrease in affinity caused by the modification of the single chain of the antigen binding region. Based on these advantages, the use of single-domain antibodies as the antigen recognition region of bispecific antibodies or chimeric antigen receptor T cells (Chimeric Antigen Receptor-T cells, CAR-T) is one of the future development trends (Serge Muyldermans. Annu. Rev. Biochem. 82: 775-797 (2013)). Single domain antibodies can recognize cell membrane proteins, or peptides derived from intracellular proteins presented to the cell surface by major histocompatibility antigens. Using the HLA-A02 complex bound to the PRAME peptide as an antigen, specific single domain antibody molecules can be screened. These candidate molecules can be used to develop bispecific antibodies, CAR-T or ADC (Antibody drug conjugate) and other biological drugs.

Summary of the invention

The object of the present invention is to provide a specific single domain antibody targeting PRAME polypeptide and corresponding specific humanized single domain antibody, bispecific antibody, chimeric antigen receptor, chimeric antigen receptor-T cell and ADC targeting PRAME polypeptide.

The present invention also aims to provide the use of the single domain antibody, humanized single domain antibody, bispecific antibody, chimeric antigen receptor, chimeric antigen receptor-T cell and ADC in treating tumors or preparing drugs for treating tumors.

In a first aspect, the present invention provides a VHH chain of a single domain antibody targeting a PRAME polypeptide, wherein the VHH chain comprises CDR1, CDR2 and CDR3 as shown in the following table:

In a preferred embodiment, the amino acid sequence of the PRAME polypeptide is: SLLQHLIGL (SEQ ID NO: 121).

In a preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence which is optionally subjected to addition, deletion, modification and/or substitution of at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and can retain high affinity binding to the PRAME polypeptide.

In a preferred embodiment, the VHH chain further comprises framework regions FR1, FR2, FR3 and FR4.

In a preferred embodiment, the amino acid sequence of the VHH chain of the single domain antibody targeting the PRAME polypeptide is shown in the following table:

In a second aspect, the present invention provides a heavy chain variable region of an antibody targeting a PRAME polypeptide, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the following table:

In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody targeting the PRAME polypeptide is shown in the following table:

In a third aspect, the present invention provides a single domain antibody targeting a PRAME polypeptide, which has the VHH chain described in the first aspect.

In a fourth aspect, the present invention provides a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide, wherein the framework regions FR1, FR2, FR3 and FR4 are humanized based on the VHH chain described in the first aspect.

In a preferred embodiment, the variable region sequence of the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide is as follows:

In a fifth aspect, the present invention provides an antibody targeting a PRAME polypeptide, wherein the antibody comprises one or more VHH chains of the single domain antibody targeting a PRAME polypeptide according to the first aspect or the VHH chain of the humanized single domain antibody targeting a PRAME polypeptide according to claim 4.

In a preferred embodiment, the antibody targeting the PRAME polypeptide comprises a monomer, a bivalent antibody, and/or a multivalent antibody.

In a sixth aspect, the present invention provides a bispecific antibody, comprising a first antibody and a second antibody, wherein the first antibody comprises the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, or the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, or the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, or the antibody targeting the PRAME polypeptide described in the fifth aspect.

In a preferred embodiment, the second antibody may bind to the same or a different antigen as the first antibody, or bind to a different epitope on the same antigen as the first antibody.

In a preferred embodiment, the second antibody is a single domain antibody, a single chain antibody or a double chain antibody.

In a preferred embodiment, the bispecific antibody comprises 2-4 single-domain antibodies targeting PRAME polypeptide; preferably, it comprises 2 single-domain antibodies targeting PRAME polypeptide; more preferably, the 2 single-domain antibodies targeting PRAME polypeptide form a single-domain antibody dimer targeting PRAME polypeptide.

In a preferred embodiment, the sequence of the bispecific antibody is shown in the following table:

In the seventh aspect, the present invention provides a fusion protein, the fusion protein comprising the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to the second aspect, the single-domain antibody targeting the PRAME polypeptide according to the third aspect, and the humanized PRAME targeting antibody according to the fourth aspect. The VHH chain of a single domain antibody of the polypeptide, or the antibody targeting the PRAME polypeptide as described in the fifth aspect, an optional linker sequence and an Fc fragment or a half-life extension domain of an immunoglobulin.

In a preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3, IgG4; preferably IgG4.

In the eighth aspect, the present invention provides a chimeric antigen receptor, which is made of the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, or the antibody targeting the PRAME polypeptide described in the fifth aspect.

In a preferred embodiment, the amino acid sequence of the chimeric antigen receptor is as follows:

In a ninth aspect, the present invention provides an immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor described in the eighth aspect.

In a preferred embodiment, the immune effector cells include but are not limited to: T cells, NK cells, TIL cells; preferably T cells.

In the tenth aspect, the present invention provides a nucleic acid molecule, which encodes the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, the antibody targeting the PRAME polypeptide described in the fifth aspect, the bispecific antibody described in the sixth aspect, the fusion protein described in the seventh aspect, or the chimeric antigen receptor described in the eighth aspect.

In the eleventh aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the tenth aspect.

In a twelfth aspect, the present invention provides a host cell, wherein the host cell comprises the expression vector described in the eleventh aspect, or the nucleic acid molecule described in the tenth aspect is integrated into its genome.

In the thirteenth aspect, the present invention provides a method for preparing the VHH chain of the single domain antibody targeting the PRAME polypeptide of the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide of the second aspect, the single domain antibody targeting the PRAME polypeptide of the third aspect, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide of the fourth aspect, the antibody targeting the PRAME polypeptide of the fifth aspect, the bispecific antibody of the sixth aspect, or the fusion protein of the seventh aspect, the method comprising the following steps:

1) culturing the host cell as described in the eleventh aspect under suitable conditions to obtain a culture containing the VHH chain of the single domain antibody targeting the PRAME polypeptide, the heavy chain variable region of the antibody targeting the PRAME polypeptide, the single domain antibody targeting the PRAME polypeptide, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide, the antibody targeting the PRAME polypeptide, the bispecific antibody or the fusion protein; and

2) Optionally, isolating or recovering the VHH chain of the single domain antibody targeting the PRAME polypeptide, the heavy chain variable region of the antibody targeting the PRAME polypeptide, the single domain antibody targeting the PRAME polypeptide, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide, the single domain antibody targeting the PRAME polypeptide, the bispecific antibody or the fusion protein from the culture.

In a fourteenth aspect, the present invention provides an immunoconjugate, the immunoconjugate comprising:

1) the VHH chain of the single domain antibody targeting the PRAME polypeptide as described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide as described in the second aspect, the single domain antibody targeting the PRAME polypeptide as described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide as described in the fourth aspect, the antibody targeting the PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, or the fusion protein as described in the seventh aspect; and

2) A conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In a preferred embodiment, the conjugated moiety is a drug or a toxin.

In a preferred embodiment, the immunoconjugate is an antibody-drug conjugate (ADC).

In a preferred embodiment, the conjugated moiety is a detectable label.

In a preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

In a preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) VHH chain of a single domain antibody targeting a PRAME polypeptide as described in the first aspect, a heavy chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, a single domain antibody targeting a PRAME polypeptide as described in the third aspect, a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide as described in the fourth aspect, an antibody targeting a PRAME polypeptide as described in the fifth aspect, a bispecific antibody as described in the sixth aspect, or a fusion protein as described in the seventh aspect.

In a preferred embodiment, the multivalency refers to the presence of multiple repeating parts in the amino acid sequence of the immunoconjugate.

