Anti-hhla2 antibodies or antigen-binding fragments thereof, pharmaceutical compositions thereof, and uses

Abstract

An anti-HHLA2 antibody or its antigen-binding fragment thereof is provided, and a pharmaceutical composition comprising the same is provided. The anti-HHLA2 antibody is preferably a nanobody comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, which can exhibit the desired antigen-binding affinity and can effectively block the HHLA2-KIR3DL3 signaling pathway by inhibiting the binding of HHLA2 to KIR3DL3, thereby exhibiting the effects of tumor treatment and enhanced immune response.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, and specifically relates to an antibody molecule capable of specifically binding to HHLA2, a pharmaceutical composition containing the antibody, and its use in anti-tumor and immune response enhancement. Background Technology

[0002] HHLA2 (Human Endogenous Retrovirus-H Long Terminal Repeat-Associating Protein 2), also known as B7-H7, is a member of the B7 molecular family and one of the most recently identified immune checkpoint molecules. The HHLA2 encoding gene is located on human chromosome 3, q13.13, and contains 414 amino acids. The HHLA2 protein consists of an N-terminal signal peptide, an extracellular domain tandemly containing IgV-IgC-IgV domains (with 6 glycosylation sites), a transmembrane region, and a 49-amino acid cytoplasmic tail, classifying it as a type I transmembrane protein. Unlike other B7 and CD28 family members, HHLA2 is not expressed in mice and rats, but has homologous proteins (90% homology) only in humans and primates such as monkeys. The HHLA2 molecule was discovered in 1999 by J. Douglas Freeman's team and identified as a member of the B7 molecular family by Yuko Ohta's research team in 2012.

[0003] In 2013, Xingxing Zang's research team confirmed the important regulatory function of HHLA2 in the immune system, demonstrating its significant inhibitory effect on the proliferation of CD4+ T and CD8+ T cells and their cytokine secretion. Meanwhile, it has been reported that while HHLA2 expression is limited in most normal human tissues, it is significantly upregulated in various tumors. Multiple studies have shown that HHLA2 is significantly increased in various solid tumors, including gastric cancer, pancreatic cancer, renal cell carcinoma, thyroid cancer, and non-small cell lung cancer, and is associated with poor tumor prognosis. More importantly, studies have found a negative correlation between HHLA2 and PD-L1 expression levels in some tumors. Therefore, immunotherapy targeting HHLA2 holds promise as a treatment option for cancer patients who have failed PD-1 / PD-L1 therapy.

[0004] Addressing the issue of limited efficacy of existing immunotherapies (such as PD-1 / PD-L1 and cell therapy) for most solid tumors, developing new therapeutic drugs based on HHLA2 holds promise for providing new options for patients with solid tumors. However, currently available anti-HHLA2 antibodies in clinical trials are all whole-length IgG antibodies, which have difficulty penetrating solid tumors with dense matrix components. Furthermore, whole-length IgG antibodies present limitations in drug development and production. Therefore, the development of new HHLA2-targeting drugs is needed to enhance their clinical application potential. Summary of the Invention

[0005] This disclosure provides anti-HHLA2 antibodies targeting HHLA2, particularly anti-HHLA2 heavy chain antibodies (HcAbs) or anti-HHLA2 nanobodies.

[0006] In one aspect, this application provides an anti-HHLA2 antibody or an antigen-binding fragment thereof, comprising:

[0007] (1) Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0008] (2) Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2 or 6; and

[0009] (3) Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3 or 7.

[0010] In another aspect, this application relates to a polynucleotide encoding the aforementioned anti-HHLA2 antibody or an antigen-binding fragment thereof.

[0011] In another aspect, this application relates to expression vectors comprising the aforementioned polynucleotides.

[0012] In another aspect, this application relates to host cells incorporating the aforementioned polynucleotides or their expression vectors.

[0013] In another aspect, this application relates to a pharmaceutical composition comprising: the above-described anti-HHLA2 antibody or its antigen-binding fragment, and optionally pharmaceutically acceptable excipients.

