Single-domain antibodies targeting PRAME polypeptides and their uses

Abstract

The present invention discloses a series of single-domain antibodies targeting PRAME polypeptides. The single-domain antibodies of the PRAME polypeptides of the present invention can bind to the PRAME polypeptides with high affinity and have good specificity, thus laying a new material basis for the development of anti-tumor drugs targeting PRAME polypeptides. The present invention also discloses bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs prepared using the single-domain antibodies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to single-domain antibodies targeting PRAME polypeptides and their uses. Background Art

[0002] PRAME (Preferentially expressed Antigen in MElanoma) is an intracellular protein encoded by the PRAME gene. PRAME is highly expressed 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. These characteristics make PRAME an ideal target for tumor-targeted therapy (Am J Surg Pathol. 2018 Nov; 42(11): 1456-1465.). After being processed by the intracellular antigen presentation system, the PRAME protein has a special polypeptide sequence: SLLQHLIGL, which can be presented to the cell surface by the major histocompatibility antigen HLA-A02 molecule. The HLA-A02 complex binding to the PRAME polypeptide can serve as a cell membrane target to develop therapies related to T cell receptor (TCR) or TCR-like antibodies.

[0003] Single-domain antibody (sdAb) is a special antibody with a relatively small molecular weight. A single-domain antibody consists of only two identical heavy chains. Compared with the molecular weight of traditional double-chain antibodies of 150-160 kDa, the molecular weight of a single-domain antibody is about 110 KD. Single-domain antibodies generally have high specificity, high affinity, low immunogenicity, and good permeability. The antigen-binding region of a single-domain antibody consists of only one chain, and the variable region (VHH) of a single-domain antibody is only 12-15 kDa. Therefore, it is very simple to modify the structure of a single-domain antibody, and there will be no problem of heavy and light chain mispairing in traditional double-chain antibodies, nor will there be a decrease in affinity caused by modifying the single chain of the antigen-binding region. Based on these advantages, using single-domain antibodies as the antigen recognition region of bispecific antibodies or chimeric antigen receptor T cells (CAR-T) is one of the future development trends (Serge Muyldermans. Annu. Rev. Biochem. 82: 775-797 (2013)). Single-domain antibodies can recognize cell membrane proteins or polypeptides derived from intracellular proteins presented to the cell surface by major histocompatibility antigens. Using the HLA-A02 complex binding to the PRAME polypeptide as an antigen, specific single-domain antibody molecules can be screened. These candidate molecules can be used to develop biologics such as bispecific antibodies, CAR-T, or ADC (Antibody drug conjugate). Summary of the Invention

[0004] The object of the present invention is to provide a specific single-domain antibody targeting the PRAME polypeptide, as well as corresponding specific humanized single-domain antibodies, bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs targeting the PRAME polypeptide, etc.

[0005] The object of the present invention is also to provide the use of the above-mentioned single-domain antibody, humanized single-domain antibody, bispecific antibody, chimeric antigen receptor, chimeric antigen receptor-T cell, and ADC in the treatment of tumors or the preparation of drugs for treating tumors.

[0006] In a first aspect, the present invention provides a VHH chain of a single-domain antibody targeting the PRAME polypeptide, and the VHH chain comprises CDR1, CDR2, and CDR3 shown in the following table:

[0007] 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 。

[0008] In a preferred embodiment, the amino acid sequence of the PRAME polypeptide is: SLLQHLIGL (SEQ ID NO: 121).

[0009] In a preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence optionally added, deleted, modified, and / or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and capable of retaining high-affinity binding to the PRAME polypeptide.

[0010] In a preferred embodiment, the VHH chain further comprises framework regions FR1, FR2, FR3, and FR4.

[0011] In a preferred embodiment, the amino acid sequence of the VHH chain of the single-domain antibody targeting the PRAME polypeptide is shown in the following table:

[0012] 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 。

[0013] In a second aspect, the present invention provides a heavy chain variable region of an antibody targeting the PRAME polypeptide, and the heavy chain variable region comprises CDR1, CDR2, and CDR3 shown in the following table

[0014] 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 。

[0015] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody targeting the PRAME polypeptide is shown in the following table:

[0016] 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 。

[0017] In a third aspect, the present invention provides a single-domain antibody targeting a PRAME polypeptide, which has the VHH chain described in the first aspect.

[0018] In a fourth aspect, the present invention provides a VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide, and the framework regions FR1, FR2, FR3, and FR4 are humanized based on the VHH chain described in the first aspect.

[0019] 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:

[0020] 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。

[0021] In a fifth aspect, the present invention provides an antibody targeting a PRAME polypeptide, and the antibody comprises one or more VHH chains of the single-domain antibody targeting a PRAME polypeptide described in the first aspect or the VHH chain of the humanized single-domain antibody targeting a PRAME polypeptide described in claim 4.

[0022] In a preferred embodiment, the antibody targeting a PRAME polypeptide comprises a monomer, a bivalent antibody, and / or a multivalent antibody.

[0023] In a sixth aspect, the present invention provides a bispecific antibody, which comprises a first antibody and a second antibody. The first antibody comprises the VHH chain of the single-domain antibody targeting a PRAME polypeptide described in the first aspect, or the heavy chain variable region of the antibody targeting a PRAME polypeptide described in the second aspect, or the single-domain antibody targeting a PRAME polypeptide described in the third aspect, the VHH chain of the humanized single-domain antibody targeting a PRAME polypeptide described in the fourth aspect, or the antibody targeting a PRAME polypeptide described in the fifth aspect.

[0024] In a preferred embodiment, the second antibody can bind to the same or different antigens as the first antibody, or bind to different epitopes of the same antigen as the first antibody.

[0025] In a preferred embodiment, the second antibody is a single-domain antibody, a single-chain antibody, or a double-chain antibody.

[0026] In a preferred embodiment, the bispecific antibody comprises 2-4 single-domain antibodies targeting a PRAME polypeptide; preferably, it comprises 2 single-domain antibodies targeting a PRAME polypeptide; more preferably, the 2 single-domain antibodies targeting a PRAME polypeptide form a dimer of the single-domain antibody targeting a PRAME polypeptide.

[0027] In a preferred embodiment, the sequence of the bispecific antibody is shown in the following table:

[0028]

[0029]

[0030] In a seventh aspect, the present invention provides a fusion protein, which comprises a VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in the first aspect, a heavy-chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, a single-domain antibody targeting a PRAME polypeptide as described in the third aspect, a VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide as described in the fourth aspect, or an antibody targeting a PRAME polypeptide as described in the fifth aspect, an optional linker sequence, and an Fc fragment of an immunoglobulin or a half-life extension domain.

[0031] In a preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3, IgG4; preferably IgG4.

[0032] In an eighth aspect, the present invention provides a chimeric antigen receptor, which is made of a VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in the first aspect, a heavy-chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, a single-domain antibody targeting a PRAME polypeptide as described in the third aspect, a VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide as described in the fourth aspect, or an antibody targeting a PRAME polypeptide as described in the fifth aspect.

[0033] In a preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown as follows:

[0034] SEQ ID NO:98 SEQ ID NO:99 SEQ ID NO:100 SEQ ID NO:101 SEQ ID NO:102。

[0035] In a ninth aspect, the present invention provides an immune effector cell, which expresses the chimeric antigen receptor as described in the eighth aspect.

[0036] In a preferred embodiment, the immune effector cell includes but is not limited to: T cells, NK cells, TIL cells; preferably T cells.

[0037] In a tenth aspect, the present invention provides a nucleic acid molecule encoding a VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in the first aspect, a heavy-chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, a single-domain antibody targeting a PRAME polypeptide as described in the third aspect, a VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide as described in the fourth aspect, an antibody targeting a PRAME polypeptide as described in the fifth aspect, a bispecific antibody as described in the sixth aspect, a fusion protein as described in the seventh aspect, or a chimeric antigen receptor as described in the eighth aspect.

[0038] In an eleventh aspect, the present invention provides an expression vector comprising the nucleic acid molecule as described in the tenth aspect.

[0039] In a twelfth aspect, the present invention provides a host cell comprising the expression vector as described in the eleventh aspect, or having the nucleic acid molecule as described in the tenth aspect integrated into its genome.

[0040] In a thirteenth aspect, the present invention provides a method for preparing a VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in the first aspect, a heavy-chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, a single-domain antibody targeting a PRAME polypeptide as described in the third aspect, a VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide as described in the fourth aspect, an antibody targeting a PRAME polypeptide as described in the fifth aspect, a bispecific antibody as described in the sixth aspect, or a fusion protein as described in the seventh aspect, the method comprising the following steps:

[0041] 1) Culturing the host cell as described in the eleventh aspect under suitable conditions to obtain a culture containing the VHH chain of the single-domain antibody targeting the PRAME polypeptide, the heavy-chain variable region of the antibody targeting the PRAME polypeptide, the single-domain antibody targeting the PRAME polypeptide, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide, the antibody targeting the PRAME polypeptide, the bispecific antibody, or the fusion protein; and

[0042] 2) Optionally, separating or recovering the VHH chain of the single-domain antibody targeting the PRAME polypeptide, the heavy-chain variable region of the antibody targeting the PRAME polypeptide, the single-domain antibody targeting the PRAME polypeptide, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide, the single-domain antibody targeting the PRAME polypeptide, the bispecific antibody, or the fusion protein from the culture.

