CN118221811A

CN118221811A - Single domain antibodies targeting PRAME polypeptides and uses thereof

Info

Publication number: CN118221811A
Application number: CN202410314701.6A
Authority: CN
Inventors: 王文博; 刘洪川; 汤贝贝; 冯爱华
Original assignee: Liling Biopharmaceutical Suzhou Co ltd
Current assignee: Liling Biopharmaceutical Suzhou Co ltd
Priority date: 2023-08-25
Filing date: 2024-03-19
Publication date: 2024-06-21

Abstract

The invention discloses a series of single domain antibodies targeting PRAME polypeptides. The single domain antibody of the PRAME polypeptide can be combined with the PRAME polypeptide with high affinity and has better specificity, thereby laying a new material foundation for the development of antitumor drugs targeting the PRAME polypeptide. The invention also discloses a bispecific antibody prepared by the single domain antibody, a chimeric antigen receptor-T cell, an ADC and the like.

Description

Single domain antibodies targeting PRAME polypeptides and uses thereof

Technical Field

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

Background

PRAME (PREFERENTIALLY EXPRESSED ANTIGEN IN MElanoma) is an intracellular protein encoded by the PRAME gene. PRAME is expressed at high levels in a variety of tumors, such as melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc., and is rarely expressed in normal human tissues, which makes PRAME an ideal tumor targeted therapeutic target (Am J Surg Pathol.2018nov;42 (11): 1456-1465.). The PRAME protein, after treatment by the intracellular antigen presenting system, has a specific polypeptide sequence: SLLQHLIGL, can be presented to the cell surface by the major histocompatibility antigen HLA-A02 molecule. HLA-A02 complexes that bind PRAME polypeptides may be used as cell membrane targets for developing T Cell Receptor (TCR) or TCR-like antibody-related therapies.

A single domain antibody (sdAb) is a specific, smaller molecular weight antibody. Single domain antibodies consist of only two identical heavy chains. Compared with the traditional double-chain antibody with the molecular weight of 150-160kDa, the molecular weight of the single-domain antibody is about 110 KD. Single domain antibodies generally have high specificity, high affinity, low immunogenicity, good permeability. The antigen binding region of a single-domain antibody consists of only one chain, and the variable region (VHH) of the single-domain antibody is only 12-15kDa, so that the structure of the single-domain antibody is very simple, the problem of mismatching of the light chain and the heavy chain of the traditional duplex antibody can not occur, and the condition of reduced affinity caused by single-chain modification of the antigen binding region can not occur. Based on these advantages, the use of single domain antibodies as bispecific antibodies or antigen recognition regions of chimeric antigen Receptor T cells (CHIMERIC ANTIGEN Receptor-T cells, CAR-T) is one of the trends in the future (Serge Muyledermans. Annu. Rev. Biochem.82:775-797 (2013)). Single domain antibodies can recognize cell membrane proteins, as well as polypeptides derived from intracellular proteins that are presented to the cell surface by major histocompatibility antigens. Specific single domain antibody molecules can be screened using HLA-A02 complexes that bind PRAME polypeptides as antigens. These candidate molecules can be used to develop bispecific antibodies, CAR-T or ADC (Antibody drug conjugate), etc. biopharmaceuticals.

Disclosure of Invention

The invention aims at providing a specific single domain antibody targeting PRAME polypeptide, and a corresponding specific humanized single domain antibody targeting PRAME polypeptide, a bispecific antibody, a chimeric antigen receptor-T cell, an ADC and the like.

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

In a first aspect, the invention provides a VHH chain of a single domain antibody that targets a PRAME polypeptide, the VHH chain comprising CDR1, CDR2 and CDR3 as shown in the following table:

CDR1	CDR2	CDR3
			SEQ ID NO:2	SEQ ID NO:3	SEQ ID NO:4
SEQ ID NO:6	SEQ ID NO:7	SEQ ID NO:8
			SEQ ID NO:10	SEQ ID NO:11	SEQ ID NO:12
SEQ ID NO:14	SEQ ID NO:15	SEQ ID NO:16
			SEQ ID NO:18	SEQ ID NO:19	SEQ ID NO:20
SEQ ID NO:22	SEQ ID NO:23	SEQ ID NO:24
			SEQ ID NO:26	SEQ ID NO:27	SEQ ID NO:28
SEQ ID NO:30	SEQ ID NO:31	SEQ ID NO:32
			SEQ ID NO:34	SEQ ID NO:35	SEQ ID NO:36
SEQ ID NO:38	SEQ ID NO:39	SEQ ID NO:40
			SEQ ID NO:42	SEQ ID NO:43	SEQ ID NO:44
SEQ ID NO:46	SEQ ID NO:47	SEQ ID NO:48
			SEQ ID NO:50	SEQ ID NO:51	SEQ ID NO:52
SEQ ID NO:54	SEQ ID NO:55	SEQ ID NO:56

。

in a preferred embodiment, the PRAME polypeptide has the amino acid sequence: SLLQHLIGL (SEQ ID NO: 121).

In a preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g. 1-3, preferably 1-2, more preferably 1) amino acid and which is capable of retaining high affinity binding to a 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 as shown in the following table:

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
	SEQ ID NO:53

。

in a second aspect, the present invention provides a heavy chain variable region of an antibody targeting a PRAME polypeptide, said heavy chain variable region comprising 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 PRAME polypeptide-targeting antibody is as shown in the following table:

。

in a third aspect, the invention provides a single domain antibody targeting a PRAME polypeptide having a VHH chain according to the first aspect.

In a fourth aspect, the invention provides a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide, the frame regions FR1, FR2, FR3 and FR4 being humanised on the basis of 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 a PRAME polypeptide is as follows:

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
	SEQ ID NO:114。

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

In preferred embodiments, the antibodies that target the PRAME polypeptide comprise monomers, bivalent antibodies, and/or multivalent antibodies.

In a sixth aspect, the invention provides a bispecific antibody comprising a first antibody comprising a VHH chain of a single domain antibody targeting a PRAME polypeptide according to the first aspect, or a heavy chain variable region of an antibody targeting a PRAME polypeptide according to the second aspect, or a single domain antibody targeting a PRAME polypeptide according to the third aspect, a humanized single domain antibody targeting a PRAME polypeptide according to the fourth aspect, or an antibody targeting a PRAME polypeptide according to the fifth aspect, and a second antibody.

In preferred embodiments, the second antibody may bind to the same or a different antigen as the first antibody, or to a different epitope of 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 polypeptides; preferably, a single domain antibody comprising 2 targeting PRAME polypeptides; more preferably, the 2 single domain antibodies targeting the PRAME polypeptide form a single domain antibody dimer targeting the PRAME polypeptide.

In a preferred embodiment, the sequences of the bispecific antibodies are shown in the following table:

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

In a preferred embodiment, the immunoglobulin is IgG1, igG2, igG3, igG4; igG4 is preferred.

In an eighth aspect, the invention provides a chimeric antigen receptor made from a VHH chain of a single domain antibody targeting a PRAME polypeptide according to the first aspect, a heavy chain variable region of an antibody targeting a PRAME polypeptide according to the second aspect, a single domain antibody targeting a PRAME polypeptide according to the third aspect, a humanized VHH chain of a single domain antibody targeting a PRAME polypeptide according to the fourth aspect, or an antibody targeting a PRAME polypeptide according to the fifth aspect.

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

SEQ ID NO:98
	SEQ ID NO:99
SEQ ID NO:100
	SEQ ID NO:101
SEQ ID NO:102。

in a ninth aspect, the invention provides an immune effector cell expressing the chimeric antigen receptor of the eighth aspect.

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

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

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

In a twelfth aspect, the invention provides a host cell comprising the expression vector of the eleventh aspect, or having integrated into its genome the nucleic acid molecule of the tenth aspect.

