Anti-b7h4 single-domain antibody and use thereof

Abstract

Provided are an anti-B7H4 single-domain antibody and the use thereof. The anti-B7H4 single-domain antibody can bind to B7H4 with high affinity, has a small molecular weight and low immunogenicity, and has prospects for use in B7H4 detection and the treatment of related diseases.

Description

Anti-B7H4 single-domain antibodies and their applications Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to anti-B7H4 single-domain antibodies and their applications. Background Technology

[0002] B7-H4 is an immunomodulatory molecule belonging to the B7 family. The expression of B7-H4 differs significantly between tumor and normal tissues, making it a promising target for tumor immunotherapy.

[0003] Several antibody drugs targeting B7-H4 have entered clinical trials, such as ADC drugs and bispecific antibody drugs. However, most of these are based on traditional B7-H4 antibodies, which have significant limitations, such as large molecular weight and weak tissue penetration. These limitations restrict the efficacy of B7-H4 antibody drugs built on traditional antibody structures. Nanobodies, on the other hand, offer unique advantages. Derived from naturally occurring heavy-chain antibodies in camelids, nanobodies possess advantages over traditional antibodies, including smaller molecular weight, stronger tissue penetration, higher stability, and ease of expression and modification. These characteristics allow nanobodies to more effectively target specific proteins on the surface of tumor cells, enhancing therapeutic efficacy. Furthermore, nanobodies offer unique advantages in constructing CAR-T cell therapies (smaller molecular weight, easier CAR transfection, etc.). Therefore, there is a need in this field to develop novel B7-H4 nanobodies. Summary of the Invention

[0004] The purpose of this invention is to provide a single-domain antibody against B7H4 and its application.

[0005] In a first aspect of the invention, an anti-B7H4 single-domain antibody is provided, wherein the complementarity-determining region (CDR) of the VHH chain of the anti-B7H4 single-domain antibody is selected from one or more of the following:

[0006] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0007] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0008] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:6;

[0009] (4) CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:6;

[0010] (5) CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:12, and CDR3 shown in SEQ ID NO:13;

[0011] (6) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0012] (7) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:6;

[0013] (8) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18;

[0014] (9) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:21;

[0015] (10) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18;

[0016] (11) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:22, and CDR3 shown in SEQ ID NO:23;

[0017] (12) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:25, and CDR3 shown in SEQ ID NO:26;

[0018] (13) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28;

[0019] (14) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:31.

[0020] In another preferred embodiment, the VHH chain of the anti-B7H4 single-domain antibody also has a frame region (FR).

[0021] In another preferred embodiment, the FR has an amino acid sequence derived from any one of SEQ ID NO:32-46.

[0022] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is as shown in any one of SEQ ID NO:32-46, or has a sequence identity of ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0023] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is shown in any one of SEQ ID NO:32-35, 40-43.

[0024] In another preferred embodiment, the single-domain antibody against B7H4 includes a monomer, a bivalent (bivalent antibody), and / or a multivalent antibody.

[0025] In another preferred embodiment, the single-domain antibody against B7H4 is a bivalent.

[0026] In another preferred embodiment, the anti-B7H4 single-domain antibody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0027] In a second aspect of the invention, an antibody against B7H4 is provided, the antibody comprising one or more VHH chains of a single-domain antibody against B7H4 as described in the first aspect of the invention.

[0028] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is as shown in any one of SEQ ID NO:32-46, or has a sequence identity of ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99%.

[0029] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is shown in any one of SEQ ID NO:32-35, 40-43.

[0030] In another preferred embodiment, the antibody against B7H4 comprises a monomer, a bivalent (bivalent antibody), and / or a multivalent antibody.

[0031] In another preferred embodiment, the divalent (or polyvalent) means that the amino acid sequence of the immunoconjugate contains two (or more) identical or different VHH chain sequences of the anti-B7H4 single-domain antibody as described in the first aspect of the present invention.

[0032] In a third aspect of the invention, a polynucleotide is provided that encodes a protein selected from the group consisting of: an anti-B7H4 single-domain antibody as described in the first aspect of the invention, or an antibody as described in the second aspect of the invention.

[0033] In another preferred embodiment, the polynucleotide is RNA, DNA, or cDNA.

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

[0035] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0036] In another preferred embodiment, the expression vector is the pcDNA3.4-hIgG1-Fc2 plasmid.

[0037] In a fifth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fourth aspect of the invention, or having a genome containing polynucleotides as described in the third aspect of the invention.

[0038] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0039] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0040] In another preferred embodiment, the host cell is a 293F cell.

[0041] In a sixth aspect of the invention, a method for generating an anti-B7H4 single-domain antibody is provided, comprising the steps of:

[0042] (a) Under conditions suitable for generating single-domain antibodies, host cells as described in the fifth aspect of the present invention are cultured to obtain a culture containing the anti-B7H4 single-domain antibody.

[0043] (b) Isolating and / or recovering the anti-B7H4 single-domain antibody from the culture; and

[0044] (c) Optionally, the anti-B7H4 single-domain antibody obtained in step (b) is purified and / or modified.

[0045] In a seventh aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0046] (a) an anti-B7H4 single-domain antibody as described in the first aspect of the invention, or an anti-B7H4 antibody as described in the second aspect of the invention; and

[0047] (b) The conjugate selected from the group consisting of: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0048] In another preferred embodiment, the radionuclide includes:

[0049] (i) a diagnostic isotope selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or

[0050] (ii) Therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0051] In another preferred embodiment, the coupling portion is a detectable marker.

[0052] In another preferred embodiment, the coupling portion is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticles.

[0053] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) VHH chain of an anti-B7H4 single-domain antibody as described in the first aspect of the invention.

[0054] In an eighth aspect of the invention, the use of an anti-B7H4 single-domain antibody as described in the first aspect of the invention, an anti-B7H4 antibody as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention, for the preparation of:

[0055] (1) Medications for the prevention and / or treatment of B7H4-related diseases;

[0056] (2) Reagents for detecting B7H4.

[0057] In another preferred embodiment, the B7H4-related diseases include aging-related diseases, cancer, or tumors.

[0058] In another preferred embodiment, the cancer or tumor is selected from the group consisting of hematologic malignancies, lymphomas, solid tumors, or combinations thereof.

[0059] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0060] In another preferred embodiment, the lymphoma is selected from the group consisting of: Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and other complex B-cell non-Hodgkin lymphomas.

[0061] In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or combinations thereof.

[0062] In another preferred embodiment, the drug is used to treat age-related diseases.

[0063] In another preferred embodiment, the age-related diseases include: infectious diseases, cardiovascular diseases, neurodegenerative diseases, autoimmune disorders, tumors, or combinations thereof.

[0064] In another preferred embodiment, the drug is administered to a human or a non-human mammal.

[0065] In another preferred embodiment, the reagent shown is a diagnostic reagent; more preferably, the diagnostic reagent is a test strip or test plate.

[0066] In another preferred embodiment, the diagnostic reagent is used to detect B7H4 or fragments thereof in a sample.

[0067] In a ninth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0068] (i) an anti-B7H4 single-domain antibody as described in the first aspect of the present invention, an anti-B7H4 antibody as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention; and

[0069] (ii) Pharmaceutically acceptable carriers.

[0070] In a tenth aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0071] (i) an anti-B7H4 single-domain antibody as described in the first aspect of the invention, or an anti-B7H4 antibody as described in the second aspect of the invention; and

[0072] (ii) Optional tag sequences to assist in expression and / or purification.

[0073] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, and a His tag.

[0074] In another preferred embodiment, the recombinant protein specifically binds to B7H4.