[0043] In a fourteenth aspect, the present invention provides an immunoconjugate comprising:

[0044] 1) The VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in the first aspect, the heavy-chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, the single-domain antibody targeting a PRAME polypeptide as described in the third aspect, the VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide as described in the fourth aspect, the antibody targeting a PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, or the fusion protein as described in the seventh aspect; and

[0045] 2) A conjugate moiety selected from: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0046] In a preferred embodiment, the conjugate moiety is a drug or a toxin.

[0047] In a preferred embodiment, the immunoconjugate is an Antibody-Drug-Conjugate (ADC).

[0048] In a preferred embodiment, the conjugate moiety is a detectable label.

[0049] In a preferred embodiment, the conjugate is selected from: a fluorescent or luminescent label, a radioactive label, an MRI (Magnetic Resonance Imaging) or CT (Computed Tomography) contrast agent, or an enzyme, radionuclide, biotoxin, cytokine (such as IL-2, etc.), antibody, antibody Fc fragment, antibody scFv fragment, gold nanoparticle / nanorod, virus particle, liposome, magnetic nanoparticle, prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0050] In a preferred embodiment, the immunoconjugate contains: a multivalent (such as divalent) VHH chain of a single-domain antibody targeting a PRAME polypeptide as described in the first aspect, the heavy-chain variable region of an antibody targeting a PRAME polypeptide as described in the second aspect, the single-domain antibody targeting a PRAME polypeptide as described in the third aspect, the VHH chain of a humanized single-domain antibody targeting a PRAME polypeptide as described in the fourth aspect, the antibody targeting a PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, or the fusion protein as described in the seventh aspect.

[0051] In a preferred embodiment, the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeated portions.

[0052] In a fifteenth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically or diagnostically effective amount of the VHH chain of the single-domain antibody targeting the PRAME polypeptide as described in the first aspect, the heavy-chain variable region of the antibody targeting the PRAME polypeptide as described in the second aspect, the single-domain antibody targeting the PRAME polypeptide as described in the third aspect, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide as described in the fourth aspect, the antibody targeting the PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, the fusion protein as described in the seventh aspect, the chimeric antigen receptor as described in the eighth aspect, the immune effector cell as described in the ninth aspect or the immunoconjugate as described in the fourteenth aspect, and optionally a pharmaceutically acceptable excipient.

[0053] In a preferred embodiment, the pharmaceutical composition is used for treating tumors, which are PRAME polypeptide-related tumors; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

[0054] In a sixteenth aspect, the present invention provides the use of the VHH chain of the single-domain antibody targeting the PRAME polypeptide as described in the first aspect, the heavy-chain variable region of the antibody targeting the PRAME polypeptide as described in the second aspect, the single-domain antibody targeting the PRAME polypeptide as described in the third aspect, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide as described in the fourth aspect, the antibody targeting the PRAME polypeptide as described in the fifth aspect, the bispecific antibody as described in the sixth aspect, the fusion protein as described in the seventh aspect, the chimeric antigen receptor as described in the eighth aspect, the immune effector cell as described in the ninth aspect or the immunoconjugate as described in the fourteenth aspect for preparing the following reagents:

[0055] 1) A reagent for detecting the PRAME polypeptide;

[0056] 2) A reagent for blocking the binding of the PRAME polypeptide to PD-L1;

[0057] 3) A drug for treating tumors.

[0058] In a preferred embodiment, the tumor is a PRAME polypeptide-related tumor; preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

[0059] In a seventeenth aspect, the present invention provides a kit, which includes:

[0060] 1) The VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the heavy-chain variable region of the antibody targeting the PRAME polypeptide according to the second aspect, the single-domain antibody targeting the PRAME polypeptide according to the third aspect, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide according to the fourth aspect, the antibody targeting the PRAME polypeptide according to the fifth aspect, the bispecific antibody according to the sixth aspect, the fusion protein according to the seventh aspect, the chimeric antigen receptor according to the eighth aspect, the immune effector cell according to the ninth aspect, the immunoconjugate according to the fourteenth aspect, or the pharmaceutical composition according to the fifteenth aspect;

[0061] 2) A container; and

[0062] 3) Optionally, an instruction manual.

[0063] In the eighteenth aspect, the present invention provides a method for detecting PRAME polypeptide protein in a sample, the method comprising the steps of:

[0064] 1) Contacting the sample to be tested with the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the heavy-chain variable region of the antibody targeting the PRAME polypeptide according to the second aspect, the single-domain antibody targeting the PRAME polypeptide according to the third aspect, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide according to the fourth aspect, the antibody targeting the PRAME polypeptide according to the fifth aspect, the bispecific antibody according to the sixth aspect, the fusion protein according to the seventh aspect, or the immunoconjugate according to the fourteenth aspect;

[0065] 2) Detecting whether an antigen-antibody complex is formed, and if a complex is formed, it indicates the presence of PRAME polypeptide protein in the sample.

[0066] In the nineteenth aspect, the present invention provides a method for treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the VHH chain of the single-domain antibody targeting the PRAME polypeptide according to the first aspect, the heavy-chain variable region of the antibody targeting the PRAME polypeptide according to the second aspect, the single-domain antibody targeting the PRAME polypeptide according to the third aspect, the VHH chain of the humanized single-domain antibody targeting the PRAME polypeptide according to the fourth aspect, the antibody targeting the PRAME polypeptide according to the fifth aspect, the bispecific antibody according to the sixth aspect, the fusion protein according to the seventh aspect, the chimeric antigen receptor according to the eighth aspect, the immune effector cell according to the ninth aspect, the immunoconjugate according to the fourteenth aspect, or the pharmaceutical composition according to the fifteenth aspect.

[0067] In a preferred embodiment, the subject includes mammals; preferably humans.

[0068] In a preferred embodiment, the disease is a disease related to PRAME polypeptide; preferably a tumor related to PRAME polypeptide; more preferably melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc.

[0069] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is shown that all 14 single-domain antibodies of the present invention can bind to the protein antigen and have high affinity;

[0071] Figure 2 It is shown that all 14 single-domain antibodies of the present invention can bind to the antigen at the cellular level and have high affinity;

[0072] Figure 3 It is shown that the single-domain antibody molecules of the present invention have good specificity;

[0073] Figure 4 It is shown that the bispecific antibody molecules of the present invention have good cell killing activity;

[0074] Figure 5 It is shown that the CAR molecules of the present invention can be highly expressed on the surface of T cells;

[0075] Figure 6 It is shown that the candidate CAR-T cells have a killing effect on target cells under different effector-to-target ratios;

[0076] Figure 7 It is shown that the CAR-T cells of the present invention can all effectively recognize T2 loaded with the target polypeptide, activate and transmit immune signals, and secrete IFN-γ cytokine;

[0077] Figure 8 It is shown that the humanized single-domain antibodies obtained by humanizing the LL-PR001 molecule can bind to the protein antigen and have high affinity;

[0078] Figure 9 It is shown that the humanized single-domain antibodies obtained by humanizing the LL-PR004 molecule can bind to the protein antigen and have high affinity. DETAILED DESCRIPTION OF THE INVENTION

[0079] After extensive and in-depth research, the inventors unexpectedly discovered a class of single-domain antibodies targeting PRAME polypeptides. The single-domain antibodies of the present invention can bind to PRAME polypeptides with high affinity and have good specificity. The present invention also provides bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs prepared using the above single-domain antibodies. Based on this, the present invention was completed.

[0080] Term Definitions

[0081] The terms used herein have the same or similar meanings as those conventionally understood by those skilled in the art. For clarity, some of the terms are defined as follows.

[0082] Single-domain antibody

[0083] In this article, "single-domain antibody", "single-structured domain antibody", "nanobody", etc. have the same or similar meanings, and all refer to a class of antibody molecules that lack the light chain of the antibody and only have the variable region of the heavy chain. The single-domain antibody is the smallest antigen-binding unit, that is, the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody that is naturally lacking in the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is then cloned to construct a single-domain antibody (VHH) composed of only one variable region of the heavy chain.

[0084] In the VHH chain of the single-domain antibody targeting PRAME polypeptides of the present invention, there are also framework regions FR1, FR2, FR3, and FR4.

[0085] Based on the VHH chain of the single-domain antibody targeting PRAME polypeptides of the present invention, the inventors also humanized the VHH chain to obtain the VHH chain of the humanized single-domain antibody targeting PRAME polypeptides.

[0086] Based on the VHH chain of the single-domain antibody targeting PRAME polypeptides or the humanized VHH chain of the present invention, the present invention also provides an antibody targeting PRAME polypeptides, which includes one or more of the VHH chains of the single-domain antibodies targeting PRAME polypeptides or the VHH chains of the humanized single-domain antibodies targeting PRAME polypeptides. The present invention also provides a bispecific antibody, which includes a first antibody and a second antibody. The first antibody can be the VHH chain of the single-domain antibody targeting PRAME polypeptides of the present invention or the humanized VHH chain. Those skilled in the art can select the second antibody in the bispecific antibody according to actual needs. For example, the second antibody can bind to the same or different antigens as the first antibody; if the second antibody binds to the same antigen as the first antibody, it is preferably bound to a different epitope. In a specific embodiment, the second antibody can be a single-domain antibody, a single-chain antibody, or a double-chain antibody.