In a fifteenth aspect, the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising a therapeutically or diagnostically effective amount of the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the second aspect, The heavy chain variable region of an antibody targeting a PRAME polypeptide, the single domain antibody targeting a PRAME polypeptide as described in the third aspect, the VHH chain of the humanized single domain antibody targeting a PRAME polypeptide as described in the fourth aspect, the antibody targeting a PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, the fusion protein as described in the seventh aspect, the chimeric antigen receptor as described in the eighth aspect, the immune effector cell as described in the ninth aspect, or the immunoconjugate as described in the fourteenth aspect, and optional pharmaceutically acceptable excipients.

In a preferred embodiment, the pharmaceutical composition is used to treat tumors, and the tumors are PRAME polypeptide-related tumors; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

In the sixteenth aspect, the present invention provides the use of the VHH chain of the single domain antibody targeting the PRAME polypeptide of the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide of the second aspect, the single domain antibody targeting the PRAME polypeptide of the third aspect, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide of the fourth aspect, the antibody targeting the PRAME polypeptide of the fifth aspect, the bispecific antibody of the sixth aspect, the fusion protein of the seventh aspect, the chimeric antigen receptor of the eighth aspect, the immune effector cell of the ninth aspect or the immunoconjugate of the fourteenth aspect for preparing the following reagents:

1) Reagents for detecting PRAME polypeptide;

2) agents that block the binding of PRAME polypeptide to PD-L1;

3) Drugs for treating tumors.

In a preferred embodiment, the tumor is a PRAME polypeptide-associated tumor; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

In a seventeenth aspect, the present invention provides a kit, comprising:

1) the VHH chain of the single domain antibody targeting the PRAME polypeptide according to the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to the second aspect, the single domain antibody targeting the PRAME polypeptide according to the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to the fourth aspect, the antibody targeting the PRAME polypeptide according to the fifth aspect, the bispecific antibody according to the sixth aspect, the fusion protein according to the seventh aspect, the chimeric antigen receptor according to the eighth aspect, the immune effector cell according to the ninth aspect, the immunoconjugate according to the fourteenth aspect, or the pharmaceutical composition according to the fifteenth aspect;

2) container; and

3) Optional instruction manual.

In the eighteenth aspect, the present invention provides a method for detecting PRAME polypeptide protein in a sample, the method comprising the steps of:

1) the sample to be tested is mixed with the VHH chain of the single domain antibody targeting the PRAME polypeptide described in the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide described in the second aspect, the single domain antibody targeting the PRAME polypeptide described in the third aspect, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide described in the fourth aspect, the antibody targeting the PRAME polypeptide described in the fifth aspect, the bispecific antibody described in the sixth aspect, the fusion protein described in the seventh aspect, or the Contacting with the immunoconjugate of aspect 14;

2) Detect whether an antigen-antibody complex is formed. If a complex is formed, it indicates that the PRAME polypeptide protein is present in the sample.

In the nineteenth aspect, the present invention provides a method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the VHH chain of the single domain antibody targeting the PRAME polypeptide of the first aspect, the heavy chain variable region of the antibody targeting the PRAME polypeptide of the second aspect, the single domain antibody targeting the PRAME polypeptide of the third aspect, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide of the fourth aspect, the antibody targeting the PRAME polypeptide of the fifth aspect, the bispecific antibody of the sixth aspect, the fusion protein of the seventh aspect, the chimeric antigen receptor of the eighth aspect, the immune effector cell of the ninth aspect, the immunoconjugate of the fourteenth aspect, or the pharmaceutical composition of the fifteenth aspect.

In a preferred embodiment, the subject comprises a mammal; preferably a human.

In a preferred embodiment, the disease is a PRAME polypeptide-related disease; preferably a PRAME polypeptide-related tumor; more preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

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 embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows that the 14 single-domain antibodies of the present invention are all able to bind to protein antigens with high affinity;

FIG2 shows that the 14 single domain antibodies of the present invention are all able to bind to antigens at the cellular level and have high affinity;

FIG3 shows that the single domain antibody molecules of the present invention have good specificity;

FIG4 shows that the bispecific antibody molecule of the present invention has good cell killing activity;

FIG5 shows that the CAR molecules of the present invention can be efficiently expressed on the surface of T cells;

FIG6 shows the killing effect of candidate CAR-T cells on target cells under different effector-target ratios;

FIG7 shows that the CAR-T cells of the present invention can effectively recognize T2 loaded with target polypeptide, activate and transmit immune signals, and secrete IFN-γ cytokine;

Figure 8 shows that the humanized single-domain antibodies obtained after humanization of the LL-PR001 molecule are all able to bind to protein antigens with high affinity;

FIG9 shows that the humanized single-domain antibodies obtained after humanization of the LL-PR004 molecule are all able to bind to protein antigens with high affinity.

DETAILED DESCRIPTION

After extensive and in-depth research, the inventors unexpectedly discovered a class of single-domain antibodies targeting PRAME polypeptide. The single domain antibody of the present invention can bind to the PRAME polypeptide with high affinity and has good specificity. The present invention also provides bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs prepared using the single domain antibody. The present invention is completed on this basis.

Definition of terms

The terms used herein have the same or similar meanings as those conventionally understood by those skilled in the art. For the sake of clarity, some of the terms are defined as follows.

Single domain antibodies

In this article, "single domain antibody", "single domain antibody", "nanoantibody" and the like have the same or similar meanings, and all refer to a type of antibody molecule that lacks the antibody light chain and only has the heavy chain variable region. A single domain antibody is the smallest antigen binding unit, that is, the smallest antigen binding fragment with complete function. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned, thereby constructing a single domain antibody (VHH) consisting of only one heavy chain variable region.

The single-domain antibody VHH chain targeting the PRAME polypeptide of the present invention further comprises framework regions FR1, FR2, FR3 and FR4.

On the basis of the single domain antibody VHH chain targeting the PRAME polypeptide of the present invention, the present inventors also humanized the VHH chain, thereby obtaining a humanized single domain antibody VHH chain targeting the PRAME polypeptide.

On the basis of the single-domain antibody VHH chain or humanized VHH chain targeting the PRAME polypeptide of the present invention, the present invention also provides an antibody targeting the PRAME polypeptide, which includes one or more VHH chains of the single-domain antibody targeting the PRAME polypeptide or the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide. The present invention also provides a bispecific antibody, the bispecific antibody includes a first antibody and a second antibody, the first antibody can be the VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, or the humanized VHH chain. Those skilled in the art can select the second antibody in the bispecific antibody according to actual needs. For example, the second antibody can bind to the same or different antigen as the first antibody; if the second antibody binds to the same antigen as the first antibody, it is preferably bound to different epitopes. In a specific embodiment, the second antibody can be a single-domain antibody, a single-chain antibody or a double-chain antibody.

Those skilled in the art can also prepare the single-domain antibody VHH chain or humanized VHH chain targeting the PRAME polypeptide of the present invention into a fusion protein, for example, a fusion protein further comprising an Fc fragment of an immunoglobulin or a half-life extension domain. The fusion protein thus obtained not only has the biological activity of the single-domain antibody VHH chain itself, but also has other characteristics conferred by the Fc fragment of the immunoglobulin, such as extended plasma half-life, reduced immunogenicity, improved stability, and the like. In a specific embodiment, the fusion protein comprises the VHH chain or humanized VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, an optional linker sequence, and an Fc fragment of an immunoglobulin. In a specific embodiment, the immunoglobulin is IgG1, IgG2, IgG3, IgG4; preferably IgG4. In a specific embodiment, the half-life extension domain is as shown in the following amino acid sequence:

The present invention includes not only complete antibodies, but also fragments, derivatives and analogs of the antibodies. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may 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 substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

The antibody of the present invention refers to a polypeptide having PRAME polypeptide protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention. Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

In addition to almost full-length polypeptides, the present invention also includes fragments of the single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to the following table.