[0075] In an eleventh aspect of the present invention, a kit is provided containing an anti-B7H4 single-domain antibody as described in the first aspect of the present invention, an anti-B7H4 antibody as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention.

[0076] In a twelfth aspect of the invention, a method for preventing and / or treating B7H4-related diseases is provided, the method comprising administering to a desired subject an anti-B7H4 single-domain antibody as described in the first aspect of the invention, an anti-B7H4 antibody as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention.

[0077] In another preferred embodiment, the object includes mammals, such as humans.

[0078] In another preferred embodiment, the B7H4-related diseases include cancer or autoimmune diseases.

[0079] In a thirteenth aspect of the present invention, a method for in vitro detection of B7H4 or fragments thereof in a sample is provided, the method comprising the steps of:

[0080] (1) In vitro, the sample is contacted with an anti-B7H4 single-domain antibody as described in the first aspect of the present invention, an anti-B7H4 antibody as described in the second aspect of the present invention, or an immunoconjugate as described in the seventh aspect of the present invention.

[0081] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of B7H4 or its fragments in the sample.

[0082] In another preferred embodiment, the detection includes diagnostic or non-diagnostic methods.

[0083] In a fourteenth aspect of the present invention, a diagnostic method for B7H4-related diseases is provided, comprising the steps of:

[0084] (i) Obtaining a sample from a diagnostic subject and contacting the sample with an anti-B7H4 single-domain antibody as described in the first aspect of the invention, an anti-B7H4 antibody as described in the second aspect of the invention, or an immunoconjugate as described in the seventh aspect of the invention; and

[0085] (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates that the subject has a B7H4-related disease.

[0086] In a fifteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, wherein the recombinant polypeptide is an anti-B7H4 single-domain antibody as described in the first aspect of the present invention or an anti-B7H4 antibody as described in the second aspect of the present invention, and the method comprises:

[0087] (a) Culture host cells as described in the fifth aspect of the invention under suitable expression conditions; and

[0088] (b) Isolate the recombinant polypeptide from the culture.

[0089] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0090] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0091] Figure 1 shows the SDS-PAGE results of the Human B7H4 recombinant protein.

[0092] Figure 2 shows the three rounds of amplification of the VHH fragment.

[0093] Figure 3 shows the results of one round of magnetic sorting of the antibody yeast display library. A: NC group; B: Original library: primary antibody: V5-FITC; C: 1MACS: primary antibody: Biotin-B7-H4-ASH, secondary antibody: SA-APC+V5-FITC.

[0094] Figure 4 shows the results of the two-round sorting of the antibody yeast display library. A: NC group; B: 1MACS: primary antibody: Bio-B7H4-ASH, secondary antibody: SA-APC+V5-FITC.

[0095] Figure 5 shows the detection of clone positivity rate after magnetic sorting and flow cytometry sorting.

[0096] Figures 6A-6F show the FACS results of antibody supernatant expressed in 293F cells specifically binding to target cells.

[0097] Figure 7 shows the SDS-PAGE results of the candidate B7H4 single-domain antibody.

[0098] Figures 8A-8F show the FACS detection results of the candidate B7H4 single-domain antibody binding to B7H4-overexpressing cells. Figure 8A corresponds to antibody 292-1-B03. Figure 8B corresponds to antibody 292-1-C09. Figure 8C corresponds to antibody 292-2-B02. Figure 8D corresponds to antibody 292-2-C06. Figure 8E corresponds to antibody 292-2-E07-1. Figure 8F corresponds to antibody PC(20502).

[0099] Figure 9 shows the binding of candidate B7H4 single-domain antibody to EC50 cells overexpressing B7H4.

[0100] Figure 10 shows the ELISA detection results of antibody binding to target protein.

[0101] Figure 11 shows the antibody affinity test results.

[0102] Figure 12 shows the three rounds of amplification of the VHH fragment.

[0103] Figures 13A, 13B, and 13C show the results of the first, second, and third panning of the phage display library, respectively.

[0104] Figures 14A-14C show the FACS detection results of antibody supernatant expressed in 293F cells specifically binding to target cells.

[0105] Figures 15A-15I show the FACS detection results of the candidate B7H4 single-domain antibody binding to B7H4-overexpressing cells. Figure 15A corresponds to antibody PC (20502). Figure 15B corresponds to antibody 6-A08. Figure 15C corresponds to antibody 1-H06. Figure 15D corresponds to antibody 1-B02. Figure 15E corresponds to antibody 8-A1. Figure 15F corresponds to antibody 8-C4. Figure 15G corresponds to antibody 8-D12. Figure 15H corresponds to antibody 8-E2. Figure 15I corresponds to antibody PC (20502).

[0106] Figure 16 shows the binding of candidate B7H4 single-domain antibody to EC50 cells overexpressing B7H4. Detailed Implementation

[0107] Through extensive and in-depth research and numerous screenings, the inventors have successfully obtained multiple single-domain antibodies against B7H4. Specifically, this invention utilizes yeast display and phage display technologies to screen an immune single-domain antibody gene library, followed by panning and identification, thereby obtaining single-domain antibodies against B7H4. Experimental results show that the single-domain antibodies against B7H4 obtained in this invention can bind to B7H4 with high affinity. Based on this, the invention was completed.

[0108] the term

[0109] As used herein, the terms "antibody of the present invention", "nanobody of the present invention", and "anti-B7H4 single-domain antibody of the present invention" have the same meaning and can be used interchangeably, all referring to the antibody that specifically recognizes and binds to B7H4 provided in the first aspect of the present invention.

[0110] Antibody

[0111] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0112] As used herein, the terms "single-domain antibody," "VHH," "nanobody," and "single-domain antibody (sdAb, or nanobody)" have the same meaning and are used interchangeably. A single-domain antibody (VHH) is constructed by cloning the variable region of the antibody heavy chain, consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0113] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0114] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the described nanobody or fragments thereof targeting B7H4.

[0115] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0116] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0117] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0118] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0119] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to B7H4, such as proteins or peptides having a heavy chain variable region. They may or may not contain initiating methionine.

[0120] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0121] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or l0 -10 M or lower affinity (KD) binding.

[0122] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0123] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0124] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0125] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., 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 (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0126] The antibody of this invention refers to a polypeptide having B7H4 binding activity and including the aforementioned CDR region. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0127] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0128] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0129] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0130] Table 1

[0131] The invention also provides a polynucleotide molecule encoding the aforementioned antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

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

[0133] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0134] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0135] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0136] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0137] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0138] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0139] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0140] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0141] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0142] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0143] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0144] 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.

[0145] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0146] B7H4

[0147] B7-H4 is an immunomodulatory molecule belonging to the B7 family and is a transmembrane glycoprotein. It possesses extracellular, transmembrane, and intracellular regions. The extracellular region can participate in interactions with other molecules, thereby exerting its functions in immunomodulation and other aspects.

[0148] In normal human tissues, the expression of B7-H4 is relatively limited and at a relatively low level. It is typically expressed in small amounts in parts of the reproductive system (such as the ovaries and endometrium), the urinary system (such as the kidneys), and some immune cells (such as macrophages). B7-H4 is highly expressed in various tumor tissues, including common malignant tumors such as breast cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, endometrial cancer, and bladder cancer.

[0149] In the tumor microenvironment, highly expressed B7-H4 inhibits T cell activation, proliferation, and cytokine production through interactions with related receptors on the surface of T cells. This allows tumor cells to evade the body's immune surveillance, creating favorable conditions for tumor growth, invasion, and metastasis. High expression of B7-H4 in tumor tissue is often associated with poor tumor prognosis. In other words, high levels of B7-H4 expression on tumor cells usually indicate a more unfavorable disease progression and a potentially shorter survival time. In conclusion, B7-H4 plays a crucial role in tumor development and progression, and its differential expression between tumor and normal tissues makes it a potential target for tumor immunotherapy.