[0014] In another aspect, this application relates to the use of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition in the preparation of a medicament for treating HHLA2-related diseases. Alternatively, this application relates to an anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for treating HHLA2-related diseases. Alternatively, this application relates to a method of treating HHLA2-related diseases in a subject of need, comprising administering the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition to the subject. Alternatively, this application relates to the use of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for treating HHLA2-related diseases.

[0015] In another aspect, this application relates to the use of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition in the preparation of a medicament for enhancing immune responses, particularly during cancer treatment. Alternatively, this application relates to an anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for enhancing immune responses. Alternatively, this application relates to a method of enhancing an immune response in a subject in need, comprising administering (e.g., a therapeutically effective amount) of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition to the subject. Alternatively, this application relates to the use of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for enhancing immune responses.

[0016] In another aspect, this application relates to the use of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment in the preparation of reagents for detecting HHLA2 protein. Alternatively, this application relates to the aforementioned anti-HHLA2 antibody or its antigen-binding fragment for detecting HHLA2 protein. Alternatively, this application relates to a method for detecting HHLA2 protein in a sample, comprising contacting the aforementioned anti-HHLA2 antibody or its antigen-binding fragment, a polynucleotide, or a pharmaceutical composition with the sample. Alternatively, this application relates to the use of the aforementioned anti-HHLA2 antibody or its antigen-binding fragment for detecting HHLA2 protein.

[0017] The anti-HHLA2 antibody or its antigen-binding fragment described in this article can exhibit the desired antigen-binding affinity, effectively block the HHLA2-KIR3DL3 signaling pathway by inhibiting the binding of HHLA2 to KIR3DL3, and can demonstrate tumor therapeutic effects and enhance immune response. Attached Figure Description

[0018] Figure 1 The preparation process of anti-HHLA2 nanobodies is shown.

[0019] Figures 2A-2BThe diagrams show the competitive sorting pattern of KIR3DL3 and the binding characteristics of the test clones M1F1-55 and M1F1-92 with biotinylated human HHLA2 and biotinylated cynomolgus monkey HHLA2, respectively.

[0020] Figures 3A-3B The heavy chain variable region (VHH) antibody-Fc fusion protein of camel-family heavy chain only antibody and human HHLA2 are shown respectively. Figure 3A ) and cynomolgus monkey-derived HHLA2 ( Figure 3B The first batch of ELISA test results were obtained by combining the results of the combination of the two.

[0021] Figures 4A-4B The VHH antibody-Fc fusion protein and human HHLA2 were shown separately. Figure 4A ) and cynomolgus monkey-derived HHLA2 ( Figure 4B The second batch of ELISA test results were combined with the results of the second batch of ELISA tests.

[0022] Figures 5A-5C The effects of VHH antibody-Fc fusion protein on HCC827 cells were shown respectively. Figure 5A ), ASPC-1-OE-B7H7 cells ( Figure 5B ) and 293T cells ( Figure 5C The first batch of results for the binding effect of ) .

[0023] Figures 6A-6B The effects of VHH antibody-Fc fusion protein on ASPC-1-OE-B7H7 cells ( Figure 6A ) and 293T cells ( Figure 6B The second batch of test results for the binding effect of )

[0024] Figures 7A-7B The binding blocking effect of the VHH antibody-Fc fusion protein on KIR3DL3 was shown in two batches of experiments.

[0025] Figures 8A-8D The effects of M1F1-55-VHH antibody and M1F1-92-VHH antibody on the PanC-1 cell line were shown respectively. Figure 8A AsPC-1 cell line Figure 8B HPAF-II cell line Figure 8C ) and SW1116 cell line ( Figure 8D The results of the in vitro killing effect determination of )

[0026] Figure 9 The results of the assay of antitumor activity of the M1F1-55-VHH antibody and M1F1-92-VHH antibody disclosed herein in tumor-bearing mice are shown.