[0087] Those skilled in the art can also prepare a fusion protein from the single-domain antibody VHH chain or humanized VHH chain targeting the PRAME polypeptide of the present invention, for example, a fusion protein further comprising an Fc fragment of an immunoglobulin or a half-life extension domain. The fusion protein obtained in this way not only has the biological activity of the single-domain antibody VHH chain itself, but also can have other characteristics conferred by the Fc fragment of the immunoglobulin, such as an extended plasma half-life, reduced immunogenicity, improved stability, and so on. In a specific embodiment, the fusion protein comprises the VHH chain or humanized VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, an optional linker sequence, and an Fc fragment of an immunoglobulin. In a specific embodiment, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4; preferably IgG4. In a specific embodiment, the half-life extension domain is shown by the following amino acid sequence: QVQLVESGGGVVQPGGSLRLSCAASGFAFRGFGMSWVRQAPGKGLEWVSSINNGGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGGPGASPSGQGTQVTVSS (SEQ ID NO: 121).

[0088] The present invention not only includes intact antibodies, but also fragments, derivatives, and analogs of the antibodies. As used herein, the terms "fragment", "derivative", and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention can be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0089] The antibody of the present invention refers to a polypeptide having the binding activity to PRAME polypeptide protein and including the above CDR regions. This term also includes variant forms of the polypeptide containing the above CDR regions and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar or close properties, the function of the protein usually will not be changed. Also, for example, adding one or several amino acids at the C-terminus and / or N-terminus usually will not change the function of the protein. This term also includes active fragments and active derivatives of the antibody of the present invention. The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained by using the antiserum against the antibody of the present invention.

[0090] In addition to the almost full-length polypeptide, the present invention also includes fragments of the single-domain antibody of the present invention. Generally, this fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids.

[0091] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids with amino acids having similar or close properties compared with the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to the following table.

[0092]

[0093]

[0094] The present invention also provides a polynucleotide molecule encoding the above antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0095] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding this polypeptide, or may also be a polynucleotide that further includes additional coding and / or non-coding sequences. The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides described in the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more. And, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0096] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination, or artificial synthesis methods. A feasible method is to use artificial synthesis to synthesize the relevant sequence, especially when the fragment length is short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination methods. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in an isolated form.

[0097] Currently, it is already possible to completely obtain the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0098] The present invention also relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins. The host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells such as Escherichia coli, Streptomyces; Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0099] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with CaCl 2 method, and the steps used are well known in the art. Another method is to use MgCl 2 . If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0100] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0101] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0102] The antibodies of the present invention can be used alone or conjugated or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modification moieties, or combinations of any of the above substances. Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0103] Therapeutic agents that can be conjugated or coupled with the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines, such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnets; 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 nanoparticles, etc.

[0104] Bispecific antibodies

[0105] Bispecific antibodies are recombinant antibodies engineered by protein engineering. Bispecific antibodies can simultaneously target two different antibody-binding epitopes, which can be from different antigens or from the same antigen. Many current studies have shown that bispecific antibodies have great therapeutic potential in treating diseases such as cancer, autoimmune diseases, and viral infections. Compared with monoclonal antibodies, the main advantage of bispecific antibodies is their ability to mediate the spatial effects of two recognition epitopes and the synergistic effect of dual targeting, resulting in biological effects that cannot be achieved by using two antibodies in combination. A special type of bispecific antibody is called a T cell engager, which can activate endogenous T cells by simultaneously binding to a target on the surface of tumor cells and T cells, leading to the lysis of tumor cells, thereby achieving the purpose of treating cancer. T cell engagers have been proven to be useful in treating cancer. Bispecific T cell engagers targeting CD20 and CD19 have been approved by the FDA for marketing (Nat Rev Clin Oncol. 2020 Jul; 17(7): 418-434.). Due to their complexity different from monoclonal antibodies, bispecific antibodies have higher technical thresholds and R & D costs.

[0106] immune cell

[0107] In this article, immune cells and immune effector cells have the same meaning and are the same as those commonly understood by those skilled in the art. They refer to cells involved in or related to immune responses, including lymphocytes and phagocytes. In a specific embodiment, the immune cells refer to lymphocytes that can recognize antigens and thus produce specific immune responses. The lymphocytes are mainly T lymphocytes, B lymphocytes, K lymphocytes, and NK lymphocytes. In addition to lymphocytes, cells involved in immune responses also include plasma cells, granulocytes, mast cells, antigen-presenting cells, and cells of the mononuclear phagocyte system (such as macrophages).

[0108] chimeric antigen receptor T cell

[0109] Chimeric antigen receptor T cell therapy is a promising cellular immunotherapy. CAR-T cells express the CAR (Chimeric Antigen Receptor) molecule, and the structure of CAR is divided into: antigen-binding region, hinge region, transmembrane domain, and intracellular signaling domain. Currently, CAR-T cells usually use the scFv (Single Chain Fragment Variables) segment, which is a single-chain modification of the antigen-binding region of monoclonal antibodies, as the antigen-binding region. However, when modifying the scFv structure, problems such as reduced affinity and altered specificity are likely to occur. At the same time, the molecular weight of scFv is relatively large and it is prone to form multimers, which affects the function of CAR (Nat Rev Cancer. 2021 Mar; 21(3): 145-161.). Therefore, CAR with an antigen-binding region of novel structure. Using single-domain antibodies to construct the CAR structure, the variable region of the single-domain antibody can be directly linked to the CAR structure, and the design is simple and convenient.

[0110] Immunoconjugate

[0111] An ADC is an antibody carrying a cytotoxic drug, which can specifically deliver the cytotoxic drug to tumor cells to achieve specific killing of tumor cells. ADC drugs targeting targets such as HER2, CD30, and Trop2 have shown good clinical efficacy and safety in clinical studies (Nat Rev Clin Oncol. 2021 Jun; 18(6): 327-344.). In recent years, several ADC drugs have been approved by the FDA for marketing. However, the current targets of ADC drugs are all cell membrane proteins, and ADC drugs targeting intracellular proteins are one of the hot directions for future development.

[0112] ADC drugs can specifically target target cells and deliver cytotoxic molecules into target cells, thereby producing a specific killing effect. The cell killing induced by cytotoxic molecules can break and improve the tumor suppressive microenvironment, and has the potential to enhance the efficacy of other therapies such as immunotherapy. The single-domain antibody of the present invention can be internalized into cells, so there is hope to develop ADC drugs based on the single-domain antibody of the present invention in the future.

[0113] The present invention also provides an immunoconjugate, which contains the VHH chain of the single-domain antibody targeting the PRAME polypeptide of the present invention, the humanized VHH chain, etc., and a conjugate part. In a specific embodiment, the conjugate part can be a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme, etc., so as to achieve purposes such as diagnosis, detection, or treatment.

[0114] In a preferred embodiment, the immunoconjugate is an antibody-drug conjugate (ADC).

[0115] Drug composition

[0116] The present invention also provides a composition. Preferably, the composition is a drug composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substances to be formulated and the disease to be treated. The formulated drug composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0117] The drug composition of the present invention can directly target the PRAME polypeptide expressed by tumor cells. Therefore, the drug composition of the present invention can be used to treat tumors. In a specific embodiment, the tumor is a PRAME polypeptide-related tumor. In a preferred embodiment, the tumor is melanoma, non-small cell lung cancer, ovarian cancer, breast cancer, etc. In addition, the drug composition of the present invention can also be used in combination with other therapeutic agents.

[0118] The drug composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the single-domain antibody (or its conjugate) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such carriers include (but not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and their combinations. The drug preparation should match the administration method. The drug composition of the present invention can be made into an injectable form, for example, prepared by a conventional method with physiological saline or an aqueous solution containing glucose and other adjuvants. Drug compositions such as injectables and solutions should be manufactured under sterile conditions.

[0119] The dosage of the active ingredient is a therapeutically effective amount, such as about 10 micrograms per kilogram of body weight per day - about 50 milligrams per kilogram of body weight. When using the drug composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight - about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also consider factors such as the administration route and the patient's health status, which are within the scope of the skills of a skilled physician.

[0120] Kit

[0121] The present invention also provides a kit containing the VHH chain of a single-domain antibody targeting the PRAME polypeptide of the present invention, or a humanized VHH chain, an antibody, a fusion protein, an immunoconjugate, etc. In a specific embodiment, the kit further includes a container, an instruction manual, a buffer, etc.

[0122] Advantages of the present invention:

[0123] 1. The single-domain antibody targeting the PRAME polypeptide of the present invention binds to the PRAME polypeptide with high affinity;

[0124] 2. The single-domain antibody targeting the PRAME polypeptide of the present invention has good specificity;

[0125] 3. Using the single-domain antibody targeting the PRAME polypeptide of the present invention, bispecific antibodies, chimeric antigen receptors, chimeric antigen receptor-T cells, and ADCs, etc. can be further prepared, thereby laying a new material foundation for the development of therapeutic or diagnostic drugs targeting the PRAME polypeptide.