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

1) Culturing the host cell of the eleventh aspect under suitable conditions, thereby obtaining a culture comprising 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 bispecific antibody or the fusion protein; and

2) Optionally, isolating or recovering from the culture 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 bispecific antibody or the fusion protein.

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

1) The VHH chain of a single domain antibody that targets a PRAME polypeptide of the first aspect, the heavy chain variable region of an antibody that targets a PRAME polypeptide of the second aspect, a single domain antibody that targets a PRAME polypeptide of the third aspect, a humanized VHH chain of a single domain antibody that targets a PRAME polypeptide of the fourth aspect, an antibody that targets a PRAME polypeptide of the fifth aspect, a bispecific antibody of the sixth aspect, or a fusion protein of the seventh aspect; and

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

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

In a preferred embodiment, the immunoconjugate is an Antibody-Drug-Conjugate (ADC).

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

In a preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.

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

In a preferred embodiment, the multivalent means that multiple repeated moieties are included in the amino acid sequence of the immunoconjugate.

In a fifteenth aspect, the invention provides 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 the first aspect, a heavy chain variable region of an antibody targeting a PRAME polypeptide according to the second aspect, a single domain antibody targeting a PRAME polypeptide according to the third aspect, a humanized single domain antibody targeting a PRAME polypeptide according to the fourth aspect, an antibody targeting a PRAME polypeptide according to the fifth aspect, a bispecific antibody according to the sixth aspect, a fusion protein according to the seventh aspect, a chimeric antigen receptor according to the eighth aspect, an immune effector cell according to the ninth aspect or an immunoconjugate according to the fourteenth aspect, and optionally a pharmaceutically acceptable excipient.

In a preferred embodiment, the pharmaceutical composition is for use in the treatment of a tumor, which is a PRAME polypeptide-related tumor; melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, and the like are preferred.

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

1) Reagents for detecting PRAME polypeptides;

2) An agent that blocks binding of the PRAME polypeptide to PD-L1;

3) A medicine for treating tumor.

In a preferred embodiment, the tumor is a PRAME polypeptide-related tumor; melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, and the like are preferred.

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

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

2) A container; and

3) Optionally instructions for use.

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

1) Contacting a test sample with a VHH chain of a single domain antibody that targets a PRAME polypeptide according to the first aspect, a heavy chain variable region of an antibody that targets a PRAME polypeptide according to the second aspect, a single domain antibody that targets a PRAME polypeptide according to the third aspect, a humanized VHH chain of a single domain antibody that targets a PRAME polypeptide according to the fourth aspect, an antibody that targets a PRAME polypeptide according to the fifth aspect, a bispecific antibody according to the sixth aspect, a fusion protein according to the seventh aspect, or an immunoconjugate according to the fourteenth aspect;

2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of a PRAME polypeptide protein in the sample.

In a nineteenth aspect, the present invention provides a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a VHH chain of a single domain antibody that targets a PRAME polypeptide of the first aspect, a heavy chain variable region of an antibody that targets a PRAME polypeptide of the second aspect, a single domain antibody that targets a PRAME polypeptide of the third aspect, a humanized single domain antibody that targets a PRAME polypeptide of the fourth aspect, an antibody that targets a PRAME polypeptide of the fifth aspect, a bispecific antibody of the sixth aspect, a fusion protein of the seventh aspect, a chimeric antigen receptor of the eighth aspect, an immune effector cell of the ninth aspect or an immunoconjugate of the fourteenth aspect, or a 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 PRAME polypeptide-related tumors; more preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows that the 14 single domain antibodies of the invention are each capable of binding protein antigens and have a higher affinity;

FIG. 2 shows that each of the 14 single domain antibodies of the invention is capable of binding antigen at the cellular level and has a higher affinity;

FIG. 3 shows that the single domain antibody molecules of the invention have better specificity;

FIG. 4 shows that the diabody molecules of the present invention have better cell killing activity;

FIG. 5 shows that the CAR molecules of the invention can be expressed efficiently on the surface of T cells;

FIG. 6 shows killing of target cells by candidate CAR-T cells under different effective target ratio conditions;

FIG. 7 shows that the CAR-T cells of the invention are both effective in recognizing T2 carrying a target polypeptide, activating and conducting immune signals, secreting IFN-gamma cytokines;

FIG. 8 shows that humanized single domain antibodies obtained by humanizing the LL-PR001 molecule can bind protein antigen and have higher affinity;

FIG. 9 shows that humanized single domain antibodies obtained by humanizing the LL-PR004 molecule can bind protein antigen and have higher affinity.

Detailed Description

The inventors have conducted extensive and intensive studies to unexpectedly find a class of single domain antibodies that target PRAME polypeptides. The single domain antibodies of the invention are capable of binding PRAME polypeptides with high affinity and have better specificity. The invention also provides bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, ADC and the like prepared by using the single domain antibodies. The present invention has been completed on the basis of this finding.

Definition of terms

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

Single domain antibodies

In this context, "single domain antibody", "nanobody" and the like have the same or similar meanings and refer to a class of antibody molecules lacking the light chain of the antibody, but only the variable region of the heavy chain. Single domain antibodies are the smallest antigen binding units, i.e. the smallest antigen binding fragments that have complete function. Typically, the variable region of the heavy chain of the antibody is cloned after the naturally deleted light and heavy chain constant region 1 (CH 1) antibodies are obtained, thereby constructing a single domain antibody (VHH) consisting of only one heavy chain variable region.

The single domain antibody VHH chain of the present invention, which targets the PRAME polypeptide, also includes framework regions FR1, FR2, FR3 and FR4.

On the basis of the single domain antibody VHH chain of the target PRAME polypeptide, the inventor also humanizes the VHH chain, so as to obtain the humanized VHH chain of the single domain antibody of the target PRAME polypeptide.

In addition to the single domain antibody VHH chain or humanized VHH chain of the subject invention that targets a PRAME polypeptide, the subject invention also provides antibodies that target a PRAME polypeptide comprising one or more of the described VHH chains of a single domain antibody that targets a PRAME polypeptide or a humanized VHH chain of a single domain antibody that targets a PRAME polypeptide. The invention also provides a bispecific antibody comprising a first antibody, which may be a VHH chain of a single domain antibody of the invention targeting a PRAME polypeptide, or a humanized VHH chain, and a second antibody. The person skilled in the art can select the second antibody of the bispecific antibody according to the actual need. For example, the second antibody may 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 preferably binds to a different epitope. In specific embodiments, the second antibody may be a single domain antibody, or may be a single chain antibody or a double chain antibody.

The person skilled in the art can also make fusion proteins from the single domain antibody VHH chains or humanized VHH chains of the target PRAME polypeptides of the invention, e.g. fusion proteins further comprising an Fc-fragment or half-life extending domain of an immunoglobulin. The fusion protein thus obtained not only has the biological activity of the single domain antibody VHH chain itself, but also has other properties imparted by the Fc fragment of an immunoglobulin, such as an extended plasma half-life, reduced immunogenicity, improved stability, etc. In a specific embodiment, the fusion protein comprises a VHH chain or a humanized VHH chain of a single domain antibody of the invention that targets a PRAME polypeptide, optionally a linker sequence, and an Fc fragment of an immunoglobulin. In specific embodiments, the immunoglobulin is IgG1, igG2, igG3, igG4; igG4 is preferred. In a specific embodiment, the half-life extending domain is as shown in the amino acid sequence ：QVQLVESGGGVVQPGGSLRLSCAASGFAFRGFGMSWVRQAPGKGLEWVSSINNGGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGGPGASPSGQGTQVTVSS(SEQ ID NO:121).

The invention includes not only whole antibodies, but also fragments, derivatives and analogues of said antibodies. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art in view of the teachings herein.