[0150] Pharmaceutical Composition

[0151] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the above-described antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0152] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0153] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0154] Single-domain antibody against B7H4

[0155] This invention provides a single-domain antibody against B7H4 that can specifically bind to B7H4.

[0156] In one aspect of the invention, an anti-B7H4 single-domain antibody is provided, characterized in that the complementarity-determining region (CDR) of the VHH chain of the anti-B7H4 single-domain antibody is selected from one or more of the following:

[0157] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0158] (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0159] (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:6;

[0160] (4) CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:6;

[0161] (5) CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:12, and CDR3 shown in SEQ ID NO:13;

[0162] (6) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6;

[0163] (7) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:6;

[0164] (8) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18;

[0165] (9) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:21;

[0166] (10) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18;

[0167] (11) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:22, and CDR3 shown in SEQ ID NO:23;

[0168] (12) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:25, and CDR3 shown in SEQ ID NO:26;

[0169] (13) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28;

[0170] (14) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:31.

[0171] In a preferred embodiment, the amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is as shown in any one of SEQ ID NO:32-46, or has ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity with it. More preferably, the amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is as shown in any one of SEQ ID NO:32-35 and 40-43.

[0172] The nanobody of the present invention exhibits high affinity for B7H4. This high affinity refers to the antibody of the present invention having an affinity of less than 10... -8 M, preferably less than 10 -9 M, more preferably less than 10 -10 M or lower affinity (KD) binds to B7H4.

[0173] The nanobodies of this invention possess the advantages of nanobodies, such as small molecular weight, rapid tissue penetration, high solubility and stability, high antigen binding specificity, and weak immunogenicity. Furthermore, due to their small molecular weight, nanobodies can recognize some hidden antigenic epitopes that monoclonal antibody drugs cannot recognize. Additionally, in the human body, nanobodies may exhibit lower immunogenicity than murine antibodies. Therefore, constructing antibody drugs or CAR-T drugs using nanobody sequences has greater advantages than constructing antibody drugs or CAR-T drugs using murine single-chain antibody sequences.

[0174] The B7H4 nanobody of the present invention can be used to construct B7H4 nanobody drugs for targeted treatment of aging-related diseases or tumors, or CAR-T drugs based on the B7H4 nanobody sequence.

[0175] In another aspect of the invention, an antibody against B7H4 is also provided, said antibody may be monovalent or polyvalent, and may comprise one or more identical or different VHH chains of the same or different single-domain antibodies against B7H4 of the present invention. Preferably, said VHH chain has the amino acid sequence shown in any one of SEQ ID NO:32-35, 40-43.

[0176] Reagent test kit

[0177] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0178] This invention also provides a detection kit for detecting B7H4 levels. The kit includes an antibody that recognizes B7H4, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffer solutions, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0179] application

[0180] As described above, the antibody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of B7H4-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis, prevention, and treatment of B7H4-related diseases.

[0181] The main advantages of this invention include:

[0182] 1) This invention provides a single-domain antibody against B7H4 with a small molecular weight and low immunogenicity, which has higher potential for detection and therapeutic applications than general monoclonal antibodies.

[0183] 2) The single-domain antibody of the present invention has a strong affinity for B7H4.

[0184] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0185] I. Screening for B7H4 single-domain antibodies using a yeast display library

[0186] Main reagents

[0187] Agar (Sigma, CAT#:A1296), Peptone (Sigma, CAT#:93926), Yeast Extract (OXOID, CAT#:LP0021), Sodium Chloride (Aladdin, CAT#:C111533), Potassium Chloride (Aladdin, CAT#:P112133), Magnesium Sulfate (Sinopharm, CAT#:10013018), Magnesium Chloride (Sinopharm, CAT#:10012818), Glucose (Sangon Biotech, CAT#:GT1991), SfiI (NEB, CAT#:R0123L), T4 DNA ligase (TaKaRa, CAT#:2011A), PrimeScript TMII 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#:6210B), NuHi Power Mix (Xinhai Biotechnology, CAT#:NH9303), 3M Sodium Acetate (pH 5.2-6) (Sigma, CAT#:126-96-5), DNA Fragment Recovery Kit (TakaRa, CAT#:9761), Gel Recovery Kit (Qiagen, CAT#:28706), PE-anti-Human IgG (eBioscience, Cat#:12-4998-82), Rabbit anti-Llama IgG (H+L) Secondary Antibody [HRP] (Novus, CAT#:NBP1-75095), EBY100 competent cells (iCareab), pDisplay yeast display vector (iCareab), NHS-biotin (APExBIO, CAT#:A8002), HRP-Streptavidin (Boster, CAT#:BA1088), HRP-Protein A (Boster, CAT#:BA1080), LVTransm transfection reagent (iCareab, Cat#:LVTran100), Streptavidin Magnetic Beads (NEB, CAT#:S1420S)

[0188] Main consumables

[0189] 50mL Falcon centrifuge tubes (Corning, CAT#352070), electroporation cuvettes (Bio-Rad 0.2cm), 1.5mL RNase-free EP tubes (QSP, CAT#:509-GRD-Q), 200μL RNase-free PCR tubes (Axygen, PCR-02D-C), T125 shake flasks (Corning, CAT#431143), 15mL Falcon centrifuge tubes (Corning, CAT#430052), 6-well plates (Corning, CAT#3516), 96-well plates (Corning, CAT#3365), His1K sensor (Sartorius, CAT#18-5120)

[0190] Main equipment

[0191] Electroporator (Eppendorf Multiporator), centrifuge (Thermo FRESCO-17), incubator (Shanghai Jinghong, DNP-9052), shaking incubator (Jingqi, CO-O6U), clean bench (Sujing Antai, SW-CJ-1FD), PCR instrument (Applied Biosystems ABI2720), biosafety cabinet (Haier, HR40-IIA2), flow cytometer (Thermo Attune Nxt flow cytometer), Thermo 3111 CO2 incubator, Fortebio (Sartorius). R2)

[0192] Experimental methods

[0193] 1. Preparation of B7H4 recombinant protein

[0194] The extracellular sequence information of Human B7H4 was retrieved from the UniProt database. His tags were added to the C-terminus. After optimization according to human codon preference, the gene was synthesized and subcloned into the pcDNA3.4 vector. After verification by Sanger sequencing, plasmid extraction was performed.

[0195] The constructed eukaryotic protein expression vector was transiently transfected into 293F cells. The protein expression supernatant was collected and the target protein was purified using a nickel column. The protein purity was detected by SDS-PAGE, and the purity was >90%.

[0196] 2. Alpaca Immunization

[0197] The recombinant protein prepared above was used to immunize alpacas at 21-day intervals. Ten days after the last immunization, peripheral blood was collected, and serum was separated and the immunization effect was detected by ELISA.

[0198] Table 2 Alpaca Immunization Procedure

[0199] 3. Detection of immune titer

[0200] Collect 5 mL of peripheral blood and incubate the centrifuge tube containing the blood sample at 37°C for 1 hour; then transfer the blood sample to 4°C and incubate overnight. Centrifuge the centrifuge tube containing the blood sample at 5000 rpm for 20 min; separate the supernatant serum and transfer it to a new sterile centrifuge tube to collect the immune serum. Dilute the target recombinant protein to a final concentration of 1 μg / mL using sterile CBS (carbonate buffer). Take a new 96-well ELISA plate and add 100 μL / well for coating overnight at 4°C. Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate 5 times with PBST. Add 100 μL of serially diluted serum (100 μL / well) and incubate at room temperature for 1 hour. The control wells are treated with PBS. Remove the liquid from the wells and wash 5 times with PBST. Add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour. Remove the liquid from the wells and wash the plate 5 times with PBST. Add 100 μL / well of TMB chromogenic buffer. Incubate at room temperature in the dark for 10-15 minutes. Add 50 μL / well of stop solution. Read the OD values ​​in the wells using a microplate reader. 450 value.