[0126] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0127] Example 1: Obtaining the single-domain antibody sequence

[0128] Two adult and healthy alpacas were selected as experimental animals for this experiment and immunized. The recombinant pMHC (peptide-Major histocompatibility complex) protein carrying the target polypeptide was used as an antigen to immunize the alpacas. A total of 4-5 immunizations were performed. The peripheral blood of the alpacas was collected at different time points to detect the immune titer. After the immunization, the peripheral blood of the alpacas was collected, the mRNA of peripheral blood PBMCs cells was extracted and reverse transcribed to obtain cDNA. Specific primers were used to amplify the cDNA to obtain PCR products with single-domain antibody gene fragments. Then, the PCR products and yeast library vectors were introduced into yeast competent cells by electroporation to prepare a yeast library. The yeast library was screened using the recombinant expressed protein antigen or T2 cells loaded with target polypeptides. In this invention, the pMHC recombinant protein carrying the target polypeptide was used as a positive antigen, and the pMHC recombinant protein carrying a negative polypeptide was used as a negative antigen. After three rounds of positive screening and three rounds of negative screening, single-domain antibody clones that specifically bind to the positive antigen but not to the negative antigen were obtained.

[0129] The single-domain antibody clones were subjected to Sanger sequencing to obtain the full-length sequences of the single-domain antibodies that specifically bind to the target protein in this invention. A total of 14 different single-domain antibody clones were obtained in this invention, which were named LL-PR001 - LL-PR014 respectively. The full-length amino acid sequences of the single-domain antibodies and the amino acid sequences of the CDR regions are shown in Table 1.

[0130] Table 1. Amino acid sequence table of candidate single-domain antibodies

[0131]

[0132]

[0133]

[0134] Example 2: Expression and purification of single-domain antibodies

[0135] In this invention, the single-domain antibodies in this invention were expressed and purified using the expression and purification methods of conventional monoclonal antibodies. First, a single-domain antibody expression vector was constructed using conventional molecular cloning techniques. After the successful construction of the vector, the above single-domain antibodies were expressed by transiently transfecting the HEK293 suspension cell line. Basic steps: The single-domain antibody gene was cloned into the expression vector pCDNA4 (Invitrogen, Cat V86220). Different single-domain antibodies were expressed by transiently transfecting the HEK293 suspension cells. After the expression was completed, the supernatant was collected, and the single-domain antibodies in this invention were purified using conventional monoclonal antibody purification methods, and finally purified single-domain antibodies were obtained.

[0136] After the purification of single-domain antibodies, the protein concentration and total protein amount of different antibodies were detected by ultraviolet spectrophotometry, and then the expression amount of each bispecific antibody was calculated according to the expression volume. The absorbance value A280 of the sample solution was read at a wavelength of 280 nm using a NanoDrop 1000, and the protein concentration of the sample was calculated by the formula C (mg / mL) = A280 / ε (ε is 1.482 mL / mg·cm-1). At the same time, the purity of different antibodies was evaluated by the conventional gel electrophoresis SDS-PAGE method. The basic steps are as follows: The sample was electrophoretically separated using an electrophoresis tank from Invitrogen and an SDS-PAGE gradient gel. The sample was diluted to about 1 mg / mL, appropriate reducing agent, loading buffer and pure water were added, and after mixing, it was heated at 70 °C for about 10 minutes. The sample loading amount was 2 - 10 μg, the electrophoresis voltage was about 200 V, the electrophoresis time was about 35 minutes, and then gel staining and decolorization were carried out respectively. After decolorization, the gel was photographed using a conventional gel imaging system and the purity of the main band was analyzed and calculated. In addition, the purity of different antibodies was evaluated by size exclusion high performance liquid chromatography (SEC-HPLC method). The basic steps are as follows: The sample was diluted to about 1.0 mg / mL, a TSKgel G3000SWXL chromatographic column was used, the column temperature was set at 25 °C, 100 mM phosphate buffer, 100 mM sodium sulfate, pH 7.0 ± 0.2 was used as the mobile phase, the injection volume was 20 - 50 μL, and isocratic elution was carried out for 20 min at a flow rate of 1.0 mL / min, and detection was carried out at a wavelength of 280 nm. The content of monomers was obtained by the peak area normalization method.

[0137] The expression amount and purity information of different single-domain antibodies are shown in Table 2. The expression amount of the single-domain antibodies of the present invention using the transient expression system is in the range of 650 - 850 mg / L, which proves that the bispecific antibody structure of the present invention has a high expression amount. The purity of the reduced SDS-PAGE and SEC-HPLC of most single-domain antibodies of the present invention is above 95%. These results prove that the single-domain structure of the present invention has a good expression amount and high purity.

[0138] Table 2. Detection results of the expression amount and purity of single-domain antibodies

[0139]

[0140] Example 3: Detection of the binding ability of single-domain antibodies to the target

[0141] In order to explore the binding ability of the candidate single-domain antibodies of the present invention to the antigen, in this example, ELISA and flow cytometry were respectively used to detect at the protein and cell levels, so as to comprehensively compare the affinity levels of the candidate molecules for the target antigen. The specific operation steps are as follows:

[0142] 3.1 Detection of the binding ability at the protein level

[0143] 1) Dilute SA protein with PBS to 0.3 μg / mL, add 100 μL per well to a 96-well transparent ELISA plate, seal the plate with a sealing film, and incubate at 37 °C for 1 h;

[0144] 2) Discard the protein, and wash the plate three times with 200 μL of PBST solution (PBS containing 0.05% Tween 20);

[0145] 3) Add the blocking solution (PBS solution containing 2% BSA) at 100 μL per well, seal the plate with a sealing film, and incubate at 37 °C for 1 h;

[0146] 4) Repeat the plate washing step 2);

[0147] 5) Dilute the biotin-labeled MHC-PRAME polypeptide complex with the blocking solution to 2 μg / ml, add 100 μL of the dilution to each well, seal the plate with a sealing film, and incubate at 37 °C for 1 h;

[0148] 6) Repeat the plate washing step 2);

[0149] 7) Dilute the candidate antibody with the blocking solution to 12 different concentrations, with the highest concentration being 0.3 μg / mL, diluted in a 3-fold gradient, add 100 μL of the dilution to each well, seal the plate with a sealing film, and incubate at 37 °C for 1 h;

[0150] 8) Repeat the plate washing step 2);

[0151] 9) Dilute SA-HRP with the blocking solution at a ratio of 1:1000, add 100 μL to each well, seal the plate with a sealing film, and incubate at 37 °C for 30 min;

[0152] 10) Repeat the plate washing step 2);

[0153] 11) Add 100 μL of TMB reaction solution to each well, seal the plate with a sealing film, react on a microplate oscillator for 10 min, then add 100 μL of 2N H 2 SO4 solution to terminate the reaction, and read the absorbance value at 450 nm with an ELISA reader.

[0154] The results are as Figure 1 shown. The results show that all 14 single-domain antibodies screened by the present invention can bind to the protein antigen and have high affinity.

[0155] 3.2 Detection of binding ability at the cellular level

[0156] 1) Prepare cells: Take an appropriate amount of T2 cells (Cell Bank of Shanghai Institute of Biochemistry and Cell Biology), adjust the cell density to 2×10 6 / ml, aliquot into 96-well U-bottom plates, 50 μL per well (about 1E5 cells);

[0157] 2) Polypeptide loading: Prepare a polypeptide with a concentration of 60 μM using 1640 medium. Add 50 μL of the polypeptide dilution to each well of the well plate and mix evenly with the cells. Incubate at 37 °C for 2 h to allow the polypeptide to be loaded onto the T2 cells. Add 200 μL of 1640 medium to resuspend the cells, centrifuge at 400 g for 5 min, and remove the supernatant;

[0158] 3) Antibody incubation: Adjust the initial concentration of the original antibody to 1 μg / mL using antibody dilution solution (PBS + 0.5% FBS), and perform a 4-fold dilution. A total of 10 concentration gradients are set. Take 100 μL of the diluted antibody and add it to the previously loaded cells. After pipetting and mixing evenly, incubate at 4 °C in the dark for 60 min;

[0159] 4) Antibody washing: Add 200 μL of antibody dilution solution to the incubated antibody-cell mixture. After centrifuging at 400 g for 5 min, discard the supernatant, resuspend the cells with 200 μL of antibody dilution solution, and repeat the washing once;

[0160] 5) Secondary antibody incubation: Dilute the fluorescent secondary antibody APC anti-human IgG Fc (BD Biosciences) at a ratio of 1:200 using antibody dilution solution. Take 100 μL and add it to the cell pellet after discarding the supernatant. After pipetting and mixing evenly, incubate at 4 °C in the dark for 30 min;

[0161] 6) Secondary antibody washing: Add 200 μL of antibody dilution solution to the incubated antibody-cell mixture. After centrifuging at 400 g for 5 min, discard the supernatant, resuspend the cells with 200 μL of antibody dilution solution, and repeat the washing once;

[0162] 7) Detection by flow cytometry: Add 100 μL of antibody dilution solution to each tube to resuspend the cells, transfer the cell suspension to a flow tube, and detect the positive labeling rate using a flow cytometer.

[0163] 8) Analyze the data using FlowJo software to obtain the mean fluorescence intensity value (MFI). Fit a dose-effect non-linear curve based on the MFI value and the antibody concentration value, and calculate the EC 50 value to evaluate the antigen affinity activity of the candidate antibody molecule.