[0027] Figures 10A-10GThe M1F1-92-VHH antibody was shown to promote IFN-γ ( Figure 10A ), TNF-α Figure 10B ), Fas( Figure 10C ), perforin Figure 10D ), GZMB Figure 10E Granulysin Figure 10F ) and FasL( Figure 10G The effect of secretion. Detailed Implementation

[0028] The concept of this application will be explained below with reference to specific embodiments; however, those skilled in the art will understand that the scope of this application is not limited thereto.

[0029] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (2012); Abbas et al., Cellular and Molecular Immunology, Elsevier Science Health Science div (2009); He Wei et al., Medical Immunology (2nd ed.), People's Medical Publishing House, 2010.

[0030] Unless otherwise stated, the terms “comprise”, “comprises”, and “comprising” or their equivalents (contain, contain, containing, include, include, including) used herein are open-ended expressions, meaning that they may cover other unspecified elements, components, and steps in addition to those listed.

[0031] Unless otherwise stated, the terms “optional” or “optionally” as used herein mean that the object or event it modifies exists or does not exist, or occurs or does not occur.

[0032] Unless otherwise stated, the term “subject” as used herein encompasses any vertebrate, such as mammals and non-mammals, including humans, non-human primates, sheep, dogs, cats, horses, cattle, chickens, pigs, rats, etc.

[0033] Unless otherwise stated, all figures used herein to represent amounts of components, measurements, or reaction conditions should be understood to be modified by the term "about" in all cases. When used with percentages, the term "about" may mean, for example, ±1%, preferably ±0.5%, more preferably ±0.1%.

[0034] In this document, unless the context clearly indicates otherwise, singular terms encompass plural referents, and vice versa. Unless the context clearly indicates otherwise, the word "or" in this document is intended to include "and".

[0035] Unless otherwise stated, the term “treatment” as used herein means that, after administration, it can inhibit, suppress, reduce, improve, slow down, alleviate or eliminate a disease or its associated symptoms, delay, slow down, stop or terminate the progression of a disease or its associated symptoms, or prevent, control or reduce the recurrence of a disease or its associated symptoms.

[0036] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or fragment thereof that specifically recognizes and binds to a particular antigen. Antibodies are classified into IgG, IgM, IgA, IgD, and IgE. A typical antibody is a tetramer, composed of two identical pairs of polypeptide chains. Each pair has one light chain and one heavy chain. The heavy chain consists of a variable region (VH) and a constant region (CH), with the constant region typically including three domains: CH1, CH2, and CH3. The light chain consists of a variable region (VL) and a constant region (CL). Both the variable regions of the heavy and light chains contain three highly variable regions: CDR1, CDR2, and CDR3.

[0037] The location of the CDR can be determined using various numbering rules well known in the art, such as Kabat, AbM, Chothia, IMGT, and Contact. Given the amino acid sequence of the antibody's variable region, those skilled in the art can determine the antibody's CDR using conventional methods. The table below shows CDRs determined according to different numbering rules.

[0038] CDR Kabat Chothia IMGT Contact HCDR1 31-35 26-32 27-38 30-35 HCDR2 50-65 52-56 56-65 47-58 HCDR3 95-102 95-102 105-117 93-101 LCDR1 24-34 24-34 27-38 30-36 LCDR2 50-56 50-56 56-65 46-55 LCDR3 89-97 89-97 89-97 89-96

[0039] The term "antigen-binding fragment" as used herein refers to an antibody moiety containing one or more CDRs capable of recognizing and specifically binding to a particular antigen, wherein the antigen-binding fragment retains substantially the same antigen-specific binding activity as the full-length antibody. The antigen-binding fragment includes, but is not limited to, CDRs, Fd fragments, etc.

[0040] The term "Fc region" in this article refers to the C-terminal region of the immunoglobulin heavy chain, which typically contains the immunoglobulin heavy chain constant region in addition to the first constant region (CH1). For IgG, the Fc region may include CH2 and CH3, as well as the hinge region between CH1 and CH2.