[0201] 4. PBMC isolation and VHH antibody fragment cloning

[0202] 100 mL of anticoagulated peripheral blood was collected, and PBMCs were isolated using lymphocyte separation medium. RNA was extracted and processed using PrimeScript. TM II. The 1st Strand cDNA Synthesis Kit was used for reverse transcription to prepare cDNA.

[0203] Prepare the following reaction mixture Mix 1 in 200 μL of PCR:

[0204] Table 3

[0205] After holding at 65℃ for 5 minutes, it is rapidly cooled on ice.

[0206] Prepare the following reaction solutions in the PCR tubes described above:

[0207] Table 4

[0208] After mixing by pipetting, dispense 80 μL / tube and place in a PCR instrument at 42°C for 1 hour, followed by heat inactivation at 70°C for 15 minutes. Finally, store the cDNA samples on ice or at -20°C for long-term storage.

[0209] Amplification of the VHH fragment:

[0210] Configure the first round of PCR reaction system (50 μL / tube): the upstream primer binds to the signal peptide, and the downstream primer binds to the CH2 region.

[0211] Table 5

[0212] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure:

[0213] Table 6

[0214] Agarose gel electrophoresis of PCR products:

[0215] PCR products were analyzed by electrophoresis using 1% agarose gel to separate fragments with a molecular weight of approximately 750 bp. PCR products were recovered using a gel extraction kit, and their concentrations were determined using NanoDrop.

[0216] Prepare the two-round PCR reaction system (50 μL / tube): the upstream primer binds to the antibody FR1 region, the downstream primer binds to the anti-Hinge and FR4 regions, and the restriction enzyme site is SfiI.

[0217] Table 7

[0218] After preparing the PCR reaction system, set up the PCR instrument according to the following procedure:

[0219] Table 8

[0220] Agarose gel electrophoresis analysis of the second-round PCR products:

[0221] PCR products were analyzed by electrophoresis using 1% agarose gel to isolate the VHH fragment with a molecular weight of approximately 400 bp. The VHH PCR product was recovered using a gel extraction kit, and its concentration was determined using NanoDrop.

[0222] 5. Construction of a single-domain antibody yeast display library

[0223] The yeast display vector pDisplay was linearized, and the enzyme digestion system is as follows:

[0224] Table 9

[0225] The pDisplay vector was digested with SfiI enzyme, 100 μL / tube, and digested overnight at 50°C.

[0226] The pDisplay vector fragment was separated using a 1% agarose gel, and a 5000 bp fragment was excised and recovered using the gel, with the concentration determined by NanoDrop. The recovered pDisplay digestion product was aliquoted into 200 μL portions in each 1.5 mL centrifuge tube, and 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL glycogen were added. The mixture was then pipetted and aspirated to mix. 880 μL of anhydrous ethanol was added, and the mixture was inverted and incubated at -80°C.

[0227] Electroconversion to construct a yeast display library:

[0228] Strawberries frozen at -80℃ were streaked onto YPD agar plates and activated at 30℃ for 3-5 days. Single colonies of competent yeast were inoculated into 50 mL of YPD medium and incubated at 250 rpm and 30℃ for 1-2 days. Competent yeast strains were then prepared. The linearized vector fragment and PCR product were mixed and added to an electroporation cuvette for electroporation. The electroporated competent yeast strains were then transfected into culture flasks and incubated at 220 rpm and 30℃ for 1 hour.

[0229] Take 20 μL of the resuspended culture, dilute it 5000 times with SDCAA, and transfer 100 μL to an SDCAA plate. Incubate for 2-3 days and calculate the volume of the culture medium. Continue incubating the remaining culture for 24 hours.

[0230] Collect the remaining bacterial culture into a 50 mL centrifuge tube and centrifuge at 3000 g for 5 min. Discard the supernatant and resuspend in 10 mL of SDCAA. Mix with 50% glycerol and the resuspension in a 1:1 ratio and store at -80°C.

[0231] 6. Yeast Display Library Magnetic Bead Sorting

[0232] Yeast cultured in SGCAA was added to a 250 mL shake flask containing 50 mL of SGCAA medium and cultured at 30 °C and 240 rpm for 16 h. After centrifugation, the supernatant was discarded, and the resuspended yeast was resuspended in 1 mL of 0.5% PBSA. The resuspended yeast was added to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 5 min, and the supernatant was discarded. The sample was washed once with 0.5% PBSA.

[0233] The streptavidin magnetic beads incubated with the antigen were washed twice with 0.5% PBSA (incubated at 4°C for 5 minutes each time), placed on a magnetic rack, and incubated for 5 minutes. The supernatant was then discarded. Yeast culture was added to the antigen-bound magnetic beads, and the mixture was incubated at 4°C for 60 minutes. The mixture was then placed on a magnetic rack for 15 minutes. The yeast culture was discarded, and the magnetic beads were washed three times with 0.5% PBSA (incubated at 4°C for 5 minutes each time). The magnetic beads were resuspended in 1 mL of SDCAA medium. 0.5–5 μL of the resuspended mixture was transferred to 100 μL of SDCAA medium and plated. The resuspended mixture was divided into two portions. One portion was added to 500 μL of 50% glycerol (stored at -80°C); the other portion was added to a shaker tube, and 2 mL of SDCAA medium was added. The tubes were incubated at 30°C and 240 rpm for 16 hours.

[0234] Transfer the bacterial culture from the shake tube to 50 mL of SDCAA medium (in a 250 mL shake flask) and incubate overnight at 30°C and 240 rpm. Measure the OD600 value of the bacterial culture. Based on the OD600, take a portion of the bacterial culture, centrifuge, resuspend in SGCAA, and transfer to 50 mL of SGCAA medium to achieve a final OD600 value of 1. Incubate overnight at 30°C and 240 rpm. Resuspend the remaining bacterial culture in a 1:1 ratio of SDCAA and 50% glycerol and store at -80°C.

[0235] 7. Two-round sorting of yeast display library

[0236] Take 1 ml of SGCAA-cultured yeast culture into a 1.5 ml centrifuge tube, centrifuge to remove the supernatant, wash twice with 1 ml PBS, resuspend with 1 ml PBS, take 100 μl of the culture into a new 1.5 ml centrifuge tube (as NC), centrifuge, and remove the supernatant.

[0237] Dilute the Biotin-antigen protein with 100 μL PBS to a concentration of 10 μg / mL, resuspend the bacterial cells in the experimental group, incubate at 4°C for 60 min by rotation, centrifuge at 3000g for 3 min, discard the supernatant, and wash twice with 1 ml PBS.

[0238] Resuspend the experimental group cells in APC-Streptavidin at a 1:1000 dilution, incubate at 4°C with rotation for 60 min, centrifuge at 3000g for 3 min, discard the supernatant, wash twice with 1 ml PBS, and resuspend in 1 ml PBS. Add the resuspended cells to flow cytometry tubes for flow cytometry separation. Prepare 1 ml of SDCAA medium in a 15 ml centrifuge tube as the collection tube. Perform flow cytometry separation using the NC group gate to separate APC-positive yeast cells.