The antibodies of the invention refer to polypeptides having PRAME polypeptide protein binding activity that include the CDR regions described above. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention. The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

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

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to the table below.

The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences. The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, 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 a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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 invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above. Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.

Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. a biotoxin; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. drug-activated enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.

Bispecific antibodies

Bispecific antibodies are recombinant antibodies engineered with proteins. Bispecific antibodies can target two different antibody binding epitopes simultaneously, which can be from different antigens or from the same antigen. Many studies have now shown that bispecific antibodies have great therapeutic potential in the treatment of tumors, autoimmune diseases, and viral infections. The major advantage of bispecific antibodies over monoclonal antibodies is that they mediate the steric effect of two recognition epitopes and the synergistic effect of dual targeting, resulting in biological effects that are not achieved by the combined use of both antibodies. A relatively specific bispecific antibody is called a T cell adapter (T CELL ENGAGER) which can achieve the purpose of treating tumors by simultaneously binding to a target on the surface of a tumor cell and a T cell, activating endogenous T cells, and causing the tumor cell to lyse. T cell adaptors have been shown to be useful in the treatment of tumors. Bispecific T cell adaptors targeting CD20 and CD19 have been marketed under FDA approval (NAT REV CLIN Oncol.2020Jul;17 (7): 418-434.). Bispecific antibodies are relatively high in technical threshold and development costs because of their complexity unlike monoclonal antibodies.

Immune cells

In this context, immune cells and immune effector cells have the same meaning and are as conventionally understood by a person skilled in the art. It refers to cells involved in or associated with an immune response, including lymphocytes and phagocytes. In particular embodiments, the immune cells are lymphocytes that recognize antigens, thereby generating 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 are 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 potential cellular immunotherapy. CAR-T cells express a CAR (CHIMERIC ANTIGEN Receptor) molecule, the structure of which is divided into: an antigen binding region, a hinge region, and a transmembrane domain, and an intracellular signaling domain. Current CAR-T cells typically use scFv (SINGLE CHAIN FRAGMENT Variables) segments derived from single chain engineering of the antigen binding region of a monoclonal antibody as the antigen binding region. However, when the scFv structure is modified, problems of reduced affinity and altered specificity are likely to occur, and scFv has a relatively large molecular weight and is likely to form a multimer, which affects the function of CAR (Nat Rev cancer.2021Mar;21 (3): 145-161.). Thus, CARs containing antigen binding regions of novel structure. The single domain antibody is used for constructing the CAR structure, so that the variable region of the single domain antibody can be directly connected to the CAR structure, and the design is simple and convenient.

Immunoconjugates

ADC is an antibody carrying cytotoxin drugs, and can be used for delivering the cytotoxin drugs to tumor cells at fixed points to realize specific killing of the tumor cells. ADC drugs targeting HER2, CD30, trop2 and other targets show good clinical effects and safety in clinical researches (NAT REV CLIN Oncol.2021Jun;18 (6): 327-344.). In recent years, a plurality of ADC drugs are approved by the FDA and marketed. However, the target of the current ADC drugs is cell membrane proteins, and the ADC drugs targeting intracellular proteins are one of the hot directions of future development.

ADC drugs can specifically target cells and deliver molecules with cytotoxic effects into target cells, thereby producing specific killing effects. Cell killing induced by cytotoxic molecules can break and improve the tumor-inhibiting microenvironment, and has the potential to improve the efficacy of other therapies such as immunotherapy and the like. The single domain antibody of the present invention can be endocytosed into cells, and therefore, it is expected that ADC drugs based on the single domain antibody of the present invention will be developed in the future.

The invention also provides an immunoconjugate comprising a VHH chain, a humanized VHH chain, etc. of a single domain antibody of the invention that targets a PRAME polypeptide, and a conjugated moiety. In particular embodiments, the coupling moiety may be a detectable label, drug, toxin, cytokine, radionuclide, enzyme, or the like, for diagnostic, detection, or therapeutic purposes, among others.

Pharmaceutical composition

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical compositions of the invention can directly target PRAME polypeptides expressed by tumor cells. Thus, the pharmaceutical composition of the present invention can be used for treating tumors. In specific embodiments, the tumor is a PRAME polypeptide-related tumor. In a preferred embodiment, the tumor is melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, or the like. In addition, the pharmaceutical compositions of the present invention may also be used in combination with other therapeutic agents.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the single domain antibodies (or conjugates thereof) of the invention described above, as well as a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions.

The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Kit for detecting a substance in a sample

The invention also provides a kit comprising a VHH chain, or a humanized VHH chain, antibody, fusion protein or immunoconjugate, etc. of a single domain antibody targeting a PRAME polypeptide of the invention. In specific embodiments, the kit further comprises a container, instructions for use, buffers, and the like.

The invention has the advantages that:

1. the single domain antibodies of the present invention that target PRAME polypeptides bind to PRAME polypeptides with high affinity;

2. The single domain antibody of the target PRAME polypeptide has better specificity;

3. The single domain antibody of the target PRAME polypeptide can be used for further preparing bispecific antibody, chimeric antigen receptor-T cell, ADC and the like, thereby laying a new material foundation for the development of therapeutic or diagnostic medicaments of the target PRAME polypeptide.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Embodiment one: single domain antibody sequence acquisition

Two adult healthy alpacas (Alpaca) were selected as the animals for immunization. The alpaca was immunized with a recombinantly expressed pMHC (peptide-Major histocompatibility complex) recombinant protein carrying the polypeptide of interest as antigen. 4-5 total immunizations were performed. And taking the peripheral blood of alpaca at different time points to detect the immune titer. And (3) taking alpaca peripheral blood after the immunization is finished, extracting mRNA of peripheral blood PBMCs cells, and carrying out reverse transcription to obtain cDNA. The cDNA was amplified using specific primers to obtain PCR products with single domain antibody gene fragments. The PCR product and yeast library vector are then introduced into yeast competent cells using electrotransformation to prepare a yeast library. The yeast library is screened using recombinantly expressed protein antigens or T2 cells loaded with the target polypeptide. The invention uses the pMHC recombinant protein carrying the target polypeptide as a positive antigen and uses the pMHC recombinant protein carrying the negative polypeptide as a negative antigen. Single domain antibody clones that specifically bound to positive antigens but not negative antigens were obtained through three positive and three negative screens.

And (3) performing sanger sequencing on the single-domain antibody clone to obtain the full-length sequence of the single-domain antibody of the specific binding target protein. The invention obtains 14 different single-domain antibody clones, which are respectively named: LL-PR001-LL-PR014. The full-length amino acid sequence and the amino acid sequence of the CDR regions of the single domain antibody are shown in Table 1.

TABLE 1 amino acid sequence listing of candidate single domain antibodies

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Embodiment two: single domain antibody expression purification

The present invention uses conventional monoclonal antibody expression and purification methods to express and purify the single domain antibodies of the present invention. First, a single domain antibody expression vector was constructed using conventional molecular cloning techniques. After successful vector construction, the single domain antibodies described above were expressed using a method of transiently transfecting HEK293 suspension cell lines. The basic steps are as follows: the single domain antibody gene was cloned into the expression vector pCDNA4 (Invitrogen, cat V86220). Different single domain antibodies were expressed using the method of transiently transfecting HEK293 suspension cells. After the expression is completed, collecting supernatant, purifying the single domain antibody by using a conventional monoclonal antibody purification method, and finally obtaining the purified single domain antibody.