[0041] The term "heavy chain antibody" or "HcAb" refers to a functional antibody that contains a heavy chain but lacks the light chain typically found in 4-chain antibodies. Cameloideas (e.g., camels, llamas, or alpacas) are known to produce HcAbs. Heavy chain antibodies lack the light chain and the heavy chain constant region 1 (CH1), containing only two heavy chains consisting of a variable region (VHH) and other constant regions. The variable region is linked to the constant region via a hinge-like structure. Each heavy chain of cameloid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3).

[0042] The antigen-binding fragment of a heavy chain antibody includes a separated CDR region, VHH, and a single-chain heavy chain antibody. By fusing with the Fc region of human IgG, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG (e.g., IgG1, IgG2, IgG3, IgG4). The terms "heavy chain variable region domain of heavy chain antibody," "VHH," and "nanobody" are used interchangeably.

[0043] The terms "identity percentage" or "sequence identity percentage" used herein are interchangeable and refer to the percentage of identical amino acid residues between two or more sequences compared, after introducing necessary gaps to maximize the number of identical amino acids between the sequences. The identity percentage between sequences can be readily determined using tools known in the art, such as BLAST.

[0044] The term “treatment” in this article means reducing or alleviating a disease or its symptoms, decreasing the frequency or rate of onset of a disease or its symptoms, reducing the risk of developing a disease or its symptoms, reducing or terminating a disease or its symptoms, causing complete or partial reversal of a disease or its symptoms, curing a disease or its symptoms, or a combination of the above.

[0045] The term “therapeutic effective dose” or “effective dose” refers to the amount or dose of a drug that is sufficient to effectively prevent or improve a disease or its symptoms and / or reduce the severity of a disease or its symptoms within a certain period of time after administration to a subject.

[0046] The term "pharmaceuticalally acceptable excipient" in this article refers to materials that are physiologically compatible with the active ingredient and the subject and are commonly used in pharmaceutical preparations to assist in the preparation, production, and storage of the drug.

[0047] In this paper, the terms “X” and “Xaa” are equivalent and refer to unspecified amino acids. The scope of an amino acid is specified by the definition in the relevant description. In order to distinguish multiple “X”s in the same amino acid sequence, the multiple Xs that appear successively are numbered (i.e., written as Xn) and their scope is defined respectively.

[0048] For purposes of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because their publications predate the filing date of this invention. All statements regarding the dates of these documents or descriptions of their contents are based on information available to the applicant of this invention and do not constitute any admission of the correctness of the dates or contents of these documents.

[0049] The following description of different implementation methods will enable those skilled in the art to better understand the technical solutions of the present invention.

[0050] Currently, all anti-HHLA2 antibodies under development are full-length antibodies, which have disadvantages such as large molecular weight and low tissue penetration. They are difficult to effectively enter the tumor to promote an immune response in solid tumors with dense fibrous stroma (especially pancreatic cancer); furthermore, genetic engineering modification is relatively complex. From a production perspective, full-length antibodies rely on mammalian cell expression systems, resulting in high costs. Nanobodies, on the other hand, are the smallest known antigen-binding units, with simple molecular structures, strong tissue penetration, and low immunogenicity. Furthermore, nanobodies are easily genetically engineered and can be multivalent through simple fusion with other protein drugs; whether used alone to develop HHLA2-KIR3DL3 signaling blockers or in combination with other targets to develop multivalent anti-tumor drugs, they represent a promising molecular type. In addition, nanobodies can be produced using various expression systems, including eukaryotic expression systems, E. coli, and yeast, offering greater flexibility in production processes. The discovery and preparation of HHLA2 nanobodies with improved affinity, stronger antitumor activity and / or higher molecular stability can further improve clinical efficacy, while having great potential for drug development and improvement and broad clinical application value.

[0051] This application provides an anti-HHLA2 antibody or its antigen-binding fragment, comprising:

[0052] (1) Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0053] (2) Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2 or 6; and

[0054] (3) Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3 or 7.

[0055] Unless otherwise stated, the CDR of the antibody described in this application is defined according to the IMGT numbering rules. However, those skilled in the art will understand that CDR sequences numbered according to any one or more other numbering rules known in the art, such as the AbM numbering rules, the Kabat numbering rules, the Chothia numbering rules, and the Contact numbering rules, also fall within the scope of protection of this application.