[0239] 8. Yeast monoclonal flow cytometry detection

[0240] After sorting, the yeast culture was plated on SDCAA plates, and single clones were picked and cultured. After induced expression for 48 h, the culture was incubated with biotin-antigen. PE-Streptavidin was used as the secondary antibody. Flow cytometry was performed after incubation. The yeast clones that bound to the target antigen were resuspended in 0.2% SDS, incubated at 95 °C for 10 min for lysis, and the supernatant was collected by centrifugation. 0.5 μL of the supernatant was used as a template for PCR amplification and assay (the remaining culture was stored at -20 °C).

[0241] 9. Construction of antibody eukaryotic expression vector

[0242] Positive yeast clones were subjected to PCR to obtain antibody sequences, which were then digested with SfiI and ligated into the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct an antibody expression vector. The eukaryotic expression vector was transiently transfected into 293F cells, and the antibody expression supernatant was collected. The binding of candidate antibodies and antigen proteins was detected by FACS.

[0243] 10. Expression and purification of candidate single-domain antibodies

[0244] Based on the ELISA results of the candidate antibodies, positive clones were selected for antibody expression and preparation.

[0245] Remove the LVTransm transfection reagent and pcDNA3.4-human IgG1Fc antibody expression vector from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Transfer 2 mL of PBS to one well of a 6-well plate, add 20 μg of antibody expression vector, mix thoroughly by pipetting, then add 60 μL of LVTransm and immediately mix by pipetting. Let stand at room temperature for 10 minutes.

[0246] Add the DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C, 5% CO2, 130 rpm.

[0247] After continuous culture for 5-7 days, the supernatant of the culture medium is collected by centrifugation, filtered through a 0.45μm filter membrane, and the filtrate is transferred to a sterile centrifuge tube for antibody purification using a Protein A column.

[0248] 11. ELISA detection of the binding of recombinant antibody to target protein

[0249] Dilute the recombinant protein to a final concentration of 1 μg / mL using sterile CBS. Take a new 96-well microplate and add 100 μL / well for coating at 4°C overnight. Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate five times with PBST.

[0250] Add the expressed recombinant antibody, transfect 50 μL of supernatant per well, or 100 μL of purified antibody (starting concentration 10 μg / mL, serially diluted 3-fold for 7 spots, 100 μL / well), and incubate at room temperature for 1 hour. Use PBS for the control wells. Remove the liquid from the wells and wash 5 times with PBST. Add 100 μL / well of HRP-Protein A antibody (1:50000 dilution) and incubate at room temperature for 1 hour. Remove the liquid from the wells and wash the plate 5 times with PBST. Add 100 μL / well of TMB chromogenic buffer. Incubate at room temperature in the dark for 10-15 minutes. Add 50 μL / well of stop solution. Read the OD450 values ​​of the wells using a microplate reader.

[0251] 12. FACS detection of the binding of B7H4 candidate antibody to overexpressing cell lines

[0252] CHO-S and CHO-S-B7H4 cell lines were resuscitated in liquid nitrogen and adjusted to the logarithmic growth phase. The cells were then divided into several fractions, each containing 2 × 10⁻⁶ cells. 5 Cells were incubated with the B7H4 candidate antibody or transfection supernatant for 1 hour at room temperature. The cells were then centrifuged at 800xg for 3 minutes at room temperature, the supernatant containing the antibody was discarded, and the cells were washed three times with PBS. Secondary antibody PE anti-human IgG (1:5000 dilution) was added, mixed thoroughly, and incubated in the dark at room temperature for 30 minutes. The cells were then centrifuged at 800xg for 3 minutes at room temperature, the supernatant containing the secondary antibody was discarded, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0253] 13. Single-domain antibody affinity detection

[0254] Antibody affinity was determined using a ForteBio OCTET R2 instrument. Candidate antibodies were immobilized using a His1K sensor at a concentration of 5 μg / ml for 200 s. The buffer was PBST (PBS + 0.02% Tween 20), and the candidate antibodies were diluted to 10, 5, 2.5, 1.25, 0.625, and 0 nM.

[0255] Affinity testing: equilibrium 60s, binding 180s, dissociation 180s, detection temperature 25℃. Kinetic characterization analysis was performed using the ForteBio OCTET R2 system.

[0256] Example 1: SDS-PAGE detection of Human B7H4 recombinant protein

[0257] Different imidazoles were used for elution, and the purified protein buffer was replaced with PBS. The purity of Human B7H4 histone was greater than 90% by SDS-PAGE, which can be used for alpaca immunization and antibody screening.

[0258] Example 2: Detection of Alpaca Immune Titer

[0259] Table 10. Alpaca Immune Potency

[0260] Serum was isolated from immunized alpacas and subjected to limiting dilutions according to the dilution gradient in Table 10. ELISA assays were then performed on pre-coated 96-well plates containing antigen. The ELISA results showed that the immune serum bound to the B7H4 recombinant protein, and the OD values ​​changed gradients with the dilution of the immune serum. A cDNA library was prepared from previously lysed PBMCs of this alpaca to construct a yeast display library.

[0261] Example 3 Amplification of VHH Fragment

[0262] Peripheral blood was collected to isolate PBMCs, and RNA was extracted. A cDNA library was prepared using a reverse transcription kit. One round of PCR was performed using single-domain antibody amplification primers, and the PCR products were obtained by agarose gel electrophoresis. The process is shown in Figure 2. The first round of PCR yielded bands of approximately 1000 bp and 750 bp, respectively. The 750 bp fragment was recovered from the gel and used as a template for the second round of PCR. The second round of PCR yielded a band of approximately 400 bp, which was the VHH fragment. The third round of PCR yielded a band of approximately 500 bp, which was the fragment with added homologous arms.

[0263] Example 4: Flow cytometry detection of streptavidin after magnetic bead sorting

[0264] After induction and display of the electroporated yeast library, the cells were fully bound to the B7H4-His-Biotin (Human) protein and magnetically sorted twice using streptavidin beads. The sorted yeast cells were plated and cultured simultaneously. After induction of expression, they were incubated with Human B7H4-His-Biotin for 1 hour. Flow cytometry analysis was performed using APC Streptavidin as the secondary antibody.

[0265] Based on the flow cytometry results, after one round of magnetic sorting, the positive rate with Biotin-Human B7H4-His was 1.480% (Figure 3), and a second round of flow cytometry sorting was arranged.

[0266] Based on the flow cytometry results, after one round of magnetic sorting and one round of flow cytometry sorting, the positive rate of Biotin-Human B7H4-His was 50.0% (Figure 5), and flow cytometry detection of yeast monoclonal antibodies was arranged.

[0267] Example 5: Yeast Monoclonal FACS Detection

[0268] From the enriched yeast display library, monoclonal cells were randomly selected, amplified and induced, and then detected using antigens to determine the binding of single-domain antibodies on the surface of monoclonal yeast cells to the target antigen.

[0269] Based on the flow cytometry results of yeast monoclonal antibodies, genomic DNA was extracted from positive clones that bound to the Human B7H4-His protein, and antibody sequences were obtained by PCR. Based on the sequencing results of the PCR products, differentially expressed clones were selected, digested, and ligated to construct a eukaryotic expression vector. The C fusion of VHH with this vector expressed hIgG1Fc. The constructed single-domain antibody eukaryotic expression vector was transiently transfected into 293F cells, and the supernatant of the expressed antibody was used for FACS and ELISA detection.