After purification of the single-domain antibody was completed, the protein concentration and total protein amount of the different antibodies were detected using ultraviolet spectrophotometry, and then the expression amount of each diabody was calculated from the expression volume. The absorbance A280 of the sample solution was read at a wavelength of 280nm using NanoDrop 1000, and the protein concentration of the sample was calculated by the formula C (mg/mL) =A280/ε (ε is 1.482mL/mg cm-1). The purity of the different antibodies was also assessed using conventional gel electrophoresis SDS-PAGE methods. The basic steps are as follows: samples were electrophoretically separated using Invitrogen electrophoresis cells and SDS-PAGE gradient gel. Diluting the sample to about 1mg/mL, adding a proper amount of reducing agent, loading buffer and pure water, mixing, heating at 70 ℃ for about 10 minutes, wherein the loading amount is 2-10 mug, the electrophoresis voltage is about 200V, the electrophoresis time is about 35 minutes, and then respectively performing gel dyeing and decolorization. After the decoloring is finished, photographing by using a conventional gel imaging system, and analyzing and calculating the purity of the main band. In addition, the purity of the different antibodies was evaluated using size exclusion high performance liquid chromatography (SEC-HPLC method). The basic steps are as follows: diluting the sample to about 1.0mg/mL, adopting a TSKgel G3000SWXL chromatographic column, setting the column temperature to 25 ℃, adopting 100mM phosphate buffer solution, 100mM sodium sulfate and pH 7.0+/-0.2 as mobile phases, adopting a sample injection volume of 20-50 mu L, carrying out isocratic elution for 20min at a flow rate of 1.0mL/min, detecting at 280nm wavelength, and adopting a peak area normalization method to obtain the content of the monomer.

The expression level and purity information of the different single domain antibodies are shown in Table 2. The expression level of the single-domain antibody of the invention using the transient expression system is in the range of 650-850mg/L, which proves that the double-antibody structure of the invention has higher expression level. The purity of the reduction SDS-PAGE and SEC-HPLC of most single domain antibodies of the invention is more than 95%. These results demonstrate that the single domain structure of the invention has better expression level and higher purity.

TABLE 2 Single domain antibody expression level and purity detection results

Embodiment III: detection of binding capacity of single domain antibodies to target

In order to explore the binding capacity of the candidate single domain antibodies of the present invention to antigens, the present example uses ELISA and flow cytometry methods to detect from both protein and cell levels, respectively, to comprehensively compare the affinity levels of candidate molecules to target antigens. The specific operation steps are as follows:

3.1 protein level binding Capacity assay

1) Diluting SA protein to 0.3 mug/mL by PBS, adding 100 mug/hole into a 96-hole transparent ELISA plate, sealing the plate, and incubating at 37 ℃ for 1h;

2) Proteins were discarded and plates were washed three times with 200 μl PBST solution (PBS containing 0.05% tween 20);

3) Adding blocking solution (PBS solution containing 2% BSA) at a concentration of 100 mu L/hole, sealing the plate, and incubating at 37 ℃ for 1h;

4) Repeating the step 2) of washing the plate;

5) Diluting the biotin-labeled MHC-PRAME polypeptide complex to 2ug/ml by using a sealing solution, adding 100 mu L of the dilution solution into each hole, sealing the plates, and then placing the plates at 37 ℃ for incubation for 1h;

6) Repeating the step 2) of washing the plate;

7) Diluting the candidate antibodies to 12 different concentrations by using a sealing solution, wherein the highest concentration is 0.3 mug/mL, carrying out 3-time gradient dilution, adding 100 mu L of the diluent into each hole, sealing the plate, and then placing the plate at 37 ℃ for incubation for 1h;

8) Repeating the step 2) of washing the plate;

9) The sealing liquid is used for sealing according to the proportion of 1: diluting SA-HRP in proportion of 1000, adding 100 mu L of the solution into each hole, sealing the plate, and then placing the plate at 37 ℃ for incubation for 30min;

10 Repeating the step 2) of washing the plate;

11 Adding 100 mu L of TMB reaction liquid into each hole, sealing the plate, placing the plate on a microplate shaker for reaction for 10min, adding 100 mu L of 2N H ₂ SO4 solution to stop the reaction, and reading the absorbance value at 450nm by an enzyme-labeling instrument.

The results are shown in FIG. 1. The results show that the 14 single-domain antibodies screened by the invention can all bind protein antigens and have higher affinity.

3.2 Detection of cell level binding Capacity

1) Preparing cells: taking proper amount of T2 cells (cell bank of Shanghai biochemical cells), adjusting cell density to 2×10 ⁶/ml, and subpackaging into 96-well U bottom plate with 50 μl (about 1E5 cells) per well;

2) Polypeptide loading: polypeptide with the concentration of 60 mu M is prepared by 1640 culture medium, the polypeptide diluent is added into an orifice plate according to 50 mu L/hole, and is uniformly mixed with cells, and the mixture is incubated for 2 hours at 37 ℃ so that the polypeptide is loaded on T2 cells. 200. Mu.L of 1640 medium is added to resuspend the cells, 400g is centrifuged for 5min, and the supernatant is removed;

3) Antibody incubation: adjusting the initial concentration of the original antibody to 1 mug/mL by using an antibody diluent (PBS+0.5% FBS), diluting 4 times, setting 10 concentration gradients altogether, adding 100 mu L of diluted antibody into the cells loaded in the front, blowing and mixing uniformly, and incubating at 4 ℃ in a dark place for 60min;

4) Antibody washing: adding 200 mu L of antibody diluent into the incubated antibody cell mixture, centrifuging for 5min at 400g, discarding the supernatant, re-suspending cells with 200 mu L of antibody diluent, and repeatedly cleaning for one time;

5) Secondary antibody incubation: diluting the fluorescent secondary antibody APC anti-human IgG Fc (BD Biosciences) with an antibody diluent according to a ratio of 1:200, adding 100 mu L into the cell sediment after supernatant discarding, blowing and mixing uniformly, and incubating for 30min at 4 ℃ in a dark place;

6) And (3) secondary antibody washing: adding 200 mu L of antibody diluent into the incubated antibody cell mixture, centrifuging for 5min at 400g, discarding the supernatant, re-suspending cells with 200 mu L of antibody diluent, and repeatedly cleaning for one time;

7) And (3) detecting: cells were resuspended by adding 100 μl of antibody dilution per tube, the cell suspension was transferred to a flow tube, and the positive labelling rate was detected by flow cytometry.

8) The data were analyzed by FlowJo software to obtain mean fluorescence intensity values (MFI), dose-effect nonlinear curves were fitted to MFI values and antibody concentration values, EC ₅₀ values were calculated, and the antigen affinity activity of candidate antibody molecules was assessed.

The results are shown in FIG. 2: the results show that the 14 single domain antibodies screened by the invention can all bind to the antigen at the cellular level and have higher affinity.

Embodiment four: specific detection of single domain antibodies for target recognition

T2 cell model loaded with different polypeptides

1) T2 cells grown to log phase were resuspended to 2x 10 ⁶/mL with complete medium;

2) Diluting the target polypeptide and the OTP polypeptide to 60 mu M by using a complete culture medium, and respectively mixing the polypeptide dilutions with a cell suspension according to a ratio of 1:1, placing the mixture in a 37 ℃ incubator for incubation for 1-2 hours, so that the polypeptide is loaded on the surface of the T2 cells;

3) After incubation, excess polypeptide was washed off with medium, cells were collected by centrifugation, cells were resuspended to 2X 10 ⁶/mL with buffer (PBS containing 2% FBS), and polypeptide-loaded T2 cells were seeded into 96-well U-bottom cell culture plates at 50. Mu.L/well;

4) Diluting candidate antibody molecules into 10 mug/mL, 2 mug/mL, 0.4 mug/mL and 0.08 mug/mL respectively by using a buffer solution, adding the antibody diluted solution into an orifice plate according to 50 mug/hole, fully and uniformly mixing the antibody diluted solution with cells, and then incubating at 4 ℃ for 1h;

5) Centrifuging 300g for 5min, removing antibody diluent, and washing cells once with 200 mu L buffer;

6) Buffer was used according to 1:200 (ex Biolegend, cat. No. 366906), adding the antibody dilution to the well plate at 100 μl/well, resuspending the cells and mixing well, incubating at 4 ℃ for 30min;

7) Centrifuging 300g for 5min, removing antibody diluent, and washing cells once with 200 mu L buffer;

8) Resuspension of cells with 100 μl of buffer, flow cytometry to detect the fluorescence binding intensity of APC channel;

9) The data were analyzed using FlowJo software to obtain mean fluorescence intensity values (MFI) and the level of non-specific binding of candidate antibody molecules to off-target polypeptides was assessed.