[0164] The results are as Figure 2 shown: The results show that the 14 single-domain antibodies screened in the present invention can all bind to the antigen at the cellular level and have high affinity.

[0165] Example 4: Specificity detection of single-domain antibody for target recognition

[0166] T2 cell model loaded with different polypeptides

[0167] 1) Take T2 cells grown to the logarithmic phase and resuspend them in complete medium to 2×10 6 / mL;

[0168] 2) Dilute the target polypeptide and OTP polypeptide to 60 μM using complete medium. Mix the polypeptide dilutions with the cell suspension evenly at a volume ratio of 1:1, and incubate in a 37°C incubator for 1 - 2 hours to load the polypeptides onto the surface of T2 cells;

[0169] 3) After incubation, wash away the excess polypeptides with medium, centrifuge to collect the cells, and resuspend the cells in buffer (PBS solution containing 2% FBS) to 2×10 6 / mL. Seed the polypeptide - loaded T2 cells into a 96 - well U - bottom cell culture plate at 50 μL / well;

[0170] 4) Dilute the candidate antibody molecules to 10 μg / mL, 2 μg / mL, 0.4 μg / mL, and 0.08 μg / mL respectively using buffer. Add the antibody dilutions to the well plate at 50 μL / well, mix well with the cells, and incubate at 4°C for 1 h;

[0171] 5) Centrifuge at 300 g for 5 min to remove the antibody dilution, and wash the cells once with 200 μL of buffer;

[0172] 6) Dilute the fluorescent secondary antibody APC anti - human IgG Fc (purchased from Biolegend, catalog number 366906) with buffer at a ratio of 1:200. Add the antibody dilution to the well plate at 100 μL / well, resuspend and mix the cells, and incubate at 4°C for 30 min;

[0173] 7) Centrifuge at 300 g for 5 min to remove the antibody dilution, and wash the cells once with 200 μL of buffer;

[0174] 8) Resuspend the cells with 100 μL of buffer and detect the fluorescence binding intensity in the APC channel using a flow cytometer;

[0175] 9) Analyze the data using FlowJo software to obtain the mean fluorescence intensity value (MFI) and evaluate the non - specific binding level of the candidate antibody molecules to the off - target polypeptides.

[0176] Table 3. List of potential off - target polypeptide sequences used in the present invention

[0177] 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)

[0178] The present invention detected the off - target situations of all candidate molecules. Figure 3 Showed the off - target analysis results of some molecules. The results are as Figure 3As shown, the single-domain antibody molecules screened by the present invention have good specificity and can be further developed.

[0179] Example 5: Design of bispecific antibody molecules based on single-domain antibodies

[0180] We selected the single-domain antibody molecule LL-PR005 as a representative to design bispecific antibody molecules with different structures. The bispecific antibody molecules designed by the present invention can simultaneously bind to the PRAME polypeptide-HLA-A02 complex and CD3. Some bispecific antibodies in the present invention integrate an Fc structure or a half-life extender structure. The amino acid sequences of the CD3 antibodies used in the bispecific antibody molecules designed by the present invention are as follows:

[0181] Heavy chain variable region:

[0182] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS(SEQ ID NO:65)

[0183] Light chain variable region:

[0184] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIK(SEQ ID NO:66)

[0185] The amino acid sequences of the half-life extender used in the bispecific antibody molecules designed by the present invention are as follows:

[0186] QVQLVESGGGVVQPGGSLRLSCAASGFAFRGFGMSWVRQAPGKGLEWVSSINNGGSDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIGGPGASPSGQGTQVTVSS(SEQ ID NO:67)

[0187] A total of 13 bispecific antibody molecules with different structures were designed in the present invention, and the amino acid sequences are shown in Table 3.

[0188] Table 4. Amino acid sequence list of bispecific antibody molecules based on single-domain antibodies designed by the present invention

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196] Example 6: Expression and Purification of Bispecific Antibody Molecules

[0197] Referring to the experimental procedures in Example 2, the bispecific antibody molecules designed in the present invention were expressed and purified, and the expression level and purity were detected. The expression level and purity information of different bispecific antibodies are shown in Table 4. The expression level of the bispecific antibodies of the present invention is in the range of 600 - 850 mg / L, demonstrating that the bispecific antibody structure of the present invention has a high expression level. The monomer purity of the bispecific antibodies of the present invention by SEC-HPLC is all above 90%. At the same time, the purity of all bispecific antibodies by reducing SDS-PAGE is all above 95%. These results prove that the bispecific antibody molecules designed in the present invention have good expression levels and high purities.

[0198] Table 5 Detection Results of Expression Level and Purity of Bispecific Antibody Molecules

[0199]

[0200] Example 7: Functional Evaluation of Bispecific Antibody Molecules

[0201] The bispecific molecule of the present invention, also known as a T cell engager, can specifically bind to the target presented by pMHC in tumor cells and the CD3 protein on the surface of T cells simultaneously, mediate the directional cruising of T cells to the vicinity of tumor cells, endogenously activate and release cytokines, resulting in the lysis of tumor cells, thereby achieving the purpose of treating tumors. To evaluate the specific recognition and killing ability of the candidate bispecific molecules expressed in Example 6 against tumor cells, in this example, target cells and CD3+ T cells were co-cultured at a certain effector-to-target ratio, and different concentrations of the candidate antibody molecules were added. By detecting the apoptosis ratio of antibody-mediated target cells and the activation and factor secretion levels of T cells; in addition, in this example, tumor cell lines NCI-H1755, HS695T, OVCAR3, U2OS, T2 with negative PRAME expression and positive HLA-A2, MCF7, and A549 cell line with negative expression of both HLA-A2 and PRAME were set as target cells to further evaluate the target-specific recognition ability of the bispecific molecule, so as to more comprehensively screen out suitable bispecific molecules for in-depth research. The basic implementation steps are as follows:

[0202] 1) Cell co-culture: Different target cells were transfected with lentivirus carrying luciferase to prepare cell lines labeled with luciferase, labeled as: NCI-H1755-GFP, HS695T-GFP, OVCAR3-GFP, MCF7-GFP, and A549-GFP. Different target cells and effector cells were resuspended in a medium (1640 medium containing 2% FBS) at concentrations of 2×10 5 / mL and 1×10 6 / mL respectively, and plated into 96-well flat-bottom opaque white plates at 25 μL / well, and temporarily incubated at 37°C;

[0203] 2) Antibody incubation: The antibody was diluted in gradients with the medium, with the highest concentration of 20 nM, diluted to 10 concentration points. 50 μL of the corresponding bispecific antibody was added to each well. After thorough mixing, centrifuge at 500 rpm for 3 minutes. Incubate the cells in an incubator at 37°C for 24 hours;

[0204] 3) Killing detection: After 24 h of co-culture of cells, the remaining luciferase activity (relative light units, RLU) of target cells was measured to detect the killing ability of T cells against target cells in the presence of different specific antibodies. The specific steps were as follows: Take out the opaque 96-well flat bottom plate after co-culture, add 100 μL of equal volume D-luciferin substrate (Thermo Fisher Scientific: 88293) to the wells, mix well and incubate in the dark for 5 min, and detect the fluorescence intensity in the microplate reader using the chemiluminescence mode. Since luciferase is only expressed in target cells, the remaining luciferase activity in the wells is directly related to the number of live target cells in the wells. In the absence of effector cells and antibodies, the maximum luciferase activity was obtained by adding the culture medium to the target cells as a control;

[0205] 4) Data analysis: The killing efficiency of the target cells corresponding to the detection wells was calculated using the maximum luciferase activity as a control, and a dose-dependent curve was fitted with the antibody addition concentration as the abscissa to estimate the EC50 value and evaluate the target cell killing level of the bispecific antibody molecule to be tested.

[0206] The results showed that the bispecific antibodies of the present invention could all mediate T cell activation and kill cells with positive target expression ( Figure 4 ).

[0207] Example VIII: Molecular design of CAR sequence targeting PRAME and construction of lentiviral vector

[0208] 8.1 Design of CAR gene sequence targeting PRAME

[0209] The CAR targeting PRAME contains a single-domain antibody sequence against human PRAME, a CD28 hinge region and transmembrane domain, a CD28 co-stimulatory signaling region, and a CD3 zeta signaling domain, which are connected in series in turn. Five single-domain antibody sequences were selected in the present invention, and five CAR structures were designed. The five different CAR molecules were named: PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, PRAME-CAR-05, and the corresponding amino acid sequences of different CARs are shown in Table 6. The five CAR gene sequences targeting PRAME designed in the present invention were synthesized by gene synthesis and subcloned into the pUC57 vector (Suzhou Genewiz Biotechnology Co., Ltd.).