The present invention also provides a polynucleotide molecule encoding the above-mentioned 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 artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that only encodes a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and/or non-coding sequences. The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods. The biological molecules (nucleic acids, proteins, etc.) involved in the present invention include biological molecules in an isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins. 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 include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with _CaCl2 . The steps used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the present invention can be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or any combination of these substances. Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.

Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines, such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. drug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

Bispecific Antibodies

Bispecific antibodies are recombinant antibodies that have been engineered through protein engineering. Bispecific antibodies can simultaneously target two different antibody binding epitopes, which can be from different antigens or from the same antigen. Many current studies have shown that bispecific antibodies have great therapeutic potential in the treatment of diseases such as tumors, autoimmune diseases, and viral infections. Compared with monoclonal antibodies, the main advantage of bispecific antibodies is that they can mediate the spatial effect of two recognition epitopes and the synergistic effect of dual targeting, producing biological effects that cannot be achieved by the combined use of two antibodies. A relatively special bispecific antibody is called a T cell engager, which can simultaneously bind to targets and T cells on the surface of tumor cells, activate endogenous T cells, and cause tumor cell lysis, thereby achieving the purpose of treating tumors. T cell engagers have been shown to be used to treat tumors. Bispecific T cell engagers targeting CD20 and CD19 have been approved by the FDA for marketing (Nat Rev Clin Oncol. 2020 Jul; 17(7): 418-434.). Because of their complexity, which is different from monoclonal antibodies, bispecific antibodies have higher technical barriers and R&D costs.

Immune cells

In this article, immune cells and immune effector cells have the same meaning and are generally understood by those skilled in the art. The same as explained above. It refers to cells involved in or related to the immune response, including lymphocytes and phagocytes. In a specific embodiment, the immune cell refers to a lymphocyte that can recognize an antigen and thus produce a specific immune response. The lymphocytes are mainly T lymphocytes, B lymphocytes, K lymphocytes and NK lymphocytes. In addition to lymphocytes, cells involved in the immune response also include plasma cells, granulocytes, mast cells, antigen presenting cells and cells of the mononuclear phagocyte system (e.g., macrophages).

Chimeric Antigen Receptor T Cells

Chimeric antigen receptor T cell therapy is a very promising cellular immunotherapy. CAR-T cells express CAR (Chimeric Antigen Receptor) molecules. The structure of CAR is divided into: antigen binding region, hinge region, transmembrane domain and intracellular signal transduction domain. Current CAR-T cells usually use scFv (Single Chain Fragment Variables) segments derived from single-chain modification of the antigen binding region of monoclonal antibodies as antigen binding regions. However, when scFv structure modification is performed, problems such as reduced affinity and changed specificity are prone to occur. At the same time, scFv has a relatively large molecular weight and is easy to form multimers, which affects the function of CAR (Nat Rev Cancer. 2021Mar; 21(3): 145-161.). Therefore, CAR containing an antigen binding region with a novel structure. Using single-domain antibodies to construct a CAR structure, the variable region of the single-domain antibody can be directly connected to the CAR structure, which is simple and convenient to design.

Immunoconjugates

ADC is an antibody carrying cytotoxic drugs, which can deliver cytotoxic drugs to tumor cells to achieve specific killing of tumor cells. ADC drugs targeting targets such as HER2, CD30 and Trop2 have shown good clinical efficacy and safety in clinical studies (Nat Rev Clin Oncol. 2021 Jun; 18(6): 327-344.). In recent years, a number of ADC drugs have been approved by the FDA for marketing. However, the targets of ADC drugs are currently cell membrane proteins, and ADC drugs targeting intracellular proteins are one of the hot directions for future development.

ADC drugs can specifically target target cells and deliver cytotoxic molecules into target cells, thereby producing a specific killing effect. Cell killing induced by cytotoxic molecules can break and improve the tumor suppressive microenvironment, and has the potential to improve the efficacy of other therapies such as immunotherapy. The single-domain antibody of the present invention can be internalized into cells, so there is hope for the development of ADC drugs based on the single-domain antibody of the present invention in the future.

The present invention also provides an immunoconjugate, which contains the VHH chain of the single domain antibody targeting the PRAME polypeptide of the present invention, the humanized VHH chain, etc. and a coupling part. In a specific embodiment, the coupling part can be a detectable marker, a drug, a toxin, a cytokine, a radionuclide or an enzyme, etc., so as to achieve the purpose of diagnosis, detection or treatment, etc.

In a preferred embodiment, the immunoconjugate is an antibody-drug conjugate (ADC).

Pharmaceutical composition

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, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated into The pharmaceutical composition is prepared in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substance being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

The pharmaceutical composition of the present invention can directly target the PRAME polypeptide expressed by tumor cells. Therefore, the pharmaceutical composition of the present invention can be used to treat tumors. In a specific embodiment, the tumor is a PRAME polypeptide-related tumor. In a preferred embodiment, the tumor is melanoma, non-small cell lung cancer, ovarian cancer or breast cancer, etc. In addition, the pharmaceutical composition of the present invention can also be used in combination with other therapeutic agents.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single domain antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 μg/kg body weight to about 50 mg/kg body weight per day. When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dosage is about 10 μg/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

Reagent test kit

The present invention also provides a kit containing the VHH chain of the single domain antibody targeting the PRAME polypeptide of the present invention, or the humanized VHH chain, antibody, fusion protein or immunoconjugate, etc. In a specific embodiment, the kit further comprises a container, instructions for use, a buffer, etc.

Advantages of the present invention:

1. The single domain antibody targeting the PRAME polypeptide of the present invention binds to the PRAME polypeptide with high affinity;

2. The single domain antibody targeting PRAME polypeptide of the present invention has good specificity;

3. The single-domain antibody targeting the PRAME polypeptide of the present invention can be further used to prepare bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs, thereby laying a new material foundation for the development of therapeutic or diagnostic drugs targeting the PRAME polypeptide.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise specified, Percentages and parts are by weight.

Example 1: Acquisition of single domain antibody sequences

Two healthy adult alpacas (Alpaca) were selected as experimental animals for immunization. The recombinantly expressed pMHC (peptide-Major histocompatibility complex) recombinant protein carrying the target polypeptide was used as an antigen to immunize the alpacas. A total of 4-5 immunizations were performed. Peripheral blood of the alpacas was taken at different time points to detect the immune titer. After the immunization, peripheral blood of the alpacas was taken, and the mRNA of the peripheral blood PBMCs cells was extracted and reverse transcribed to obtain cDNA. The cDNA was amplified using specific primers to obtain a PCR product with a single-domain antibody gene fragment. Then, the PCR product and the yeast library vector were introduced into yeast competent cells by electroporation to prepare a yeast library. The yeast library was screened using recombinantly expressed protein antigens or T2 cells loaded with target polypeptides. The present invention uses a pMHC recombinant protein carrying the target polypeptide as a positive antigen and a pMHC recombinant protein carrying a negative polypeptide as a negative antigen. After three rounds of positive screening and three rounds of negative screening, a single-domain antibody clone that specifically binds to the positive antigen but not to the negative antigen was obtained.

The single domain antibody clones were subjected to Sanger sequencing to obtain the full-length sequence of the single domain antibody that specifically binds to the target protein of the present invention. A total of 14 different single domain antibody clones were obtained in the present invention, and they were named: LL-PR001-LL-PR014. The full-length amino acid sequence of the single domain antibody and the amino acid sequence of the CDR region are shown in Table 1.