TABLE 3 potential off-target polypeptide sequence listing for use in the invention

Sequence number	Polypeptide name	Polypeptide sequence
			1	Target	SLLQHLIGL(SEQ ID NO:57)
2	OTP-1	KLYQHEINL(SEQ ID NO:58)
			3	OTP-2	FLPIHLLGL(SEQ ID NO:59)
4	OTP-3	YLDGHLITT(SEQ ID NO:60)
			5	OTP-4	ILAMHLIDV(SEQ ID NO:61)
6	OTP-5	SLLGHVIRL(SEQ ID NO:62)
			7	OTP-6	SLADRLIGV(SEQ ID NO:63)
8	OTP-7	ALMYHTITL(SEQ ID NO:64)

The invention detects off-target conditions of all candidate molecules. FIG. 3 shows the results of a partial molecular off-target analysis. As shown in FIG. 3, the single domain antibody molecules screened by the invention have better specificity and can be further developed.

Fifth embodiment: single-domain antibody-based diabody design

We selected the single domain antibody molecule LL-PR005 as representative and designed diabody molecules of different structures. The double antibody molecule designed by the invention can combine with PRAME polypeptide-HLA-A 02 complex and CD3. Some bispecific antibodies of the invention incorporate an Fc structure or half life extender structure. The amino acid sequence of the CD3 antibody used by the double antibody molecule designed by the invention:

Heavy chain variable region:

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS(SEQ ID NO:65)

Light chain variable region:

QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIK(SEQ ID NO:66)

The half life extender amino acid sequence of the designed diabody molecule is as follows:

QVQLVESGGGVVQPGGSLRLSCAASGFAFRGFGMSWVRQAPGKGLEWVSSINNGGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGGPGASPSGQGTQVTVSS(SEQ ID NO:67)

the invention designs 13 kinds of diabody molecules with different structures, and the amino acid sequence is shown in table 3.

TABLE 4 double antibody molecular amino acid sequence table based on single domain antibody designed by the invention

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Example six: expression purification of diabody molecules

Reference example two experimental procedures, the double antibody molecule designed by the invention is expressed and purified, and the expression quantity and purity are detected. The expression level and purity information of the different diabodies are shown in Table 4. The expression quantity of the double antibody is in the range of 600-850mg/L, and the double antibody structure has higher expression quantity. The monomer purity of SEC-HPLC of the double antibody is above 90%. Meanwhile, the purity of the reduction SDS-PAGE of all the double antibodies is over 95 percent. These results prove that the designed diabody molecule has better expression quantity and higher purity.

TABLE 5 results of determination of the expression level and purity of diabody molecules

Embodiment seven: functional evaluation of diabody molecules

The double antibody molecule, also called as a T cell adapter (T CELL ENGAGER), can be used for treating tumors by simultaneously and specifically combining a target presented by pMHC in tumor cells and CD3 protein on the surface of the T cells, mediating the directional cruising of the T cells to the vicinity of the tumor cells and endogenous activating and releasing cytokines to cause the cracking of the tumor cells. In order to evaluate the specific recognition and killing ability of the candidate diabody molecules expressed in the sixth embodiment to tumor cells, the present embodiment co-cultures target cells and cd3+ T cells according to a certain effective target ratio, and adds candidate antibody molecules with different concentrations, and detects the apoptosis proportion of the target cells and the activation and factor secretion level of the T cells mediated by the antibodies; in addition, in the embodiment, tumor cell lines NCI-H1755, HS695T, OVCAR and U2OS with different antigen expression abundances and PRAME expression negative HLA-A2 positive T2, MCF7 and HLA-A2 and PRAME expression negative A549 cell lines are set as target cells, so that the target specificity recognition capability of the double antibody molecules can be further evaluated, and suitable double antibody molecules can be more comprehensively screened for deep research. The basic implementation steps are as follows:

1) Co-culturing cells: different target cells are transfected by using lentiviruses with luciferase, and a cell line marked with the luciferase is prepared, and the markers are as follows: NCI-H1755-GFP, HS695T-GFP, OVCAR3-GFP, MCF7-GFP and A549-GFP. Different target cells and effector cells were resuspended in medium (1640 medium with 2% FBS) at concentrations of 2X 10 ⁵/mL and 1X 10 ⁶/mL, respectively, plated at 25. Mu.L/well into 96-well flat bottom opaque plates, respectively, and incubated temporarily at 37 ℃;

2) Antibody incubation: the antibody was diluted in a gradient of medium, up to 20nM, to 10 concentration points. 50. Mu.L of the corresponding bispecific antibody was added to each well. After thoroughly mixing, the mixture was centrifuged at 500rpm for 3 minutes. Incubating the cells in a 37 ℃ incubator for 24 hours;

3) Killing detection: after 24h of cell co-culture, the remaining luciferase activity (relative light units, RLU) of the target cells was measured to determine the killing capacity of T cells against the target cells in the presence of antibodies of different specificities. The method comprises the following specific steps: taking out the opaque 96-well flat bottom plate after co-culture, adding 100 mu L of an equal volume of D-luciferin substrate (Thermo FISHER SCIENTIFIC: 88293) into the well, uniformly mixing, and developing for 5min in a dark place, and detecting the fluorescence intensity by using a chemiluminescent mode in an enzyme-labeled instrument. Since luciferase is expressed only in target cells, the remaining luciferase activity in the well is directly related to the number of viable target cells in the well. Obtaining maximum luciferase activity by adding the culture medium to the target cells in the absence of effector cells and antibodies as a control;

4) Data analysis: and calculating the killing efficiency of the target cells corresponding to the detection hole by taking the maximum luciferase activity as a control, fitting a dose-dependent curve by taking the antibody addition concentration as an abscissa, estimating an EC50 value, and evaluating the killing level of the target cells of the double antibody molecules to be detected.

The results show that the bispecific antibodies of the invention can well mediate T cell activation and kill cells positive for target expression (figure 4).

Example eight: PRAME-targeted CAR sequence molecular design and lentiviral vector construction

8.1 Design of PRAME-targeted CAR Gene sequences

PRAME-targeted CARs comprise a single domain antibody sequence against human PRAME, a CD28 hinge region and transmembrane domain, a CD28 co-stimulatory signaling region and a CD3zeta signaling domain, connected in a sequential tandem fashion. Five single domain antibody sequences are selected, and five CAR structures are designed in total. The 5 different CAR molecules were individually named: the amino acid sequences corresponding to PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, PRAME-CAR-05, and the different CARs are shown in Table 6. Five PRAME-targeted CAR gene sequences designed in the present invention were genosynthesized and subcloned into pUC57 vector (su Jin Weizhi biotechnology limited).