[0210] Table 6. Amino acid sequence table of CAR targeting PRAME

[0211]

[0212]

[0213] 8.2 Construction of CAR Lentiviral Vectors

[0214] Design primers and amplify 5 different CAR molecules from the pUC57 vector by PCR respectively. When designing primers, homologous arms of the lentiviral vector need to be added to the 5' ends of the forward and reverse primers. After the amplified PCR products are detected by agarose gel electrophoresis, they are respectively subjected to gel recovery and purification (Nanjing Novoprotein Science & Technology Co., Ltd., product number DC301) to obtain DNA fragments. The DNA fragments recovered by enzymatic digestion are cloned into the lentiviral vector by homologous recombination. The lentiviral vector needs to be digested with restriction endonucleases BamHI (NEB: R3136V) and SalI (NEB: R3138V) and then recovered and purified. In this way, 5 different recombinant plasmids are obtained: 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. Send the 5 lentiviral vectors to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing verification. The sequencing primers are: Lenti-seqF: TTGAGTTTGGATCTTGGTTC (SEQ ID NO:103), Lenti-seqR: CAGCAACCAGGATTTATACA (SEQ ID NO:104). After sanger sequencing verification, all 5 lentiviral vector plasmids are constructed correctly.

[0215] Example 9: Preparation and Functional Evaluation of PRAME-Targeted CAR-T Cells

[0216] 9.1 Preparation of Lentivirus

[0217] The lentiviral plasmids verified by sequencing are respectively transformed into Escherichia coli stbl3 (purchased from Yeasen Biotechnology Co., Ltd.). The next day, pick monoclonal colonies from the transformed plate into a shaking tube containing 2 ml of liquid LB medium. The medium already contains kanamycin (50 μg / ml). Incubate at 37°C with shaking at 220 rpm for 8 h. Pipette 1 ml from the activated bacterial solution and inoculate it into 250 ml of liquid LB medium containing kanamycin. Incubate at 37°C with shaking at 220 rpm for 12 - 16 h. Use the large-scale plasmid extraction kit NucleoBond Xtra Midi Plus (MN, product number: 740412.50) to extract plasmids according to the experimental procedures provided by the kit. After plasmid extraction, use Nanodrop (Thermo Fisher Scientific) to measure the plasmid concentration and verify it by sanger sequencing. At the same time, detect the content of supercoiled plasmids by DNA agarose gel.

[0218] After the cryopreserved 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were taken out of liquid nitrogen, they were thawed in a 37°C water bath, the tube mouth was wiped with 75% alcohol, and then transferred to a 15 ml centrifuge tube containing 10 ml of pre-warmed DMEM complete medium (90% DMEM + 10% FBS + 1% penicillin / streptomycin). Gently blow to mix evenly, centrifuge at 400 g for 4 min, and then aspirate and discard the supernatant. Add another 10 ml of DMEM complete medium, gently blow to mix evenly, and then inoculate into a T25 or T75 culture flask, and culture in a cell culture incubator at 37°C containing 5% CO 2 2. The cells were cultured. When the cell density reached more than 80% the next day, the cells were passaged and then continued to be cultured. The 293T cells cultured for more than 3 generations could be used to package lentivirus. The specific steps were as follows:

[0219] 1) On the first day, inoculate 293T cells: Inoculate the cells at about 1.0×10^7 cells / T175 flask (cultured with 40 mL of medium), and when the cell density reaches 90% the next day, transfection can be carried out.

[0220] 2) On the second day, plasmid transfection: Before transfection, change the medium to DMEM medium with 10% FBS but without double antibiotics. First, prepare the plasmid complex: Add the following plasmids into 1.5 ml of Opti-MEM (Thermo Fisher Scientific; 31985-070) and mix evenly: 18 μg of psPAX2 plasmid (Addgene; catalog number: 12260), 9 μg of pMD2.G plasmid (Addgene; catalog number: 12259), 18 μg of lentiviral vector plasmid. The lentiviral plasmids were: p-lenti-PRAME-CAR-01, p-lenti-PRAME-CAR-02, p-lenti-PRAME-CAR-03, lenti-PRAME-CAR-04, lenti-PRAME-CAR-05. Then prepare the transfection reagent complex: According to the mass ratio of plasmid to PEI of 1:3, add 67.5 μL (2 mg / mL) of PEI (polysciences: 24765) into 1.5 mL of Opti-MEM and mix evenly, and let it stand at room temperature for 5 min; then add the transfection reagent complex dropwise into the plasmid complex, mix evenly and let it stand for 20 min. Finally, slowly drop the transfection complex into the 293T cell culture flask, gently mix evenly, and continue to culture in a cell culture incubator at 37°C containing 5% CO 2 2. The cells were cultured.

[0221] 3) On the fourth day, harvest the virus: Collect the culture medium supernatant 48 h after transfection, and centrifuge at 2000 rpm for 10 min to remove cell debris. Filter the supernatant using a 0.45 μM filter membrane (Millex-HV, catalog number SLHVR33RB), transfer the filtrate to a dedicated centrifuge tube and balance it. Ultracentrifuge at 25000 rpm for 2 h using an ultracentrifuge. After pouring off the supernatant, resuspend the lentivirus with 1 ml of X-VIVO-15 medium, aliquot the lentivirus and store it in an ultra-low temperature freezer at -80 °C. Prepare lentiviruses containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, and PRAME-CAR-05 respectively according to this procedure.

[0222] 9.2 Preparation of CAR-T cells and detection of CAR molecule expression

[0223] The prepared lentiviruses containing PRAME-CAR-01, PRAME-CAR-02, PRAME-CAR-03, PRAME-CAR-04, and PRAME-CAR-05 were used to infect primary human T cells respectively to prepare CAR-T cells carrying different CAR genes. The CAR-T cells carrying 5 CAR genes were named: PRAME-CAR-T-01, PRAME-CAR-T-02, PRAME-CAR-T-03, PRAME-CAR-T-04, and PRAME-CAR-T-05 respectively, and the untransfected T cells were used as negative controls and named NT. The specific steps are as follows:

[0224] 1) Resuscitate CD3+ T cells derived from healthy human peripheral blood (Miaoshun (Shanghai) Biotechnology Co., Ltd.), resuspend the cells with T cell medium containing 300 IU / mL IL-2 to make the density 1×10 6 / mL, add the T cell activator CD3 / CD28 magnetic beads (ACRO Biosystems, catalog number: MBS-C001) according to the ratio of cells to magnetic beads of 1:1, mix well, and then inoculate the cells in a 6-well plate for culture;

[0225] 2) 24 hours after T cell activation, count the T cells and inoculate them into a new 24-well plate. Inoculate the cells at 500 ul per well and 5×10 5 cells / well. After inoculation, add 100 μL of lentivirus solution carrying different CAR genes to infect the T cells respectively. The T cells without adding the virus solution were used as the negative control NT, and the cells were placed in an incubator for continued culture.

[0226] 3) After 48 hours of lentivirus infection, aspirate the cells from the culture wells, remove the magnetic beads by magnetic adsorption using a magnetic stand, and centrifuge to collect the cells, which are then resuspended in fresh T cell medium.

[0227] 4) At the same time, take 100 μL of the cell suspension with lentivirus and the negative control respectively, centrifuge to collect the cells, resuspend the cells with 100 μL of FACS buffer, add the APC-labeled pMHC protein complex tetramer to each tube of cells, mix well, and incubate in the dark at 4 °C for 30 minutes;

[0228] 5) Wash the cells twice with PBS, then resuspend the cells with 100 μL of FACS buffer, and detect the expression efficiency of the 4 kinds of CARs on T cells by flow cytometry.

[0229] The results show that the 5 CAR molecules of the present invention can all be successfully expressed on T cells, and the expression efficiencies are relatively consistent, being 30 - 45% ( Figure 5 ).

[0230] 9.3 Detection of the killing ability of CAR-T cells

[0231] In this experiment, 2 kinds of HLA-A2 and PRAME target expression double-positive cell lines were used as target cells, namely non-small cell lung cancer NCI-H1755 and melanoma HS695T. 2 kinds of A549 and 293T cells with negative expression of PRAME and HLA-A2 were used as negative cells respectively. The 5 kinds of CAR-T cells prepared in Example 2 above were co-cultured with the target cells respectively to detect the killing effect and evaluate the biological functions of different CAR-Ts. The specific steps are as follows:

[0232] 1) Transfect different target cells with a lentivirus solution expressing GFP luciferase (GenBank: AAR29591.1) to obtain cell lines labeled with luciferase, labeled as: NCI-H1755-GFP-luc, HS695T-GFP-luc, 293T-GFP-luc, and A549-GFP-luc;

[0233] 2) Inoculate NCI-H1755-GFP-luc, HS695T-GFP-luc, 293T-GFP-luc, and A549-GFP cells into a 96-well flat-bottom opaque cell culture plate at a cell concentration of 1×10 5 / mL, 50 μL / well, and temporarily place them in an incubator at 37 °C.

[0234] 3) Adjust the positive ratio of 5 kinds of CAR-T cells to 15% with CT, set 4 effector-to-target ratios of 5:1, 2.5:1, 1:1 and 0.5:1, and inoculate 5 different CAR-T cells and control T cells into target cells at 50 μL / well respectively for co-culture. After thorough mixing, place them in an incubator at 37 °C and incubate overnight;

[0235] 4) Take out the opaque 96-well flat bottom plate after co-culture, add 100 μL of equal volume D-luciferin substrate (Thermo Fisher Scientific: 88293) to the wells, mix well and react in the dark for 10 minutes, and detect the fluorescence intensity with a microplate reader in the chemiluminescence mode. Since luciferase is only expressed in target cells, the remaining luciferase activity in the wells is directly related to the number of live target cells. Add the culture medium to the target cells to obtain the maximum luciferase activity as a control, and calculate the apoptosis ratio of target cells by subtracting the fluorescence signal value of live cells, which is the killing effect of CAR-T cells on target cells. The results are as Figure 6 shown.