Table 1. Amino acid sequence list of candidate single domain antibodies

Example 2: Single domain antibody expression and purification

The present invention uses conventional monoclonal antibody expression and purification methods to express and purify the single-domain antibodies in the present invention. First, a single-domain antibody expression vector is constructed using conventional molecular cloning technology. After the vector is successfully constructed, the single-domain antibody is expressed by transient transfection of HEK293 suspension cell lines. Basic steps: Clone the single-domain antibody gene into the expression vector pCDNA4 (Invitrogen, Cat V86220). Use the method of transient transfection of HEK293 suspension cells to express different single-domain antibodies. After the expression is completed, the supernatant is collected, and the single-domain antibody of the present invention is purified using a conventional monoclonal antibody purification method to finally obtain a purified single-domain antibody.

After the single-domain antibody purification is completed, the protein concentration and total protein amount of different antibodies are detected by ultraviolet spectrophotometry, and then the expression amount of each double antibody is calculated according to the expression volume. The absorbance value A280 of the sample solution is read at a wavelength of 280nm using NanoDrop 1000, and the protein concentration of the sample is calculated by the formula C (mg/mL) = A280/ε (ε is 1.482mL/mg·cm-1). At the same time, the purity of different antibodies is evaluated using the conventional gel electrophoresis SDS-PAGE method. The basic steps are as follows: The samples are electrophoretically separated using the Invitrogen electrophoresis tank and SDS-PAGE gradient gel. The sample is diluted to about 1mg/mL, and an appropriate amount of reducing agent, loading buffer and pure water are added. After mixing, it is heated at 70℃ for about 10 minutes, the sample amount is 2-10μg, the electrophoresis voltage is about 200V, and the electrophoresis time is about 35 minutes. After that, the gel is stained and destained respectively. After decolorization, a conventional gel imaging system is used to take pictures and analyze and calculate the purity of the main band. In addition, the purity of different antibodies was evaluated by size exclusion high performance liquid chromatography (SEC-HPLC). The basic steps are as follows: dilute the sample to about 1.0 mg/mL, use a TSKgel G3000SWXL column, set the column temperature to 25°C, use 100 mM phosphate buffer, 100 mM sodium sulfate, pH 7.0 ± 0.2 as the mobile phase, the injection volume is 20-50 μL, at a flow rate of 1.0 mL/min, isocratic elution for 20 min, detection at a wavelength of 280 nm, and the peak area normalization method is used to obtain the monomer content.

The expression and purity information of different single domain antibodies are shown in Table 2. The expression of the single domain antibody of the present invention using a transient expression system is in the range of 650-850 mg/L, proving that the dual antibody structure of the present invention has a higher expression level. The purity of the reduced SDS-PAGE and SEC-HPLC of most single domain antibodies of the present invention is above 95%. These results prove that the single domain structure of the present invention has a good expression level and a high purity.

Table 2. Expression level and purity test results of single domain antibodies

Example 3: Detection of the binding ability of single domain antibodies to targets

In order to explore the binding ability of the candidate single domain antibody of the present invention to the antigen, this example uses ELISA and flow cytometry methods to detect at the protein and cell levels, so as to comprehensively compare the affinity level of the candidate molecules to the target antigen. The specific operation steps are as follows:

3.1 Protein level binding capacity detection

1) Dilute SA protein to 0.3 μg/mL with PBS, add 100 μL/well to a 96-well transparent ELISA plate, seal the plate with a sealing film, and incubate at 37°C for 1 hour;

2) Discard the protein and wash the plate three times with 200 μL PBST solution (PBS containing 0.05% Tween20);

3) Add blocking solution (PBS solution containing 2% BSA) at 100 μL/well, seal the plate with sealing film and incubate at 37°C for 1 hour;

4) Repeat the plate washing step 2);

5) Dilute the biotinylated MHC-PRAME polypeptide complex to 2ug/ml with blocking solution, add 100μL of diluent to each well, seal the plate with sealing film and incubate at 37°C for 1h;

6) Repeat the plate washing step 2);

7) Dilute the candidate antibody to 12 different concentrations with blocking solution, with the highest concentration being 0.3 μg/mL, and dilute 3-fold. Add 100 μL of diluent to each well, seal the plate with sealing film, and incubate at 37°C for 1 hour;

8) Repeat the plate washing step 2);

9) Dilute SA-HRP with blocking solution at a ratio of 1:1000, add 100 μL to each well, seal the plate with sealing film and incubate at 37°C for 30 min;

10) Repeat the plate washing step 2);

11) Add 100 μL of TMB reaction solution to each well, seal the plate with a sealing film and place it on a microplate oscillator for 10 minutes, then add 100 μL of 2N H ₂ SO 4 solution to terminate the reaction, and read the absorbance at 450 nm with an enzyme reader.

The results are shown in Figure 1. The results show that the 14 single-domain antibodies screened by the present invention can bind to protein antigens with high affinity.

3.2 Cell-level binding ability detection

1) Prepare cells: Take an appropriate amount of T2 cells (cell bank of Shanghai Institute of Biochemistry and Cell Biology), adjust the cell density to 2×10 ⁶ /ml, and dispense into 96-well U-bottom plates, 50 μL per well (about 1E5 cells);

2) Peptide loading: Prepare a 60 μM peptide with 1640 medium, add 50 μL/well of the peptide dilution to the well plate and mix evenly with the cells, incubate at 37°C for 2 hours to load the peptide onto the T2 cells. Add 200 μL 1640 medium to resuspend the cells, centrifuge at 400 g for 5 minutes, and remove the supernatant;

3) Antibody incubation: Use antibody diluent (PBS + 0.5% FBS) to adjust the initial concentration of the original antibody to 1 μg/mL, dilute 4 times, and set 10 concentration gradients in total. Take 100 μL of the diluted antibody and add it to the previously loaded cells. After pipetting and mixing, incubate at 4°C in the dark for 60 min.

4) Antibody washing: Add 200 μL of antibody diluent to the incubated antibody-cell mixture, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells with 200 μL of antibody diluent, and repeat the washing once;

5) Secondary antibody incubation: Dilute the fluorescent secondary antibody APC anti-human IgG Fc (BD Biosciences) at 1:200 with antibody diluent, take 100 μL and add it to the cell pellet after discarding the supernatant, pipette to mix, and incubate at 4°C in the dark for 30 min.

6) Secondary antibody washing: Add 200 μL of antibody diluent to the incubated antibody-cell mixture, centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells with 200 μL of antibody diluent, and repeat the washing once;

7) Detection on the flow cytometer: Add 100 μL of antibody diluent to each tube to resuspend the cells, transfer the cell suspension to the flow cytometer, and detect the positive labeling rate by flow cytometry.

8) Analyze the data using FlowJo software to obtain the mean fluorescence intensity (MFI) value, fit the dose-effect nonlinear curve based on the MFI value and the antibody concentration value, calculate the _EC50 value, and evaluate the antigen affinity activity of the candidate antibody molecule.

The results are shown in FIG2 : The results show that the 14 single-domain antibodies screened by the present invention are all able to bind to antigens at the cellular level and have high affinity.