[0270] FACS results are shown in Figures 6A-6F. Candidate clones 1-B03, 1-E05, 1-H04, 2-B02, 2-C06, 1-C09, 292-1-H09, 292-2-E07-1, 292-3-D06-2, 292-4-F06, and 292-4-F06-1 were bound to CHO-S-B7-H4 cells. The antibodies were then expressed, purified, and validated. OD450 values ​​(μg / ml) detected by ELISA are shown in Table 11.

[0271] Table 11 OD450 detected by ELISA

[0272] Candidate clones with significant sequence differences (292-2-B02, 292-2-C06, 292-1-B03, 292-1-C09, and 292-2-E07-1) were selected for antibody preparation and subsequent validation.

[0273] Example 6: SDS-PAGE Detection of Candidate Single-Domain Antibodies

[0274] The constructed single-domain antibody expression vector was transiently transfected into 293F cells, and the recombinant antibody was purified using Protein A magnetic beads. SDS-PAGE was then performed to determine the purity of the candidate antibody. The results, shown in Figure 7, indicate that the molecular weight of the candidate antibody met expectations, and the purity was >90%, allowing for further experimental procedures.

[0275] Example 7 Single-domain antibody FACS detection

[0276] Table 12

[0277] FACS results (Figures 8A-8F, Figure 9) show that candidate antibodies 1-B03, 1-C09, and 2-B02 all exhibit strong binding activity to CHO-S-B7H4.

[0278] Example 8: ELISA detection and affinity assay of single-domain antibody binding to target protein

[0279] The ELISA test results are shown in Tables 13 and 14 and Figure 10. The antibody affinity test results are shown in Figure 11.

[0280] Table 13

[0281] Table 14

[0282] II. Screening for B7H4 single-domain antibodies using phage display library

[0283] Main reagents

[0284] Agar (Sigma, CAT#A1296), Peptone (Sigma, CAT#93926), Yeast Extract (OXOID, CAT#:LP0021), Sodium Chloride (Aladdin, CAT#:C111533), Potassium Chloride (Aladdin, CAT#:P112133), Magnesium Sulfate (Sinopharm, CAT#:10013018), Magnesium Chloride (Sinopharm, CAT#:10012818), Glucose (Sangon Biotech, CAT#:GT1991), SfiI (NEB, CAT#:R0123L), T4 DNA ligase (TaKaRa, CAT#:2011A), PrimeScript TMII 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#:6210B), NuHi Power Mix (XinHai Bio, CAT#:NH9303), 3M Sodium Acetate (pH 5.2-6) (Sigma, CAT#:126-96-5), DNA Fragment Recovery Kit (TakaRa, CAT#:9761), Gel Recovery Kit (Qiagen, CAT#:28706), Tiangen Plasmid Extraction Kit (Tiangen, CAT#:DP117), HRP-Anti-M13 (iCarTab), PE-anti-Human IgG (eBioscience, Cat#:12-4998-82), Rabbit anti-Llama IgG (H+L) Secondary Antibody [HRP] (Novus, CAT#NBP1-75095), SS320 competent cells (iCarTab), pDisplay phage display vector (iCarTab), HRP-Streptavidin (Boster, CAT#:BA1088), HRP-Protein A (Boster, BA1080), PBS (Gbico, CAT#14190-250), DMEM (Gbico, CAT#41965-062), RPMI1640 (Gbico, CAT#61870044), FBS (Gbico, CAT#10099-141), Genomic DNA Purification Kit (Lifetech, CAT#K0512)

[0285] Main consumables

[0286] 50mL Falcon centrifuge tubes (Corning, CAT#352070), electroporation cuvettes (Bio-Rad 0.2cm), 1.5mL RNase-free EP tubes (QSP, CAT#:509-GRD-Q), 200μL RNase-free PCR tubes (Axygen, PCR-02D-C), T125 shake flasks (Corning, CAT#431143), 15mL Falcon centrifuge tubes (Corning, CAT#430052), 6-well plates (Corning, CAT#3516), 96-well plates (Corning, CAT#3365)

[0287] Main equipment

[0288] Electroporator (Eppendorf Multiporator), centrifuge (Thermo FRESCO-17), incubator (Shanghai Jinghong DNP-9052), shaking incubator (Jingqi CO-O6U), clean bench (Sujing Antai SW-CJ-1FD), PCR instrument (Applied Biosystems ABI2720), biosafety cabinet (Haier HR40-IIA2), flow cytometer (Thermo Attune Nxt flow cytometer), Thermo 3111 CO2 incubator

[0289] Experimental methods

[0290] 1. Preparation of B7H4 recombinant protein

[0291] The method used is the same as the experimental method used in the yeast display library described above.

[0292] 2. Alpaca Immunization

[0293] The recombinant protein prepared above was used to immunize alpacas at 21-day intervals. Ten days after the last immunization, peripheral blood was collected, and serum was separated and the immunization effect was detected by ELISA.

[0294] 3. Detection of immune titer

[0295] The method used is the same as the experimental method used in the yeast display library described above.

[0296] 4. PBMC isolation and VHH antibody fragment cloning

[0297] The method used is the same as the experimental method used in the yeast display library described above.

[0298] 5. Construction of phage display library

[0299] 5.1 Construction of Single-Domain Antibody Phage Display Vector

[0300] The pDisplay vector and the VHH PCR gel recovery product obtained above were digested with SfiI, and the digestion system is as follows:

[0301] Table 15

[0302] Aliquot the above enzyme digestion system into 100 μL / tube and place them on a PCR instrument at 50°C overnight for enzyme digestion.

[0303] Table 16

[0304] The above enzyme digestion system was aliquoted into 100 μL tubes and incubated overnight at 50°C on a PCR instrument. The pDisplay vector fragment was separated using a 1% agarose gel, and a 5000 bp fragment was extracted for gel recovery. Simultaneously, the PCR digestion products were purified using a DNA fragment recovery kit, and the concentration was determined using NanoDrop. The digested pDisplay vector and VHH fragment were ligated using T4 ligase overnight at 16°C.

[0305] 5.2 Electroporation of Escherichia coli using phage ligation products

[0306] Prepare electroporation cuvettes, ligation products, and competent cells, and pre-chill them on ice. Add the pre-chilled library construction and ligation products to the competent cells and place them on ice for 1 min. Add 300 μL of DNA / competent cell mixture to each cuvette and place them on ice. Perform electroporation at 2500 V for 5 ms. Immediately after electroporation, resuspend the cells in SOC medium equilibrated to room temperature and incubate at 37°C with shaking for 1 hour. Take 15 mL of the bacterial suspension for phage rescue directly, and add an equal volume of 50% glycerol to the remaining 5 mL of electroporation product. Mix thoroughly and store at -80°C.

[0307] Separately, take 20 μL of bacterial culture and dilute it in 980 μL of 2YT medium. Then, take 100 μL of the diluted product and dilute it again in 900 μL of 2YT medium. Take 50 μL of this diluted product and spread it evenly on an LB agar plate containing ampicillin. Incubate overnight at 37°C. The next day, remove the plate and calculate the number of clones produced by each ligation, and calculate the library capacity. Simultaneously, pick 20 single clones from the plate and transfer them to 2YT medium containing ampicillin. Incubate at 37°C with shaking for approximately 6-8 hours, then send the bacterial culture for sequencing (using universal sequencing primer M13R) to calculate library diversity.