TABLE 6 PRAME-targeting CAR amino acid sequence Listing

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8.2 Construction of CAR lentiviral vectors

Designing primers, namely amplifying 5 different CAR molecules from a pUC57 vector by using PCR, carrying homologous arms of lentiviral vectors on 5' ends of forward and reverse primers during primer design, and respectively performing gel recovery and purification (Nanjinouzan biotechnology Co., ltd., product number DC 301) on amplified PCR products after agarose gel electrophoresis detection to obtain DNA fragments. The DNA fragment recovered by the digestion is cloned to a lentiviral vector by homologous recombination, and the lentiviral vector is digested and recovered by restriction enzymes BamHI (NEB: R3136V) and SalI (NEB: R3138V) for purification. The 5 different recombinant plasmids ：p-lenti-PRAME-CAR-01、p-lenti-PRAME-CAR-02、p-lenti-PRAME-CAR-03、p-lenti-PRAME-CAR-04、p-lenti-PRAME-CAR-05. obtained by this method sent 5 lentiviral vectors to sequencing verification by biotechnology limited in su Jin Weizhi, sequencing primers were: lenti-seqF: TTGAGTTTGGATCTTGGTTC (SEQ ID NO: 103), lenti-seqR: CAGCAACCAGGATTTATACA (SEQ ID NO: 104), and by sanger sequencing, it was verified that all 5 lentiviral vector plasmids were constructed correctly.

Example nine: PRAME-targeted CAR-T cell preparation and functional evaluation

9.1 Preparation of lentiviruses

Sequencing confirmed that the correct lentiviral plasmid was transformed into E.coli stbl3 (purchased from the company Limited of the Saint Biotech Co., ltd.). The next day, single clones were picked from the transformed plates into shake tubes of 2ml liquid LB medium, which already contained kanamycin (50 ug/ml), at 37℃at 220rpm, and shake cultured for 8h. 1ml of the activated bacterial liquid is sucked up and inoculated into 250ml of liquid LB medium containing kanamycin, and the liquid LB medium is cultured for 12-16 hours by shaking at 37 ℃ and 220 rpm. Plasmid extraction was performed using a large extraction kit NucleoBond Xtra Midi Plus (MN, cat# 740412.50) according to the protocol provided by the kit. Plasmid concentration was determined using Nanodrop (Thermo FISHER SCIENTIFIC) after plasmid extraction and verified by sanger sequencing, while supercoiled plasmid content was detected by DNA agarose gel.

Frozen 293T cells (from the cell bank of the national academy of sciences) were removed from the liquid nitrogen, thawed in a 37℃water bath, and the tube was wiped with 75% alcohol, transferred to a 15ml centrifuge tube into which 10ml of pre-warmed DMEM complete medium (90% DMEM+10% FBS+1% penicillin/streptomycin) had been added, gently swirled, and centrifuged at 400g for 4min, and the supernatant was aspirated. 10ml of DMEM complete medium was added, gently swirled and inoculated into T25 or T75 flasks and cultured in a cell incubator containing 5% CO ₂ at 37 ℃. The 293T cells cultured for more than 3 passages can be used for packaging the lentivirus after the cells are continuously cultured after the passage when the cell density reaches more than 80% in the next day. The method comprises the following specific steps:

1) The first day, 293T cells were seeded: cells were seeded in about 1.0X10-7/T175 flasks (40 mL medium) and transfected the next time the cell density reached 90%.

2) The following day, plasmid transfection: the medium was changed to DMEM medium with 10% fbs but without double antibodies prior to transfection. First, preparing a plasmid complex: the following plasmids were added to 1.5ml Opti-MEM (Thermo FISHER SCIENTIFIC; 31985-070) and mixed well: 18 μg psPAX plasmid (Addgene; cat. No. 12260), 9 μg pMD2.G plasmid (Addgene; cat. No. 12259), 18 μg lentiviral vector plasmid. Lentiviral plasmids ：p-lenti-PRAME-CAR-01、p-lenti-PRAME-CAR-02、p-lenti-PRAME-CAR-03、lenti-PRAME-CAR-04、lenti-PRAME-CAR-05. respectively followed by preparation of transfection reagent complexes: adding 67.5 mu L (2 mg/mL) of PEI (polysciences: 24765) into 1.5mL of Opti-MEM according to the mass ratio of the plasmid to the PEI of 1:3, uniformly mixing, and standing at room temperature for 5min; then the transfection reagent compound is added into the plasmid compound drop by drop, and the mixture is kept stand for 20min after being mixed evenly. Finally, the transfection complex is slowly dripped into a 293T cell culture flask, gently mixed, and continuously cultured in a cell culture box containing 5% CO ₂ at 37 ℃.

3) Fourth day, virus is collected: the culture supernatant was harvested 48h after transfection and centrifuged at 2000rpm for 10min to remove cell debris. The supernatant was filtered using a 0.45. Mu.M filter (Millex-HV, cat. SLHVR RB) and the filtrate was transferred to a special centrifuge tube for trimming. Ultracentrifugation was performed using an ultracentrifuge 25000rpm for 2h. After the supernatant was decanted, 1ml of X-VIVO-15 medium was used to resuspend the lentivirus, and the lentivirus was stored in an ultra-low temperature refrigerator at-80℃after sub-packaging. Lentiviruses containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, PRAME-CAR-05 were prepared according to this procedure, respectively.

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

The prepared slow viruses containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04 and PRAME-CAR-05 respectively infect primary human T cells, and CAR-T cells carrying different CAR genes are prepared. CAR-T cells carrying 5 CAR genes were respectively named: PRAME-CAR-T-01, PRAME-CAR-T-02, PRAME-CAR-T-03, PRAME-CAR-T-04, PRAME-CAR-T-05, untransfected T cells were used as negative controls and named NT. The method comprises the following specific steps:

1) Resuscitates CD3+ T cells (Miaoshun (Shanghai) Biotechnology Co., ltd.) from peripheral blood of healthy persons, and resuspensions the cells with T cell medium containing 300IU/mL IL-2 to a density of 1X 10 ⁶/mL, according to cell and magnetic beads 1:1, adding a T cell activator CD3/CD28 magnetic bead (ACRO Biosystems, product number: MBS-C001) in a ratio, fully and uniformly mixing, and inoculating the cells into a 6-hole plate for culture;

2) After T cells are activated for 24 hours, counting the T cells and inoculating the T cells into a new 24-well plate, inoculating the cells according to 500ul of 5 multiplied by 10 ⁵ cells/well, respectively adding 100 mu L of slow virus liquid carrying different CAR genes after the inoculation is finished to infect the T cells, taking the T cells without virus liquid as negative control NT, and placing the cells into an incubator for continuous culture.

3) After 48 hours of slow virus infection, cells are sucked out from the culture holes, magnetic beads are removed by using a magnetic frame in a magnetic adsorption mode, and the cells are collected by centrifugation and resuspended in fresh T cell culture medium.

4) Simultaneously taking 100 mu L of cell suspension added with slow virus and negative control, centrifugally collecting cells, re-suspending the cells by using 100 mu LFACS buffer, respectively adding APC-labeled pMHC protein complex tetramer into each tube of cells, uniformly mixing, and incubating for 30 minutes at 4 ℃ in a dark place;

5) Cells were washed 2 times with PBS and resuspended with 100 μl FACS buffer, and the expression efficiency of 4 CARs on T cells was flow tested.

The results show that all 5 CAR molecules of the invention can be successfully expressed on T cells with a relatively uniform expression efficiency of 30-45% (fig. 5).