Example 4: Specificity detection of single domain antibodies for target recognition

T2 cell model loaded with different peptides

1) Take T2 cells grown to the logarithmic phase and resuspend them to 2×10 ⁶ /mL in complete culture medium;

2) Use complete culture medium to dilute the target peptide and OTP peptide to 60 μM, mix the peptide dilution with the cell suspension at a volume ratio of 1:1, and incubate at 37°C incubator for 1-2 hours to load the peptide onto the T2 cell surface;

3) After the incubation is completed, the excess polypeptide is washed away with culture medium, the cells are collected by centrifugation, the cells are resuspended to 2×10 ⁶ /mL with buffer (PBS solution containing 2% FBS), and the polypeptide-loaded T2 cells are inoculated into a 96-well U-bottom cell culture plate at 50 μL/well;

4) Dilute the candidate antibody molecules with buffer to 10 μg/mL, 2 μg/mL, 0.4 μg/mL, 0.08 μg/mL, add the antibody dilution into the well plate at 50 μL/well, mix thoroughly with the cells, and incubate at 4°C for 1 h;

5) Centrifuge at 300 g for 5 min, remove the antibody diluent, and wash the cells once with 200 μL buffer;

6) Dilute the fluorescent secondary antibody APC anti-human IgG Fc (purchased from Biolegend, catalog number 366906) with buffer at a ratio of 1:200, add the antibody dilution to the well plate at 100 μL/well, resuspend the cells and mix well, and incubate at 4°C for 30 min;

7) Centrifuge at 300 g for 5 min, remove the antibody diluent, and wash the cells once with 200 μL buffer;

8) Resuspend the cells with 100 μL buffer and detect the fluorescence binding intensity of the APC channel by flow cytometry;

9) Analyze the data using FlowJo software to obtain the mean fluorescence intensity (MFI) and evaluate the nonspecific binding level of the candidate antibody molecules to the off-target peptides.

Table 3. Potential off-target polypeptide sequence list used in the present invention

The present invention detects the off-target situation of all candidate molecules. Figure 3 shows the off-target analysis results of some molecules. As shown in Figure 3, the single domain antibody molecules screened by the present invention have good specificity and can be further developed.

Example 5: Design of bispecific antibodies based on single domain antibodies

We selected the single-domain antibody molecule LL-PR005 as a representative to design bispecific antibodies with different structures. The bispecific antibody molecules designed in the present invention can bind to the PRAME polypeptide-HLA-A02 complex and CD3 at the same time. Some bispecific antibodies in the present invention integrate Fc structures or half life extender structures. The amino acid sequence of the CD3 antibody used in the bispecific antibody molecules designed in the present invention is:

Heavy chain variable region:

Light chain variable region:

The half life extender amino acid sequence used in the bispecific antibody molecule designed in the present invention is:

The present invention designed a total of 13 bispecific antibody molecules with different structures, and the amino acid sequences are shown in Table 3.

Table 4. Amino acid sequence table of the dual-antibody molecules based on single-domain antibodies designed in the present invention

Example 6: Expression and purification of bispecific antibodies

Referring to the experimental steps of Example 2, the bispecific antibody molecules designed by the present invention were expressed and purified, and the expression level and purity were detected. The expression level and purity information of different bispecific antibodies are shown in Table 4. The expression level of the bispecific antibody of the present invention is in the range of 600-850 mg/L, which proves that the bispecific antibody structure of the present invention has a higher expression level. The monomer purity of the bispecific antibody of the present invention by SEC-HPLC is above 90%. At the same time, the purity of the reduced SDS-PAGE of all bispecific antibodies is above 95%. These results prove that the bispecific antibody molecules designed by the present invention have good expression levels and high purity.

Table 5 Expression level and purity test results of bispecific antibodies

Example 7: Functional evaluation of bispecific antibodies

The bispecific antibody molecule described in the present invention, also known as a T cell engager, can simultaneously and specifically bind to the target presented by pMHC in the tumor cell and the CD3 protein on the surface of the T cell, thereby mediating the directional cruising of T cells to the vicinity of the tumor cells, endogenously activating and releasing cytokines, leading to the lysis of the tumor cells, thereby achieving the purpose of treating the tumor. In order to evaluate the specific recognition and killing ability of the candidate bispecific antibody molecules expressed in Example 6 on tumor cells, this example co-cultures the target cells with CD3+T cells at a certain effect-target ratio, and adds different concentrations of candidate antibody molecules to detect the antibody-mediated target cell apoptosis ratio and T cell activation and factor secretion levels; in addition, this example sets tumor cell lines NCI-H1755, HS695T, OVCAR3, U2OS with different antigen expression abundances, PRAME expression negative HLA-A2 positive T2, MCF7 and HLA-A2 and PRAME negative A549 cell lines as target cells, which can further evaluate the target-specific recognition ability of the bispecific antibody molecules, thereby more comprehensively screening out suitable bispecific antibody molecules for in-depth research. The basic implementation steps are as follows:

1) Cell co-culture: Use lentivirus carrying luciferase to transfect different target cells to prepare fluorescently labeled Enzyme cell lines, labeled as: NCI-H1755-GFP, HS695T-GFP, OVCAR3-GFP, MCF7-GFP and A549-GFP. Different target cells and effector cells were resuspended in culture medium (1640 culture medium containing 2% FBS) at a concentration of 2×10 ⁵ /mL and 1×10 ⁶ /mL, respectively, and plated into 96-well flat-bottom opaque white plates at 25 μL/well, and temporarily placed at 37°C for incubation;

2) Antibody incubation: dilute the antibody in a gradient manner with the culture medium, with the highest concentration being 20 nM, and dilute to 10 concentration points. Add 50 μL of the corresponding bispecific antibody to each well. Mix thoroughly and centrifuge at 500 rpm for 3 minutes. Incubate the cells at 37°C for 24 hours;

3) Killing detection: After 24 hours of cell co-culture, the remaining luciferase activity (relative light unit, RLU) of the target cells was measured to detect the killing ability of T cells on target cells in the presence of different specific antibodies. The specific steps are: take out the opaque 96-well flat-bottom plate after co-culture, add 100 μL of an equal volume of D-luciferin substrate (Thermo Fisher Scientific: 88293) to the well, mix well, protect from light and develop color for 5 minutes, and detect the fluorescence intensity in the chemiluminescence mode of the microplate reader. Since luciferase is only expressed in target cells, the remaining luciferase activity in the well is directly related to the number of live target cells in the well. In the absence of effector cells and antibodies, the maximum luciferase activity is obtained by adding culture medium to the target cells as a control;

4) Data analysis: The maximum luciferase activity was used as a control to calculate the target cell killing efficiency corresponding to the detection well, and the dose-dependent curve was fitted with the antibody addition concentration as the horizontal axis to estimate the EC50 value and evaluate the target cell killing level of the tested bispecific antibody molecule.

The results showed that the bispecific antibodies of the present invention can mediate T cell activation and kill cells expressing positive target sites ( FIG. 4 ).

Example 8: Molecular design of CAR sequence targeting PRAME and construction of lentiviral vector

8.1 Design of CAR gene sequences targeting PRAME

The CAR targeting PRAME comprises a single domain antibody sequence of anti-human PRAME, a CD28 hinge region and a transmembrane domain, a CD28 co-stimulatory signal transduction region, and a CD3zeta signal transduction domain, which are connected in series. The present invention selects five single domain antibody sequences and designs a total of five CAR structures. The five different CAR molecules are named as: PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, PRAME-CAR-05, and the amino acid sequences corresponding to different CARs are shown in Table 6. The five CAR gene sequences targeting PRAME designed by the present invention are subjected to gene synthesis and subcloned into the pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.).