[0308] 6. Preparation of phage library and phage precipitation

[0309] The electroporated conversion product was diluted with 2YT and adjusted to OD. 600 The concentration was approximately 0.2. Ampicillin was added to a final concentration of 100 μg / mL and the mixture was placed in a constant temperature shaker at 37°C and 225 rpm until the OD value reached approximately 0.2. 600 Stop when the value reaches 0.5. Add M13KO7, shake well, and let stand at 37°C for 30 min, then incubate at 37°C and 225 rpm for 1 h. M13KO7 volume = 10 x volume x OD 600 x 5x 10 8 / M13KO7 titer. Centrifuge the bacterial culture at 6000 rpm for 10 min, resuspend in 2YT-AK medium, and incubate overnight at 25°C and 200 rpm. Centrifuge the bacterial culture at 10000 rpm for 15 min. Discard the precipitate, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG / NaCl to the tube, mix well, and incubate at 4°C for 2 h. Centrifuge the precipitated phage supernatant at 10000 rpm and 4°C for 30 min. Discard the supernatant, and resuspend the precipitate (phage) in 1 mL of sterile PBS from each 50 mL centrifuge tube. Transfer the resuspended phage to a 1.5 mL EP tube and centrifuge at 12000 g and 4°C for 5 min. Transfer the supernatant to a new 1.5 mL EP tube, add 250 μL of PEG / NaCl to each tube, mix well, and incubate at 4°C for 10 min. Centrifuge at 12000g for 10 min, discard the supernatant, and resuspend in 1 mL PBS. Centrifuge at 12000g for 5 min, discard the precipitate, and transfer the supernatant to a new 1.5 mL EP tube. Centrifuge at 12000g for 5 min, and transfer the supernatant to a new 1.5 mL EP tube to obtain the original phage library.

[0310] Take 10 μL of the precipitate and add it to 90 μL of 2YT culture medium, and record this as 10 μL. -1 Dilute 10 times to 10 in succession. -9 Take 10 -7 10 -8 10 -9 Three gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD. 600 ER2738 was mixed at 0.5 and placed in a 37°C water bath for 10 minutes. Each 100 μL was coated onto an LB-AMP solid plate and incubated overnight at 37°C. The spots were counted the next day to determine the titer.

[0311] Titer calculation: Select plates with a number of spots between 30 and 300, take the average of two plates, multiply the number of spots by the dilution factor, and then multiply by 100 to obtain the titer.

[0312] 7. Selection of phage display libraries

[0313] Recombinant proteins were panned, and then the phage display library was incubated with the recombinant proteins. The recombinant phages bound to the target antigen were eluted using TEA and amplified. After 3-4 rounds of panning, single clones were selected for sequencing.

[0314] Take approximately 6 x 10 11150 μL of the Phage library was diluted to approximately 1 mL with 1% PBSA and added to an ELISA plate coated with the target protein. The plate was incubated at 4°C for 1 h. The bound phage was eluted with 600 μL of 1xTEA for 10 min. The eluted product was transferred to a pre-blocked EP tube and neutralized with 300 μL of Tris-HCl.

[0315] Take 10 μL of the output product and add it to 90 μL of 2YT medium, label it as 100, and then dilute it 10-fold to 10⁻⁶ each time. -2 Take 10 1 10 0 10 -1 10 -2 Four gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD. 600 ER2738(10) with a value of 0.5 1 The undiluted product (20 μL) was directly added to ER2738, mixed well, placed in a 37°C water bath, and left to stand for 10 min. Each 100 μL was coated onto an LB-AMP solid plate and incubated overnight at 37°C. The spots were counted the next day to determine the titer.

[0316] Titer calculation: Select plates with a number of spots between 30 and 300, take the average value of two plates, multiply the number of spots by the dilution factor, and then multiply by the elution volume.

[0317] 8. Phage ELISA

[0318] Dispense 500 μL of 2YT-Amp medium into each well of a 96-well deep-well plate. Pick single colonies from the output plate and incubate at 37°C and 225 rpm until OD. 600 Up to 0.5; wells E12 and F12 are used as blank controls, with no clones selected and only culture medium added; wells G12 and H12 are used as positive controls, with no clones selected and the binding of antigen and positive antibody detected.

[0319] Simultaneously, the antigen was coated onto an ELISA plate with CBS at a concentration of 1 μg / mL, 100 μL / well, at 37°C for 2 hours. Separately, a 96-well deep-well plate was prepared, and 500 μL of 2YT-A culture medium was dispensed into each well. OD values ​​were then sequentially aspirated using a pipette. 600 Add 10 μL of 0.5% bacterial culture to a freshly dispensed 96-well plate and incubate overnight at 37°C and 225 rpm. This is the bacterial culture for sequencing.

[0320] To OD 600 Add M13KO7 to a bacterial culture solution with a concentration of 0.5, mix well, and incubate at 37°C for 15 minutes. The volume of M13KO7 = 10 x volume x OD. 600 x 5x 108 / M13KO7 titer. After infection, place the bacterial culture on a shaker and incubate at 37°C, 225 rpm for 45 min. Centrifuge the bacterial culture at 4000 rpm for 10 min, discard the supernatant, resuspend in 2YT-AK medium (800 μL per well), and incubate overnight at 30°C, 210 rpm. Simultaneously, discard the antigen from the ELISA plate, wash three times with PBST, and block with 250 μL / well of 3% MPBS, incubating overnight at 4°C; also block an additional blank plate as a BLANK. The next day, the 96-well deep-well plate was placed in a centrifuge and centrifuged at 4000 rpm for 10 min. The milk in the ELISA plate was discarded, and the plate was washed 4 times with 200 μL PBST. 50 μL PBST was added to each well, followed by 50 μL of the centrifuged phage supernatant. The plate was incubated at 4℃ for 1 h. The supernatant was discarded, and the plate was washed 5 times with PBST. 100 μL of HRP-Anti M13 secondary antibody was diluted with PBST in each well and incubated at 4℃ for 45 min. The secondary antibody was washed away, and the plate was washed 5 times with PBST. TMB was added for color development at room temperature for 10 min, and hydrochloric acid was used to stop the reaction. The readings were taken, and clones with an S / N ratio greater than 6 were selected and sent for testing with the culture medium.

[0321] 9. Construction of expression carriers

[0322] The method used is the same as the experimental method used in the yeast display library described above.

[0323] 10. Expression and purification of candidate single-domain antibodies

[0324] The method used is the same as the experimental method used in the yeast display library described above.

[0325] 11. FACS detection of the binding of recombinant antibodies to target proteins

[0326] CHO and CHO-B7H4 cell lines were resuscitated in liquid nitrogen and adjusted to the logarithmic growth phase. Each cell line was divided into a subset of 3 x 10⁵ cells. The expressed antibody was incubated separately on the target cells, and after thorough mixing, the cells were incubated at room temperature for 1 hour. The cells were centrifuged at 800 x g for 5 minutes at room temperature, the supernatant containing the antibody was removed, and the cells were washed three times with PBS. 100 μL of PE-labeled anti-human IgG (1:500 dilution) was added, and after thorough mixing, the cells were incubated at room temperature in the dark for 30 minutes. The cells were centrifuged at 800 x g for 5 minutes at room temperature, the supernatant containing the secondary antibody was removed, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS for flow cytometry analysis.

[0327] Example 1: Alpaca Immune Potency Detection

[0328] The purity of the Human B7H4 recombinant protein was greater than 90% as determined by SDS-PAGE, making it suitable for use in alpaca immunization and antibody screening.

[0329] Serum was isolated from immunized alpacas and subjected to limiting dilutions according to the dilution gradient in Table 17. ELISA assays were then performed on pre-coated 96-well plates. The ELISA results showed that the immune serum bound to the B7H4 recombinant protein, and the OD value changed gradients with the serum dilutions, achieving an immunogenic titer of 1:64K or higher. 100 ml of peripheral blood was collected to prepare a cDNA library for antibody display library construction.