9.3 Detection of CAR-T cell killing Capacity

The experiment adopts 2 HLA-A2 and PRAME target expression double positive cell lines as target cells, which are non-small cell lung cancer NCI-H1755 and melanoma HS695T respectively. 2 PRAME and HLA-A2 expressing negative A549 and 293T cells are respectively adopted as negative cells, 5 CAR-T cells prepared in the second embodiment are respectively subjected to co-culture with target cells to detect killing effect, and the biological functions of different CAR-T cells are evaluated. The method comprises the following specific steps:

1) Different target cells were transfected with slow virus liquid expressing GFP luciferase (GenBank: AAR 29591.1) to obtain a luciferase-labeled cell line, labeled: 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 seeded at a cell concentration of 1X 10 ⁵/mL, 50. Mu.L/well into 96-well flat bottom opaque cell culture plates and temporarily placed at 37℃for incubation;

3) Positive ratios of 5 CAR-T cells were adjusted to 15% with CT, set 5:1, 2.5:1, 1:1 and 0.5:1, respectively inoculating 5 different CAR-T cells and control T cells into target cells according to 50 mu L/hole, co-culturing, fully mixing, and placing in a 37 ℃ incubator for overnight incubation;

4) The opaque 96-well flat bottom plate after co-cultivation was removed and 100 μl of an equal volume of D-luciferin substrate was added to the wells (Thermo FISHER SCIENTIFIC:88293 Mixing uniformly, and carrying out light-proof reaction for 10 minutes, and detecting the fluorescence intensity by using a chemiluminescence mode in an enzyme-labeled instrument. Since luciferase is expressed only in target cells, the remaining luciferase activity in the wells is directly related to the number of live target cells, the maximum luciferase activity is obtained by adding the culture medium to the target cells as a control, and the apoptosis ratio of the target cells is calculated by subtracting the fluorescence signal value of the live cells, namely the killing effect of CAR-T cells on the target cells, and the result is shown in FIG. 6.

The result shows that the 5 CAR molecules can mediate T cells to kill 3 target positive cells, wherein NCI-H1755 cells have the strongest killing effect.

9.4 Detection of CAR-T cytokine secretion

To further evaluate the level of target cell-specific activation of CAR-T cells and release of cytokines according to the present invention, T cells in culture supernatants were assayed for secretion of IFN- γ and IL-2 cytokines after co-culture with 5 CAR-T cells prepared in example two and target cells in example four at an effective target ratio of 1:1 for 24 hours. The method comprises the following specific steps:

1) Taking target cells NCI-H1755, HS695T, A549 and 293T, re-suspending to 1X 10 ⁶/mL by using a complete culture medium, and inoculating the cell suspension into a 96-well U-bottom deep hole plate according to 100 mu L/well;

2) 5 CAR-T and control T cells were counted and added to different target cells for co-culture at a 1:1 effective target ratio, i.e., cell concentration of 1X 10 ⁶/mL, 100. Mu.L/well;

3) After the above cells were co-cultured overnight, 50 μl of culture supernatant was collected, transferred to a new U-bottom 96-well plate, and the cells were cultured using ELISA kit (Thermo FISHER SCIENTIFIC; goods No. 88-7316) for the secretion of IFN-gamma and IL-2 cytokines in T cells, plate preparation and detection of supernatant cytokines were performed according to the instructions provided for the kit;

4) Data analysis was performed using GRAPHPAD PRISM software, a dose-dependent curve was fitted, and EC ₅₀ values were calculated.

The results are shown in FIG. 7. The results show that the candidate CAR-T cells can effectively recognize T2 loaded with target polypeptides, activate and transmit immune signals and secrete IFN-gamma cytokines, wherein the biological activity of the CAR-T-01 molecule is relatively better. From the results, the CAR sequence has better affinity activity and biological function, which suggests that the CAR molecule has further development and application values.

Example ten: single domain antibody humanization

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

1) The sequences of the single domain antibody candidate molecules LL-PR001 and LL-PR004 were entered into the IMGT database for antibody sequence alignment. According to the sequence comparison result of the database, selecting IGHV3-23 x 04 as an LL-PR001 humanized female parent carrier, and selecting IGHV3-23 x 02 as an LL-PR004 humanized female parent carrier;

2) Grafting the CDR region of the LL-PR001 single domain antibody to IGHV3-23 x 04, and grafting the CDR region of the LL-PR004 single domain antibody to IGHV3-23 x 02;

3) The humanized antibody after transplantation is back mutated to ensure affinity of the humanized antibody with reference to the prior art document.

By the method, ten candidate humanized single-domain antibodies are designed by taking LL-PR001 as a humanized female parent, and the antibody sequences are shown in Table 7. Six candidate humanized single domain antibodies were designed using LL-PR004 as the humanized female parent, the antibody sequences are shown in Table 8.

TABLE 7 humanized Single-domain antibody sequences designed with LL-PR001 as the humanized female parent

TABLE 8 humanized Single-domain antibody sequence designed with LL-PR004 as the humanized female parent

Example eleven: humanized single domain antibody molecule expression and functional identification

11.1 Expression of humanized Single Domain antibody molecules

The humanized antibody molecule is subjected to codon optimization to obtain a nucleotide sequence, and the full-length gene of the humanized antibody molecule is constructed into a single-domain antibody expression vector through gene synthesis. After successful vector construction, the single domain antibodies described above were expressed using the method of transiently transfecting HEK293 suspension cell lines, for specific procedures reference example two.

The expression levels and purities of the different humanized single domain antibodies are shown in Table 9. The expression level of the humanized single-domain antibody using the transient expression system is in the range of 700-900mg/L, which proves that the humanized single-domain antibody has higher expression level. And the purity of the reduced SDS-PAGE and SEC-HPLC of most humanized single domain antibodies is above 95%. These results demonstrate that the humanized single domain antibodies of the invention have better expression levels and higher purity.

TABLE 9 detection of expression level and purity of humanized Single-Domain antibodies

11.2 Protein level binding Capacity assay

The binding capacity of the humanized molecules to the target antigen was detected by ELISA, thereby judging the affinity of the humanized molecules. Specific experimental procedure reference example three 3.1. The results show that 10 candidate humanized single domain antibodies (huLL-PR 001-1 to huLL-PR 001-10) obtained by humanizing the LL-PR001 molecule are all capable of binding protein antigens and all have higher affinity (the results are shown in FIG. 8). The affinity of the molecules after partial humanization was slightly reduced, but still had a higher affinity. Can be applied to the research and development of subsequent biological medicine projects such as double antibody, CAR-T, ADC, RDC and the like. Similarly, 6 candidate humanized single domain antibodies (huLL-PR 004-1 to huLL-PR 004-6) obtained by humanizing and modifying the LL-PR004 molecule can be combined with protein antigen, and have higher affinity. The six affinities after humanization were slightly lower than the parent molecule, but still had higher affinities (results are shown in fig. 9). Can be applied to the research and development of subsequent biological medicine projects such as double antibody, CAR-T, ADC, RDC and the like.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A VHH chain of a single domain antibody that targets a PRAME polypeptide, the VHH chain comprising CDR1, CDR2 and CDR3 as shown in the following table:

。

2. The VHH chain of a single domain antibody that targets a PRAME polypeptide of claim 1, wherein the PRAME polypeptide has the amino acid sequence: SLLQHLIGL (SEQ ID NO: 121).

3. The VHH chain of a single domain antibody that targets a PRAME polypeptide according to claim 1, wherein the amino acid sequence of the VHH chain of the single domain antibody that targets a PRAME polypeptide is selected from the group consisting of ：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.

4. A heavy chain variable region of an antibody that targets a PRAME polypeptide, said heavy chain variable region comprising CDR1, CDR2, and CDR3 as set forth in the following table:

。

5. The heavy chain variable region of an antibody targeting a PRAME polypeptide of claim 4, wherein the amino acid sequence of the heavy chain variable region of the antibody targeting a PRAME polypeptide is selected from the group consisting of ：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.

6. A single domain antibody targeting a PRAME polypeptide having a VHH chain according to any one of claims 1-3.

7. A humanized VHH chain of a single domain antibody targeting a PRAME polypeptide, the frame regions FR1, FR2, FR3 and FR4 being humanized based on the VHH chain of any one of claims 1-3.

8. The humanized single domain antibody directed against a PRAME polypeptide of claim 7, wherein the variable region sequence of the humanized single domain antibody directed against a PRAME polypeptide is selected from the group consisting of ：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.