[0236] The results show that all 5 CAR molecules of the present invention can mediate T cell killing of cells positive for 3 targets, and the killing effect on NCI-H1755 cells is the strongest.

[0237] 9.4 Detection of CAR-T cell factor secretion

[0238] In order to further evaluate the level of specifically activating the CAR-T cells of the present invention by target cells and releasing cytokines, after co-culturing 5 kinds of CAR-T cells prepared in Example 2 and the target cells in Example 4 at an effector-to-target ratio of 1:1 for 24 hours, detect the levels of IFN-γ and IL-2 cytokines secreted by T cells in the culture supernatant. The specific steps are as follows:

[0239] 1) Take target cells NCI-H1755, HS695T, A549 and 293T, resuspend them in complete medium to 1×10 6 / mL, and inoculate the cell suspension into a 96-well U-bottom deep well plate at 100 μL / well;

[0240] 2) After counting 5 kinds of CAR-T cells and control T cells, add them to different target cells for co-culture at an effector-to-target ratio of 1:1, that is, the cell concentration is 1×10 6 / mL and 100 μL / well;

[0241] 3) After the above cell co - culture overnight, 50 μL of the culture supernatant was collected and transferred to a new U - bottom 96 - well plate. The secretion of IFN - γ and IL - 2 cytokines in T cells was detected using an ELISA kit (Thermo Fisher Scientific; catalog number 88 - 7316). The preparation of the plate and the detection of the supernatant cytokines were carried out according to the instructions provided by the kit;

[0242] 4) Graphpad Prism software was used for data analysis, fitting a dose - dependent curve, and calculating the EC 50 value.

[0243] The results are as Figure 7 shown. The results indicate that the candidate CAR - T cells can all effectively recognize T2 loaded with the target polypeptide, activate and transduce immune signals, and secrete IFN - γ cytokine. Among them, the biological activity of the CAR - T - 01 molecule is relatively better. Considering the above results, the CAR sequence described in the present invention has good affinity and biological functions, suggesting that the CAR molecule of the present invention has the value of further development and application.

[0244] Example Ten: Humanization of Single - Domain Antibodies

[0245] In the present invention, two single - domain antibody candidate molecules, LL - PR001 and LL - PR004, were humanized respectively. The basic steps are as follows:

[0246] 1) The sequences of the single - domain antibody candidate molecules LL - PR001 and LL - PR004 were input into the IMGT database for antibody sequence alignment. According to the sequence alignment results in the database, IGHV3 - 23*04 was selected as the humanized parental vector for LL - PR001, and IGHV3 - 23*02 was selected as the humanized parental vector for LL - PR004;

[0247] 2) The CDR regions of the LL - PR001 single - domain antibody were transplanted into IGHV3 - 23*04, and the CDR regions of the LL - PR004 single - domain antibody were transplanted into IGHV3 - 23*02;

[0248] 3) Referring to the existing technical literature, back - mutations were performed on the transplanted humanized antibodies to ensure the affinity of the humanized antibodies.

[0249] Through the above - mentioned method, ten candidate humanized single - domain antibodies were designed with LL - PR001 as the humanized parent, and the antibody sequences are shown in Table 7. Six candidate humanized single - domain antibodies were designed with LL - PR004 as the humanized parent, and the antibody sequences are shown in Table 8.

[0250] Table 7. Humanized single - domain antibody sequences designed with LL - PR001 as the humanized parent

[0251]

[0252]

[0253] Table 8. Humanized single-domain antibody sequences designed with LL-PR004 as the humanized parent

[0254]

[0255] Example XI: Expression and functional identification of humanized single-domain antibody molecules

[0256] 11.1 Expression of humanized single-domain antibody molecules

[0257] The humanized antibody molecule was first codon-optimized to obtain a nucleotide sequence, and the full-length gene of the humanized antibody molecule was constructed into a single-domain antibody expression vector by gene synthesis. After the vector construction was successful, the above single-domain antibody was expressed by transient transfection of the HEK293 suspension cell line. The specific steps refer to Example II.

[0258] The expression levels and purities of different humanized single-domain antibodies are shown in Table 9. The expression level of the humanized single-domain antibody of the present invention using the transient expression system is in the range of 700-900 mg / L, which proves that the humanized single-domain antibody of the present invention has a high expression level. And the purity of the reduced SDS-PAGE and SEC-HPLC of most humanized single-domain antibodies is above 95%. These results prove that the humanized single-domain antibody of the present invention has a good expression level and a high purity.

[0259] Table 9. Detection of expression level and purity of humanized single-domain antibodies

[0260]

[0261]

[0262] 11.2 Detection of binding ability at the protein level

[0263] The binding ability of the humanized molecule to the target antigen was detected by ELISA to judge the affinity of the humanized molecule. The specific experimental operation steps refer to 3.1 of Example III. The results showed that all 10 candidate humanized single-domain antibodies (huLL-PR001-1 to huLL-PR001-10) obtained by humanization of the LL-PR001 molecule were able to bind the protein antigen and all had high affinities (the results are as Figure 8As shown). After partial humanization, the affinity of the molecule decreased slightly, but still had a high affinity. It can be applied to the research and development of subsequent biopharmaceutical projects such as bispecific antibodies, CAR-T, ADC, and RDC. Similarly, the 6 candidate humanized single-domain antibodies (huLL-PR004-1 to huLL-PR004-6) obtained after humanization of the LL-PR004 molecule can also bind to the protein antigen and have a high affinity. The affinity of the six after humanization is slightly lower than that of the parent molecule, but still has a high affinity (the results are as Figure 9 shown). It can be applied to the research and development of subsequent biopharmaceutical projects such as bispecific antibodies, CAR-T, ADC, and RDC.

[0264] All documents mentioned in this invention are cited in this application for reference as if each document was cited separately for reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A single-domain antibody targeting a PRAME polypeptide, the single-domain antibody having a VHH chain, the VHH chain comprising a CDR1 as shown in SEQ ID NO: 2, a CDR2 as shown in SEQ ID NO: 3, and a CDR3 as shown in SEQ ID NO: 4; a CDR1 as shown in SEQ ID NO: 6, a CDR2 as shown in SEQ ID NO: 7, and a CDR3 as shown in SEQ ID NO: 8; a CDR1 as shown in SEQ ID NO: 10, a CDR2 as shown in SEQ ID NO: 11, and a CDR3 as shown in SEQ ID NO: 12; a CDR1 as shown in SEQ ID NO: 14, a CDR2 as shown in SEQ ID NO: 15, and a CDR3 as shown in SEQ ID NO: 16; a CDR1 as shown in SEQ ID NO: 18, a CDR2 as shown in SEQ ID NO: 19, and a CDR3 as shown in SEQ ID NO: 20; a CDR1 as shown in SEQ ID NO: 22, a CDR2 as shown in SEQ ID NO: 23, and a CDR3 as shown in SEQ ID NO: 24; a CDR1 as shown in SEQ ID NO: 25, a CDR2 as shown in SEQ ID NO: 26, and a CDR3 as shown in SEQ ID NO: 27; : the CDR1 shown in SEQ ID NO: 26, the CDR2 shown in SEQ ID NO: 27, and the CDR3 shown in SEQ ID NO: 28; the CDR1 shown in SEQ ID NO: 30, the CDR2 shown in SEQ ID NO: 31, and the CDR3 shown in SEQ ID NO: 32; the CDR1 shown in SEQ ID NO: 34, the CDR2 shown in SEQ ID NO: 35, and the CDR3 shown in SEQ ID NO: 36; the CDR1 shown in SEQ ID NO: 38, the CDR2 shown in SEQ ID NO: 39, and the CDR3 shown in SEQ ID NO: 40; the CDR1 shown in SEQ ID NO: 42, the CDR2 shown in SEQ ID NO: 43, and the CDR3 shown in SEQ ID NO: 44; the CDR1 shown in SEQ ID NO: 46, the CDR2 shown in SEQ ID NO: 47, and the CDR3 shown in SEQ ID NO: 48; the CDR1 shown in SEQ ID NO: 50, the CDR2 shown in SEQ ID NO: 51, and the CDR3 shown in SEQ ID NO: or, the CDR1 shown in SEQ ID NO: 54, the CDR2 shown in SEQ ID NO: 55, and the CDR3 shown in SEQ ID NO:

56.

2. The single domain antibody targeting PRAME polypeptide according to claim 1, characterized in that The amino acid sequence of the PRAME polypeptide is: SLLQHLIGL.

3. The single domain antibody targeting PRAME polypeptide according to claim 1, characterized in that The amino acid sequence of the VHH chain of the single-domain antibody targeting the 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 humanized single domain antibody targeting PRAME polypeptide, wherein the framework regions FR1, FR2, FR3 and FR4 are humanized based on the single domain antibody according to any one of claims 1 to 3.

5. The humanized single domain antibody targeting PRAME polypeptide according to claim 4, characterized in that The variable region sequence of the humanized single-domain antibody targeting the 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.