[0330] Table 17

[0331] Example 2: Amplification of the VHH Fragment

[0332] Peripheral blood was collected, total RNA was extracted, and reverse transcribed into cDNA. Two rounds of PCR were performed using single-domain antibody amplification primers, and the PCR products were analyzed by agarose gel electrophoresis. The first round of PCR yielded bands of approximately 1000 bp and 750 bp, respectively. The 750 bp fragment was recovered and used as the template for the second round of PCR. The second round of PCR yielded a band of approximately 400 bp, which was the VHH fragment. The pDisplay vector and the obtained VHH fragment were digested with SfiI, and the PCR products were recovered from the gel. The digested pDisplay vector and VHH fragment were ligated using T4 ligase and incubated overnight at 16°C before transformation into *E. coli*.

[0333] Example 3: Diversity Analysis of Phage Display Library

[0334] Randomly selected single clones were sequenced to analyze the diversity of the constructed phage display library. Sequencing results showed that the empty vector rate and antibody repetition rate of the phage display library were no higher than 5%. The *E. coli* library size was 2.69 * 102 9 .

[0335] Example 4: Phage Display Library Selection and Antibody Validation

[0336] 4.1 Phage Display Library Selection

[0337] The recombinant protein of the target antigen was co-incubated with a single-domain antibody phage display library to enrich phages that specifically bind to the target antigen. After four enrichment cycles, the input and output of each cycle were statistically analyzed, and the enrichment factor for each cycle was calculated.

[0338] First panning: 96-well plate solid-phase coating, panning antigen: B7-H4-His

[0339] Table 18

[0340] Second panning: 96-well plate solid-phase coating, panning antigen: B7-H4-His

[0341] Table 19

[0342] Third screening: magnetic bead screening, screening for antigen: B7-H4-His

[0343] Table 20

[0344] 4.2 Phage ELISA

[0345] Output products were selected for phage ELISA detection. The target protein was coated onto the phage, and the recombinant phage supernatant was used for ELISA detection. The control group consisted of directly blocked wells. Clones with an S / N ratio greater than 5 were selected for sequencing, and antibody sequences were analyzed. A eukaryotic expression vector was constructed for validation.

[0346] The results of the three rounds of selection are shown in Figures 13A-13C.

[0347] 4.3 Validation of candidate antibody transfection supernatant

[0348] The FACS results are shown in Figures 14A-14C. Clones 1-B02, 1-H06, and 6-A08 all specifically bind to CHO-S / B7-H4.

[0349] 4.4 Candidate Antibody FACS Detection

[0350] According to FACS detection (Figures 15A-15I), a total of 7 B7H4 positive antibody sequences were obtained, among which 6-A08, 1-B02, 8-C4, and 8-E2 bound strongly to the CHO-S / B7-H4 cell line. EC50 values ​​are shown in Figure 16 and Table 21.

[0351] Table 21

[0352] The positive control antibody 20502 used in this application is the B7-H4 antibody numbered 20502 described in patent application CN111094352A.

[0353] Amino acid sequences of single-domain antibodies were screened from the yeast display library.

[0354] >SDAB2224-292-1-B03

[0355] >SDAB2224-292-1-C09

[0356] >SDAB2224-292-2-B02

[0357] >SDAB2224-292-2-C06

[0358] >SDAB2224-292-2-E07-1

[0359] >SDAB2224-292-1-H09

[0360] >SDAB2224-292-3-D06-2

[0361] >SDAB2224-292-4-F06-1

[0362] Amino acid sequences of single-domain antibodies were screened from the phage display library.

[0363] >8-C4

[0364] >8-E2

[0365] >1-B02

[0366] >6-A08

[0367] >8-A1

[0368] >1-H06

[0369] >8-D12

[0370] Table 22 Antibody CDR Sequences

[0371] Table 23 Abbreviations

[0372] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-B7H4 single-domain antibody, characterized in that, The complementarity-determining region (CDR) of the VHH chain of the anti-B7H4 single-domain antibody is selected from one or more of the following groups: (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; (2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; (3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:6; (4) CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:6; (5) CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:12, and CDR3 shown in SEQ ID NO:13; (6) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; (7) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:6; (8) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18; (9) CDR1 shown in SEQ ID NO:19, CDR2 shown in SEQ ID NO:20, and CDR3 shown in SEQ ID NO:21; (10) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18; (11) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:22, and CDR3 shown in SEQ ID NO:23; (12) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:25, and CDR3 shown in SEQ ID NO:26; (13) CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28; (14) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:

31.

2. The anti-B7H4 single-domain antibody of claim 1, wherein, The amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is as shown in any one of SEQ ID NO:32-46, or has ≥80%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity with it. 3.The anti-B7H4 single-domain antibody of claim 1, wherein, The amino acid sequence of the VHH chain of the anti-B7H4 single-domain antibody is shown in any one of SEQ ID NO:32-35 and 40-43.

4. An antibody against B7H4, characterized in that, The antibody comprises the VHH chain of one or more single-domain antibodies against B7H4 as described in any one of claims 1-3.

5. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-4. The polynucleotide encodes a protein selected from the group consisting of: an anti-B7H4 single-domain antibody as described in any one of claims 1-3, or an antibody as described in claim 4.

6. An expression vector, characterized by, The expression vector contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6, or its genome is integrated with the polynucleotide as described in claim 5. 8.A method for producing an anti-B7H4 single-domain antibody, characterized in that, The method includes the following steps: (a) Under conditions suitable for generating single-domain antibodies, the host cells as described in claim 7 are cultured to obtain a culture containing the anti-B7H4 single-domain antibody; (b) Isolating and / or recovering the anti-B7H4 single-domain antibody from the culture; and (c) Optionally, the anti-B7H4 single-domain antibody obtained in step (b) is purified and / or modified.

9. An immunoconjugate, comprising, The immunoconjugate contains: (a) the anti-B7H4 single-domain antibody as described in any one of claims 1-3, or the anti-B7H4 antibody as described in claim 4; and (b) The conjugate selected from the group consisting of: detectable markers, drugs, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

10. Use of the anti-B7H4 single-domain antibody according to any one of claims 1-3, the antibody against B7H4 according to claim 4, or the immunoconjugate according to claim 9, characterized in that, Used for preparation: (1) Medications for the prevention and / or treatment of B7H4-related diseases; (2) Reagents for detecting B7H4.

11. A pharmaceutical composition, characterized by, The pharmaceutical composition contains: (i) the anti-B7H4 single-domain antibody as described in any one of claims 1-3, the anti-B7H4 antibody as described in claim 4, or the immunoconjugate as described in claim 9; and (ii) Pharmaceutically acceptable carriers.

12. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the anti-B7H4 single-domain antibody as described in any one of claims 1-3, or the anti-B7H4 antibody as described in claim 4; and (ii) Optional tag sequences to assist in expression and / or purification.

13. A kit characterized in that, The kit contains an anti-B7H4 single-domain antibody as described in any one of claims 1-3, an anti-B7H4 antibody as described in the second aspect of the present invention, or an immunoconjugate as described in claim 9.

14. A method for preventing and / or treating B7H4-related diseases, the method comprising administering to a subject a single-domain anti-B7H4 antibody as described in any one of claims 1-3, an anti-B7H4 antibody as described in claim 4, or an immunoconjugate as described in claim 9.

15. A method for in vitro detection of B7H4 or fragments thereof in a sample, the method comprising the steps of: (1) in vitro, contacting the sample with the anti-B7H4 single-domain antibody according to any one of claims 1-3, the antibody against B7H4 according to claim 4, or the immunoconjugate according to claim 9; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of B7H4 or a fragment thereof in the sample.

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