Table 6. Amino acid sequence list of CAR targeting PRAME

8.2 Construction of CAR Lentiviral Vector

Primers were designed to use PCR to amplify five different CAR molecules from the pUC57 vector. When designing primers, the homology arms of the lentiviral vector should be added to the 5' ends of the forward and reverse primers. The amplified PCR products were detected by agarose gel electrophoresis and then gel-recovered and purified (Nanjing Novozyme Biotechnology Co., Ltd., catalog number DC301) to obtain DNA fragments. The DNA fragments recovered by enzyme digestion were cloned into the lentiviral vector by homologous recombination. The lentiviral vector needs to be digested with restriction endonucleases BamHI (NEB: R3136V) and SalI (NEB: R3138V) for recovery and purification. Five different recombinant plasmids were obtained in this way: p-lenti-PRAME-CAR-01, p-lenti-PRAME-CAR-02, p-lenti-PRAME-CAR-03, p-lenti-PRAME-CAR-04, and p-lenti-PRAME-CAR-05. The five lentiviral vectors were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification. The sequencing primers were: Lenti-seqF: TTGAGTTGGATCTTGGTTC (SEQ ID NO: 103), Lenti-seqR: CAGCAACCAGGATTTATACA (SEQ ID NO: 104). Sanger sequencing verified that the five lentiviral vector plasmids were constructed correctly.

Example 9: Preparation and functional evaluation of CAR-T cells targeting PRAME

9.1 Preparation of Lentivirus

The correct lentiviral plasmids verified by sequencing were transformed into Escherichia coli stbl3 (purchased from Yisheng Biotechnology Co., Ltd.). The next day, single clones were picked from the transformed plates and placed in a 2ml liquid LB culture tube containing kanamycin (50ug/ml) and cultured on a shaker at 37℃220rpm for 8h. 1ml of the activated bacterial solution was inoculated into 250ml liquid LB culture medium containing kanamycin and cultured on a shaker at 37℃220rpm for 12-16h. The plasmid was extracted using the NucleoBond Xtra Midi Plus kit (MN, catalog number: 740412.50) according to the experimental procedure provided by the kit. After the plasmid was extracted, the plasmid concentration was determined using Nanodrop (Thermo Fisher Scientific) and verified by sanger sequencing. The supercoiled plasmid content was detected by DNA agarose gel.

The frozen 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were taken out of liquid nitrogen, thawed in a 37°C water bath, and then the tube mouth was wiped with 75% alcohol and transferred to a tube containing 10 ml of preheated DMEM complete medium (90% DMEM +

10% FBS + 1% penicillin / streptomycin) in a 15ml centrifuge tube, gently blow it evenly, centrifuge it at 400g for 4 minutes, and then discard the supernatant. Then add 10ml DMEM complete medium, gently blow it evenly, and inoculate it into a T25 or T75 culture flask, and culture it in a cell culture incubator at 37℃ with 5% _CO2 . The next day, when the cell density reaches more than 80%, the cells are subcultured and continued to be cultured. 293T cells cultured for more than 3 generations can be used to package lentivirus. The specific steps are:

1) On the first day, inoculate 293T cells: inoculate cells at a rate of about 1.0×10^7 cells/T175 flask (cultured in 40 mL of culture medium), and transfect when the cell density reaches 90% on the second day.

2) The next day, plasmid transfection: Before transfection, the culture medium was replaced with DMEM medium containing 10% FBS but without dual antibodies. Prepare the plasmid complex first: add the following plasmids into 1.5 ml Opti-MEM (Thermo Fisher Scientific; 31985-070) and mix well: 18 μg of psPAX2 plasmid (Addgene; Catalog No.: 12260), 9 μg of pMD2.G plasmid (Addgene; Catalog No.: 12259), 18 μg of lentiviral vector plasmid. The lentiviral plasmids are: p-lenti-PRAME-CAR-01, p-lenti-PRAME-CAR-02, p-lenti-PRAME-CAR-03, lenti-PRAME-CAR-04, lenti-PRAME-CAR-05. Then prepare the transfection reagent complex: according to the mass ratio of plasmid to PEI of 1:3, add 67.5μL (2mg/mL) of PEI (polysciences: 24765) to 1.5mL Opti-MEM, mix well, and let stand at room temperature for 5min; then add the transfection reagent complex dropwise to the plasmid complex, mix well and let stand for 20min. Finally, slowly drip the transfection complex into the 293T cell culture flask, gently mix, and continue to culture in a cell culture incubator at 37℃ with ₅ % CO2.

3) On the fourth day, virus collection: 48 hours after transfection, collect the culture supernatant and centrifuge at 2000rpm for 10 minutes to remove cell debris. Use a 0.45μM filter membrane (Millex-HV, catalog number SLHVR33RB) to filter the supernatant and transfer the filtrate to a dedicated centrifuge tube for balancing. Use an ultracentrifuge at 25000rpm for 2 hours. After discarding the supernatant, use 1ml of X-VIVO-15 The lentivirus was resuspended in the culture medium, and the lentivirus was aliquoted and stored in an ultra-low temperature refrigerator at -80°C. According to the process, lentivirus containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, and PRAME-CAR-05 were prepared respectively.

9.2 Preparation of CAR-T cells and detection of CAR molecule expression

The prepared lentivirus containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, and PRAME-CAR-05 were used to infect primary human T cells to prepare CAR-T cells carrying different CAR genes. The CAR-T cells carrying the five CAR genes were named PRAME-CAR-T-01, PRAME-CAR-T-02, PRAME-CAR-T-03, PRAME-CAR-T-04, and PRAME-CAR-T-05, and the untransfected T cells were used as negative controls and named NT. The specific steps are as follows:

1) CD3+T cells from healthy human peripheral blood (Miaoshun (Shanghai) Biotechnology Co., Ltd.) were resuspended in T cell culture medium containing 300 IU/mL IL-2 to a density of 1×10 ⁶ /mL, and T cell activator CD3/CD28 magnetic beads (ACRO Biosystems, catalog number: MBS-C001) were added at a ratio of 1:1 between cells and magnetic beads. After thorough mixing, the cells were inoculated in 6-well plates for culture;

2) After 24 hours of T cell activation, the T cells were counted and inoculated into a new 24-well plate, with 500ul per well and 5×10 ⁵ cells/well. After the inoculation, 100μL of lentivirus solution carrying different CAR genes was added to infect the T cells. T cells without virus solution were used as negative control NT, and the cells were placed in the incubator for further culture.

3) After 48 hours of infection with lentivirus, the cells were aspirated from the culture wells, the magnetic beads were removed by magnetic adsorption using a magnetic stand, and the cells were collected by centrifugation and resuspended in fresh T cell culture medium.

4) Take 100 μL of cell suspension with lentivirus and negative control at the same time, collect cells by centrifugation, resuspend cells with 100 μL FACS buffer, add APC-labeled pMHC protein complex tetramer to each tube of cells, mix well, and incubate at 4°C in the dark for 30 minutes;

5) Wash the cells twice with PBS, resuspend the cells in 100 μL FACS buffer, and detect the expression efficiency of the four CARs on T cells by flow cytometry.

The results showed that the five CAR molecules described in the present invention were all successfully expressed on T cells, with relatively consistent expression efficiencies of 30-45% ( FIG. 5 ).