6. An antibody targeting a PRAME polypeptide, said antibody comprising one or more of the single domain antibodies targeting a PRAME polypeptide according to any one of claims 1 to 3 or the humanized single domain antibodies targeting a PRAME polypeptide according to claim 4 or 5.

7. The antibody targeting PRAME polypeptide according to claim 6, characterized in that The antibody targeting PRAME polypeptide includes monomeric or multivalent antibodies.

8. The antibody targeting PRAME polypeptide according to claim 6, characterized in that The antibody targeting PRAME polypeptide is a bivalent antibody.

9. A bispecific antibody, comprising a first antibody and a second antibody, wherein the first antibody comprises the single-domain antibody targeting the PRAME polypeptide described in any one of claims 1-3, the humanized single-domain antibody targeting the PRAME polypeptide described in claim 4 or 5, or the antibody targeting the PRAME polypeptide described in any one of claims 6-8.

10. The bispecific antibody according to claim 9, characterized in that The second antibody may bind to the same or a different antigen as the first antibody, or bind to a different epitope on the same antigen as the first antibody.

11. The bispecific antibody according to claim 10, characterized in that The second antibody is a single domain antibody, a single chain antibody or a double chain antibody.

12. The bispecific antibody according to claim 10, characterized in that The bispecific antibody comprises 2-4 single domain antibodies targeting PRAME polypeptide.

13. The bispecific antibody according to claim 12, characterized in that The bispecific antibody comprises two single domain antibodies targeting the PRAME polypeptide.

14. The bispecific antibody according to claim 13, characterized in that The two single domain antibodies targeting PRAME polypeptide form a single domain antibody dimer targeting PRAME polypeptide.

15. The bispecific antibody according to claim 9, wherein The sequences of the bispecific antibodies are as follows: chain A as shown in SEQ ID NO: 68 and chain B as shown in SEQ ID NO: 69; chain A as shown in SEQ ID NO: 70 and chain B as shown in SEQ ID NO: 71; chain A as shown in SEQ ID NO: 72 and chain B as shown in SEQ ID NO: 73; chain A as shown in SEQ ID NO: 74, chain B as shown in SEQ ID NO: 75, and chain C as shown in SEQ ID NO: 76; chain A as shown in SEQ ID NO: 77, chain B as shown in SEQ ID NO: 78, and chain C as shown in SEQ ID NO: 79; chain A as shown in SEQ ID NO: 80, chain B as shown in SEQ ID NO: 81, and chain C as shown in SEQ ID NO: 82; chain A as shown in SEQ ID NO: 83, chain B as shown in SEQ ID NO: 84, and chain C as shown in SEQ ID NO: 85; chain A as shown in SEQ ID NO: 86 and chain B as shown in SEQ ID NO: Chain A as shown in SEQ ID NO: 88 and chain B as shown in SEQ ID NO: 89; Chain A as shown in SEQ ID NO: 90 and chain B as shown in SEQ ID NO: 91; Chain A as shown in SEQ ID NO: 92 and chain B as shown in SEQ ID NO: 93; Chain A as shown in SEQ ID NO: 94 and chain B as shown in SEQ ID NO: 95; or, Chain A as shown in SEQ ID NO: 96 and chain B as shown in SEQ ID NO:

97.

16. A fusion protein, comprising the single domain antibody targeting PRAME polypeptide according to any one of claims 1-3, the humanized single domain antibody targeting PRAME polypeptide according to claim 4 or 5, or the antibody targeting PRAME polypeptide according to any one of claims 6-8, and a linker sequence and an Fc fragment of an immunoglobulin.

17. The fusion protein according to claim 16, characterized in that The immunoglobulin is IgG1, IgG2, IgG3 or IgG4.

18. The fusion protein according to claim 16, characterized in that The immunoglobulin is IgG4.

19. A chimeric antigen receptor, which is made of the single domain antibody targeting PRAME polypeptide according to any one of claims 1-3, the humanized single domain antibody targeting PRAME polypeptide according to claim 4 or 5, or the antibody targeting PRAME polypeptide according to any one of claims 6-8.

20. The chimeric antigen receptor according to claim 19, wherein The amino acid sequence of the chimeric antigen receptor is selected from the group consisting of SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO:

102.

21. An immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor according to claim 19 or 20.

22. The immune effector cell according to claim 21, characterized in that The immune effector cells include T cells, NK cells or TIL cells.

23. The immune effector cell according to claim 21, characterized in that The immune effector cells are T cells.

24. A nucleic acid molecule encoding the single domain antibody targeting the PRAME polypeptide according to any one of claims 1-3, the humanized single domain antibody targeting the PRAME polypeptide according to claim 4 or 5, the antibody targeting the PRAME polypeptide according to any one of claims 6-8, the bispecific antibody according to any one of claims 9-15, the fusion protein according to any one of claims 16-18, or the chimeric antigen receptor according to claim 19 or 20.

25. An expression vector comprising the nucleic acid molecule of claim 24.

26. A host cell, comprising the expression vector of claim 25, or the nucleic acid molecule of claim 24 integrated into its genome.

27. A method for preparing a single domain antibody targeting a PRAME polypeptide according to any one of claims 1-3, a humanized single domain antibody targeting a PRAME polypeptide according to claim 4 or 5, an antibody targeting a PRAME polypeptide according to any one of claims 6-8, a bispecific antibody according to any one of claims 9-15, a fusion protein according to any one of claims 16-18, or a chimeric antigen receptor according to claim 19 or 20, the method comprising the following steps: 1) Cultivating the host cell of claim 26 under suitable conditions to obtain a culture containing the single domain antibody targeting PRAME polypeptide, humanized single domain antibody targeting PRAME polypeptide, antibody targeting PRAME polypeptide, bispecific antibody, fusion protein or chimeric antigen receptor; and 2) isolating or recovering the single domain antibody targeting PRAME polypeptide, humanized single domain antibody targeting PRAME polypeptide, antibody targeting PRAME polypeptide, bispecific antibody, fusion protein or chimeric antigen receptor from the culture.

28. An immunoconjugate comprising: 1) The single domain antibody targeting PRAME polypeptide according to any one of claims 1 to 3, the humanized single domain antibody targeting PRAME polypeptide according to claim 4 or 5, the antibody targeting PRAME polypeptide according to any one of claims 6 to 8, the bispecific antibody according to any one of claims 9 to 15, or the fusion protein according to any one of claims 16 to 18; and 2) A conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine or an enzyme.

29. The immunoconjugate of claim 28, wherein The coupling moiety is a radionuclide.

30. A pharmaceutical composition comprising a therapeutically or diagnostically effective amount of a single domain antibody targeting a PRAME polypeptide as described in any one of claims 1-3, a humanized single domain antibody targeting a PRAME polypeptide as described in claim 4 or 5, an antibody targeting a PRAME polypeptide as described in any one of claims 6-8, a bispecific antibody as described in any one of claims 9-15, a fusion protein as described in any one of claims 16-18, a chimeric antigen receptor as described in claim 19 or 20, an immune effector cell as described in any one of claims 21-23, or an immunoconjugate as described in claim 28 or 29, and a pharmaceutically acceptable excipient.

31. The pharmaceutical composition according to claim 30, characterized in that The pharmaceutical composition is used for treating tumors, and the tumor is a PRAME polypeptide-related tumor.

32. The pharmaceutical composition according to claim 31, characterized in that The tumor is melanoma, non-small cell lung cancer, ovarian cancer or breast cancer.

33. Use of the single domain antibody targeting PRAME polypeptide according to any one of claims 1-3, the humanized single domain antibody targeting PRAME polypeptide according to claim 4 or 5, the antibody targeting PRAME polypeptide according to any one of claims 6-8, the bispecific antibody according to any one of claims 9-15, the fusion protein according to any one of claims 16-18, the chimeric antigen receptor according to claim 19 or 20, the immune effector cell according to any one of claims 21-23, the immunoconjugate according to claim 28 or 29, or the pharmaceutical composition according to any one of claims 30-32 in the preparation of an agent for detecting PRAME polypeptide or a drug for treating tumors, The tumor is melanoma or non-small cell lung cancer.

34. A kit, comprising: 1) The single domain antibody targeting PRAME polypeptide according to any one of claims 1-3, the humanized single domain antibody targeting PRAME polypeptide according to claim 4 or 5, the antibody targeting PRAME polypeptide according to any one of claims 6-8, the bispecific antibody according to any one of claims 9-15, the fusion protein according to any one of claims 16-18, the chimeric antigen receptor according to any one of claims 19 or 20, the immune effector cell according to any one of claims 21-23, the immunoconjugate according to claim 28 or 29, or the pharmaceutical composition according to any one of claims 30-32; 2) container; and 3) Instructions for use.

35. A method for detecting PRAME polypeptide in a sample, the method comprising the steps of: 1) contacting the sample to be tested with the single domain antibody targeting PRAME polypeptide according to any one of claims 1-3, the humanized single domain antibody targeting PRAME polypeptide according to claim 4 or 5, the antibody targeting PRAME polypeptide according to any one of claims 6-8, the bispecific antibody according to any one of claims 9-15, the fusion protein according to any one of claims 16-18, or the immunoconjugate according to claim 28 or 29; 2) Detect whether an antigen-antibody complex is formed. If a complex is formed, it indicates that the PRAME polypeptide is present in the sample; The method is for non-diagnostic purposes.

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