9. An antibody targeting a PRAME polypeptide, said antibody comprising one or more VHH chains of a single domain antibody targeting a PRAME polypeptide according to any of claims 1-3 or a humanized single domain antibody targeting a PRAME polypeptide according to claim 7 or 8.

10. The PRAME polypeptide-targeting antibody of claim 9, wherein the PRAME polypeptide-targeting antibody comprises a monomer, a bivalent antibody, and/or a multivalent antibody.

11. A bispecific antibody comprising a first antibody comprising a VHH chain of a single domain antibody that targets a PRAME polypeptide according to any one of claims 1-3, or a heavy chain variable region of an antibody that targets a PRAME polypeptide according to claim 4 or 5, or a single domain antibody that targets a PRAME polypeptide according to claim 6, a humanized single domain antibody that targets a PRAME polypeptide according to claim 7 or 8, or an antibody that targets a PRAME polypeptide according to claim 9 or 10, and a second antibody.

12. The bispecific antibody of claim 11, wherein said second antibody can bind to the same or different antigen as the first antibody, or to a different epitope of the same antigen as the first antibody.

13. The bispecific antibody of claim 11, wherein said second antibody is a single domain antibody, a single chain antibody, or a diabody.

14. The bispecific antibody of claim 11, wherein said bispecific antibody comprises 2-4 single domain antibodies targeting PRAME polypeptides; preferably, a single domain antibody comprising 2 targeting PRAME polypeptides; more preferably, the 2 single domain antibodies targeting the PRAME polypeptide form a single domain antibody dimer targeting the PRAME polypeptide.

15. The bispecific antibody of claim 11, wherein the bispecific antibody has the sequence shown in the following table:

Chain A Chain B Chain C SEQ ID NO:68 SEQ ID NO:69 SEQ ID NO:70 SEQ ID NO:71 SEQ ID NO:72 SEQ ID NO:73 SEQ ID NO:74 SEQ ID NO:75 SEQ ID NO:76 SEQ ID NO:77 SEQ ID NO:78 SEQ ID NO:79 SEQ ID NO:80 SEQ ID NO:81 SEQ ID NO:82 SEQ ID NO:83 SEQ ID NO:84 SEQ ID NO:85 SEQ ID NO:86 SEQ ID NO:87 SEQ ID NO:88 SEQ ID NO:89 SEQ ID NO:90 SEQ ID NO:91 SEQ ID NO:92 SEQ ID NO:93 SEQ ID NO:94 SEQ ID NO:95 SEQ ID NO:96 SEQ ID NO:97

。

16. A fusion protein comprising the VHH chain of a single domain antibody targeting a PRAME polypeptide of any one of claims 1-3, the heavy chain variable region of an antibody targeting a PRAME polypeptide of claim 4 or 5, a single domain antibody targeting a PRAME polypeptide of claim 6, the humanized VHH chain of a single domain antibody targeting a PRAME polypeptide of claim 7 or 8, or an antibody targeting a PRAME polypeptide of claim 9 or 10, optionally a linker sequence and an Fc fragment or half-life extending domain of an immunoglobulin.

17. The fusion protein of claim 16, wherein the immunoglobulin is IgG1, igG2, igG3, igG4; igG4 is preferred.

18. A chimeric antigen receptor made from the VHH chain of the single domain antibody targeting a PRAME polypeptide of any one of claims 1-3, the heavy chain variable region of the antibody targeting a PRAME polypeptide of claim 4 or 5, the single domain antibody targeting a PRAME polypeptide of claim 6, the humanized VHH chain of the single domain antibody targeting a PRAME polypeptide of claim 7 or 8, or the antibody targeting a PRAME polypeptide of claim 9 or 10.

19. The chimeric antigen receptor according to claim 18, wherein the amino acid sequence of the chimeric antigen receptor is selected from the group consisting of: 98, 99, 100, 101 and 102.

20. An immune effector cell that expresses the chimeric antigen receptor of claim 18 or 19.

21. The immune effector cell of claim 20, wherein the immune effector cell comprises, but is not limited to: t cells, NK cells, TIL cells; t cells are preferred.

22. A nucleic acid molecule encoding 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 or a chimeric antigen receptor according to claim 18 or 19.

23. An expression vector comprising the nucleic acid molecule of claim 22.

24. A host cell comprising the expression vector of claim 23, or having integrated on its genome the nucleic acid molecule of claim 22.

25. A method of making a VHH chain of a single domain antibody that targets a PRAME polypeptide of any one of claims 1-3, a heavy chain variable region of an antibody that targets a PRAME polypeptide of claim 4 or 5, a single domain antibody that targets a PRAME polypeptide of claim 6, a humanized single domain antibody that targets a PRAME polypeptide of claim 7 or 8, an antibody that targets a PRAME polypeptide of claim 9 or 10, a bispecific antibody of any one of claims 11-15, a fusion protein of claim 16 or 17, or a chimeric antigen receptor of claim 18 or 19, the method comprising the steps of:

1) Culturing the host cell of claim 24 under suitable conditions, thereby obtaining a culture comprising 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 bispecific antibody, the fusion protein or the chimeric antigen receptor; and

2) Optionally, isolating or recovering from the culture 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 bispecific antibody, the fusion protein or the chimeric antigen receptor.

26. An immunoconjugate, the immunoconjugate comprising:

1) A VHH chain of a single domain antibody targeting a PRAME polypeptide according to any one of claims 1-3, 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 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 11-15 or a fusion protein according to claim 16 or 17; and

27. 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 1-3, 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 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 11-15, 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.

28. The pharmaceutical composition of claim 27, wherein the pharmaceutical composition is for use in treating a tumor, the tumor being a PRAME polypeptide-related tumor; melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, and the like are preferred.

29. Use of a VHH chain of a single domain antibody targeting a PRAME polypeptide according to any one of claims 1-3, 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 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 11-15, 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, an immunoconjugate according to claim 26 or a pharmaceutical composition according to claim 27 or 28 for the preparation of:

1) Reagents for detecting PRAME polypeptides;

2) An agent that blocks binding of the PRAME polypeptide to PD-L1;

3) A medicine for treating tumor.

30. The use of claim 29, wherein the tumor is a PRAME polypeptide-related tumor; melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, and the like are preferred.

31. A kit, comprising:

1) A VHH chain of a single domain antibody that targets a PRAME polypeptide according to any one of claims 1-3, a heavy chain variable region of an antibody that targets a PRAME polypeptide according to claim 4 or 5, a single domain antibody that targets a PRAME polypeptide according to claim 6, a humanized single domain antibody that targets a PRAME polypeptide according to claim 7 or 8, an antibody that targets a PRAME polypeptide according to claim 9 or 10, a bispecific antibody according to any one of claims 11-15, a fusion protein according to any one of claims 16 or 17, a chimeric antigen receptor according to claim 18 or 19, an immune effector cell according to claim 20 or 21, an immunoconjugate according to claim 26 or a pharmaceutical composition according to claim 27 or 28;

2) A container; and

3) Optionally instructions for use.

32. A method of detecting PRAME polypeptide proteins in a sample, the method comprising the steps of:

1) Contacting a test sample with a VHH chain of a single domain antibody that targets a PRAME polypeptide according to any one of claims 1-3, a heavy chain variable region of an antibody that targets a PRAME polypeptide according to claim 4 or 5, a single domain antibody that targets a PRAME polypeptide according to claim 6, a humanized single domain antibody that targets a PRAME polypeptide according to claim 7 or 8, an antibody that targets a PRAME polypeptide according to claim 9 or 10, a bispecific antibody according to any one of claims 11-15, a fusion protein according to claim 16 or 17, or an immunoconjugate according to claim 26;