9.3 Detection of CAR-T cell killing ability

This experiment uses two HLA-A2 and PRAME target expression double positive cell lines as target cells, namely non-small cell lung cancer NCI-H1755 and melanoma HS695T. Two PRAME and HLA-A2 negative A549 and 293T cells are used as negative cells, and the five CAR-T cells prepared in Example 2 above are co-cultured with target cells to detect the killing effect and evaluate the biological functions of different CAR-T. The specific steps are as follows:

1) Use lentiviral solution expressing GFP luciferase (GenBank: AAR29591.1) to transfect different target cells to obtain cell lines labeled with luciferase, labeled as: NCI-H1755-GFP-luc, HS695T-GFP-luc, 293T-GFP-luc and A549-GFP-luc;

2) NCI-H1755-GFP-luc, HS695T-GFP-luc, 293T-GFP-luc and A549-GFP cells were inoculated into 96-well flat-bottom opaque cell culture plates at a cell concentration of 1×10 ⁵ /mL and 50 μL/well, and temporarily placed at 37°C for incubation;

3) Use CT to adjust the positive ratio of the five CAR-T cells to 15%, set 4 effector-target ratios of 5:1, 2.5:1, 1:1 and 0.5:1, and inoculate the five different CAR-T cells and control T cells into the target cells at 50 μL/well for co-culture. After fully mixing, place in a 37°C incubator and incubate overnight;

4) Take out the opaque 96-well flat-bottom plate after co-culture, add 100 μL of an equal volume of D-luciferin substrate (Thermo Fisher Scientific: 88293) to the wells, mix well and react in the dark for 10 minutes, and detect the fluorescence intensity in the chemiluminescence mode of the microplate reader. Since luciferase is only expressed in target cells, the remaining luciferase activity in the well is directly related to the number of live target cells. The culture medium is added to the target cells to obtain the maximum luciferase activity as a control, and the target cell apoptosis ratio is calculated by deducting the live cell fluorescence signal value, which is the killing effect of CAR-T cells on target cells. The results are shown in Figure 6.

The results showed that the five CAR molecules described in the present invention can mediate T cell killing of three target-positive cells, among which NCI-H1755 cells have the strongest killing effect.

9.4 Detection of CAR-T cell factor secretion

In order to further evaluate the level of target cell-specific activation of the CAR-T cells of the present invention and the release of cytokines, the five CAR-T cells prepared in Example 2 and the target cells in Example 4 were co-cultured at a 1:1 effector-target ratio for 24 hours, and the levels of IFN-γ and IL-2 cytokines secreted by T cells in the culture supernatant were detected. The specific steps are as follows:

1) Take target cells NCI-H1755, HS695T, A549 and 293T, resuspend them to 1×10 ⁶ /mL with complete culture medium, and inoculate the cell suspension into a 96-well U-bottom deep-well plate at 100 μL/well;

2) After counting the five CAR-T cells and control T cells, they were added to different target cells for co-culture at a 1:1 effector-target ratio, i.e., a cell concentration of 1×10 ⁶ /mL, 100 μL/well;

3) After the above cells were co-cultured overnight, 50 μL of the culture supernatant was collected and transferred to a new U-bottom 96-well plate. The secretion of IFN-γ and IL-2 cytokines in T cells was detected using an ELISA kit (Thermo Fisher Scientific; Cat. No. 88-7316). The plate preparation and supernatant cytokine detection were performed according to the instructions provided by the kit.

4) Graphpad Prism software was used for data analysis, dose-dependent curve fitting, and _EC50 value calculation.

The results are shown in Figure 7. The results show that the candidate CAR-T cells can effectively recognize T2 loaded with target peptides, activate and transmit immune signals, and secrete IFN-γ cytokines, among which the CAR-T-01 molecule has relatively better biological activity. Based on the above results, the CAR sequence described in the present invention has good affinity activity and biological function, indicating that the CAR molecule of the present invention has the value of further development and application.

Example 10: Humanization of single domain antibodies

The present invention humanizes two single-domain antibody candidate molecules, LL-PR001 and LL-PR004, respectively. Basic steps:

1) The sequences of the single-domain antibody candidate molecules LL-PR001 and LL-PR004 were input into the IMGT database for antibody sequence alignment. Based on the database sequence alignment results, IGHV3-23*04 was selected as the LL-PR001 humanized parent vector, and IGHV3-23*02 was selected as the LL-PR004 humanized parent vector;

2) The CDR region of the LL-PR001 single domain antibody was transplanted to IGHV3-23*04, and the CDR region of the LL-PR004 single domain antibody was transplanted to IGHV3-23*02;

3) Refer to the prior art literature to perform back mutation on the transplanted humanized antibody to ensure the affinity of the humanized antibody force.

By the above method, LL-PR001 was used as the humanized parent to design ten candidate humanized single domain antibodies, and the antibody sequences are shown in Table 7. LL-PR004 was used as the humanized parent to design six candidate humanized single domain antibodies, and the antibody sequences are shown in Table 8.

Table 7. Humanized single domain antibody sequences designed based on LL-PR001 humanized parent

Table 8. Humanized single domain antibody sequences designed based on LL-PR004 humanized parent

Example 11: Expression and functional identification of humanized single domain antibody molecules

11.1 Expression of humanized single domain antibody molecules

The humanized antibody molecule is first codon optimized to obtain a nucleotide sequence, and the full-length gene of the humanized antibody molecule is constructed into a single-domain antibody expression vector by gene synthesis. After the vector is successfully constructed, the single-domain antibody is expressed by transient transfection of HEK293 suspension cell lines. The specific steps are referred to Example 2.

The expression levels and purities of different humanized single domain antibodies are shown in Table 9. The expression levels of the humanized single domain antibodies of the present invention using the transient expression system are in the range of 700-900 mg/L, proving that the humanized single domain antibodies of the present invention have a high expression level. And the purity of the reduced SDS-PAGE and SEC-HPLC of most humanized single domain antibodies is above 95%. These results prove that the humanized single domain antibodies of the present invention have a good expression level and a high purity.

Table 9. Expression and purity detection of humanized single domain antibodies

11.2 Protein level binding capacity detection

The binding ability of the humanized molecule to the target antigen is detected by ELISA to determine the affinity of the humanized molecule. For specific experimental operation steps, refer to Example 3 3.1. The results show that the 10 candidate humanized single domain antibodies (huLL-PR001-1 to huLL-PR001-10) obtained after the humanization of the LL-PR001 molecule can bind to the protein antigen and have a high affinity (the results are shown in Figure 8). The affinity of the molecule after partial humanization has decreased slightly, but it still has a high affinity. It can be applied to the research and development of subsequent bispecific antibodies, CAR-T, ADC and RDC and other biopharmaceutical projects. Similarly, the six candidate humanized single domain antibodies (huLL-PR004-1 to huLL-PR004-6) obtained after the humanization of the LL-PR004 molecule can also bind to the protein antigen and have a high affinity. The six affinities after humanization are slightly lower than those of the parent molecule, but still have a high affinity (the results are shown in Figure 9). It can be applied to the subsequent research and development of biologics projects such as bispecific antibodies, CAR-T, ADC and RDC.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. 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 claims attached to this application.

Claims (32)

A VHH chain of a single domain antibody targeting a PRAME polypeptide, wherein the VHH chain comprises CDR1, CDR2 and CDR3 as shown in the following table:
The VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in claim 1, wherein the amino acid sequence of the PRAME polypeptide is: SLLQHLIGL (SEQ ID NO: 121). The VHH chain of a single domain antibody targeting a PRAME polypeptide as described in claim 1 is characterized in that the amino acid sequence of the VHH chain of a single domain antibody targeting a PRAME polypeptide is selected from the following group: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 53. A heavy chain variable region of an antibody targeting a PRAME polypeptide, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in the following table:

The heavy chain variable region of the antibody targeting the PRAME polypeptide as described in claim 4 is characterized in that the amino acid sequence of the heavy chain variable region of the antibody targeting the PRAME polypeptide is selected from the following group: SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 53. A single domain antibody targeting a PRAME polypeptide, comprising the VHH chain according to any one of claims 1 to 3. A humanized VHH chain of a single-domain antibody targeting a PRAME polypeptide, wherein the framework regions FR1, FR2, FR3 and FR4 are humanized based on the VHH chain described in any one of claims 1 to 3. The VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide as described in claim 7 is characterized in that the variable region sequence of the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide is selected from the following group: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 and SEQ ID NO: 114. An antibody targeting a PRAME polypeptide, the antibody comprising one or more VHH chains of the single domain antibody targeting a PRAME polypeptide according to any one of claims 1 to 3 or the VHH chains of the humanized single domain antibody targeting a PRAME polypeptide according to claim 7 or 8. The antibody targeting the PRAME polypeptide according to claim 9, characterized in that the antibody targeting the PRAME polypeptide comprises a monomer, a bivalent antibody, and/or a multivalent antibody. A bispecific antibody, comprising a first antibody and a second antibody, wherein the first antibody comprises the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, or the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, or the single-domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide according to claim 7 or 8, or the antibody targeting the PRAME polypeptide according to claim 9 or 10. The bispecific antibody of claim 11, wherein the second antibody can bind to the same or different antigen as the first antibody, or bind to a different epitope of the same antigen as the first antibody. The bispecific antibody of claim 11, wherein the second antibody is a single domain antibody, a single chain antibody or a double chain antibody. The bispecific antibody according to claim 11, characterized in that the bispecific antibody comprises 2-4 single domain antibodies targeting the PRAME polypeptide; preferably, it comprises 2 single domain antibodies targeting the PRAME polypeptide; more preferably, the 2 single domain antibodies targeting the PRAME polypeptide form a single domain antibody dimer targeting the PRAME polypeptide. The bispecific antibody according to claim 11, characterized in that the sequence of the bispecific antibody is shown in the following table:

A fusion protein, the fusion protein comprising the VHH chain of the single domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to claim 7 or 8, or the antibody targeting the PRAME polypeptide according to claim 9 or 10, an optional linker sequence and an Fc fragment of an immunoglobulin or a half-life extension domain. The fusion protein according to claim 16, characterized in that the immunoglobulin is IgG1, IgG2, IgG3, IgG4; preferably IgG4. A chimeric antigen receptor, which is made of the VHH chain of the single domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to claim 7 or 8, or the antibody targeting the PRAME polypeptide according to claim 9 or 10. The chimeric antigen receptor as described in claim 18 is characterized in that the amino acid sequence of the chimeric antigen receptor is selected from the following group: SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101 and SEQ ID NO:102. An immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor according to claim 18 or 19. The immune effector cell according to claim 20, characterized in that the immune effector cell includes but is not limited to: T cells, NK cells, TIL cells; preferably T cells. A nucleic acid molecule encoding the VHH chain of a single domain antibody targeting a PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of an antibody targeting a PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting a PRAME polypeptide according to claim 6, the VHH chain of a humanized single domain antibody targeting a PRAME polypeptide according to claim 7 or 8, the antibody targeting a PRAME polypeptide according to claim 9 or 10, the bispecific antibody according to any one of claims 11 to 15, the fusion protein according to claim 16 or 17, or the chimeric antigen receptor according to claim 18 or 19. An expression vector comprising the nucleic acid molecule of claim 22. A host cell, the host cell comprising the expression vector according to claim 23, or a The nucleic acid molecule according to claim 22 is incorporated. A method for preparing the VHH chain of a single domain antibody targeting a PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of an antibody targeting a PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting a PRAME polypeptide according to claim 6, the VHH chain of a humanized single domain antibody targeting a PRAME polypeptide according to claim 7 or 8, the antibody targeting a PRAME polypeptide according to claim 9 or 10, the bispecific antibody according to any one of claims 11 to 15, the fusion protein according to claim 16 or 17, or the chimeric antigen receptor according to claim 18 or 19, the method comprising the following steps: 1) Cultivating the host cell of claim 24 under suitable conditions to obtain a culture containing the VHH chain of the single domain antibody targeting the PRAME polypeptide, the heavy chain variable region of the antibody targeting the PRAME polypeptide, the single domain antibody targeting the PRAME polypeptide, the humanized VHH chain of the single domain antibody targeting the PRAME polypeptide, the antibody targeting the PRAME polypeptide, the bispecific antibody, the fusion protein or the chimeric antigen receptor; and 2) Optionally, isolating or recovering the VHH chain of the single domain antibody targeting the PRAME polypeptide, the heavy chain variable region of the antibody targeting the PRAME polypeptide, the single domain antibody targeting the PRAME polypeptide, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide, the single domain antibody targeting the PRAME polypeptide, the bispecific antibody, the fusion protein or the chimeric antigen receptor from the culture. An immunoconjugate comprising: 1) the VHH chain of the single domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to claim 7 or 8, the antibody targeting the PRAME polypeptide according to claim 9 or 10, the bispecific antibody according to any one of claims 11 to 15, or the fusion protein according to claim 16 or 17; and 2) A conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme. A pharmaceutical composition comprising a therapeutically or diagnostically effective amount of a VHH chain of a single domain antibody targeting a PRAME polypeptide according to any one of claims 1 to 3, a heavy chain variable region of an antibody targeting a PRAME polypeptide according to claim 4 or 5, a single domain antibody targeting a PRAME polypeptide according to claim 6, a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide according to claim 7 or 8, an antibody targeting a PRAME polypeptide according to claim 9 or 10, a bispecific antibody according to any one of claims 11 to 15, a fusion protein according to claim 16 or 17, a chimeric antigen receptor according to claim 18 or 19, an immune effector cell according to claim 20 or 21, or an immunoconjugate according to claim 26, and optionally a pharmaceutically acceptable excipient. The pharmaceutical composition according to claim 27, characterized in that the pharmaceutical composition is used to treat a tumor, and the tumor is a PRAME polypeptide-related tumor; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc. The VHH chain of the single domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to claim 7 or 8, Use of the antibody targeting the PRAME polypeptide of claim 9 or 10, the bispecific antibody of any one of claims 11-15, the fusion protein of claim 16 or 17, the chimeric antigen receptor of claim 18 or 19, the immune effector cell of claim 20 or 21, the immunoconjugate of claim 26 or the pharmaceutical composition of claim 27 or 28 for preparing the following reagents: 1) Reagents for detecting PRAME polypeptide; 2) agents that block the binding of PRAME polypeptide to PD-L1; 3) Drugs for treating tumors. The use according to claim 29, characterized in that the tumor is a PRAME polypeptide-associated tumor; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc. A kit, comprising: 1) the VHH chain of the single domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to claim 7 or 8, the antibody targeting the PRAME polypeptide according to claim 9 or 10, the bispecific antibody according to any one of claims 11 to 15, the fusion protein according to claim 16 or 17, the chimeric antigen receptor according to claim 18 or 19, the immune effector cell according to claim 20 or 21, the immunoconjugate according to claim 26, or the pharmaceutical composition according to claim 27 or 28; 2) container; and 3) Optional instruction manual. A method for detecting PRAME polypeptide protein in a sample, the method comprising the steps of: 1) contacting the sample to be tested with the VHH chain of the single domain antibody targeting the PRAME polypeptide according to any one of claims 1 to 3, the heavy chain variable region of the antibody targeting the PRAME polypeptide according to claim 4 or 5, the single domain antibody targeting the PRAME polypeptide according to claim 6, the VHH chain of the humanized single domain antibody targeting the PRAME polypeptide according to claim 7 or 8, the antibody targeting the PRAME polypeptide according to claim 9 or 10, the bispecific antibody according to any one of claims 11 to 15, the fusion protein according to claim 16 or 17, or the immunoconjugate according to claim 26; 2) Detect whether an antigen-antibody complex is formed. If a complex is formed, it indicates that the PRAME polypeptide protein is present in the sample.