[0056] In some embodiments, the anti-HHLA2 antibody or its antigen-binding fragment comprises:

[0057] (1) Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0058] (2) Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2; and

[0059] (3) Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3.

[0060] In some embodiments, the anti-HHLA2 antibody or its antigen-binding fragment comprises:

[0061] (1) Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0062] (2) Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:6; and

[0063] (3) Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:7.

[0064] In some embodiments, the anti-HHLA2 antibody may be a camel-derived antibody or a humanized antibody.

[0065] In some embodiments, the anti-HHLA2 antibody described in this application is a nanobody or a heavy chain antibody.

[0066] In some embodiments, the anti-HHLA2 antibody described in this application is a nanobody. In some embodiments, the nanobody has the following composition: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0067] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0068] FR1 comprises the amino acid sequence shown in SEQ ID NO:9 or 13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0069] FR2 comprises the amino acid sequence shown in SEQ ID NO:17, 10 or 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0070] FR3 comprises the amino acid sequence shown in SEQ ID NO: 18, 19, 20, 11 or 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0071] FR4 comprises an amino acid sequence shown in SEQ ID NO:21, 22, 12 or 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0072] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0073] FR1 comprises the amino acid sequence shown in SEQ ID NO:9 or 13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0074] FR2 comprises the amino acid sequence shown in SEQ ID NO:10 or 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0075] FR3 comprises the amino acid sequence shown in SEQ ID NO:11 or 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and

[0076] FR4 comprises the amino acid sequence shown in SEQ ID NO:12 or 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0077] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0078] FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0079] Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0080] FR2 comprises the amino acid sequence shown in SEQ ID NO:10 or 17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0081] Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2;

[0082] FR3 comprises the amino acid sequence shown in SEQ ID NO:18, 19, 20 or 11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0083] Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and

[0084] FR4 comprises the amino acid sequence shown in SEQ ID NO:21 or 22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0085] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0086] FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0087] Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0088] FR2 comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0089] Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2;

[0090] FR3 comprises the amino acid sequence shown in SEQ ID NO:18, 20 or 11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0091] Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and

[0092] FR4 comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0093] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0094] FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0095] Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0096] FR2 comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0097] Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2;

[0098] FR3 comprises the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0099] Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and

[0100] FR4 comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0101] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0102] FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0103] Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0104] FR2 comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0105] Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2;

[0106] FR3 comprises the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0107] Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and

[0108] FR4 comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0109] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0110] FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0111] Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0112] FR2 comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0113] Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2;

[0114] FR3 comprises the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0115] Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and

[0116] FR4 comprises the amino acid sequence shown in SEQ ID NO:12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0117] In some embodiments, the nanobody or its antigen-binding fragment comprises:

[0118] FR1 comprises the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0119] Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1;

[0120] FR2 comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0121] Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:6;

[0122] FR3 comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it;

[0123] Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:7; and

[0124] FR4 comprises the amino acid sequence shown in SEQ ID NO:16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0125] In some preferred embodiments, the anti-HHLA2 antibody or its antigen-binding fragment described in this application comprises a VHH (Variable domain of heavy chain of heavy chain antibody). Preferably, the VHH comprises an amino acid sequence shown in SEQ ID NO: 23, 24, 25, 26, 27, 4 or 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity with it.

[0126] In some preferred embodiments, the VHH comprises the amino acid sequence shown in SEQ ID NO:23, 24, 25, 26, or 27, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity with it. In some specific embodiments, amino acids differing from those shown in SEQ ID NO:23, 24, 25, 26, or 27 are predominantly (or all) present in the FR (backbone region).

[0127] In some preferred embodiments, the anti-HHLA2 antibody or its antigen-binding fragment described in this application comprises VHH (Variable domain of heavy chain of heavy chain antibody). Preferably, the VHH comprises an amino acid sequence shown in SEQ ID NO:4 or 8, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity with it.

[0128] In some specific embodiments, amino acids that differ from the amino acid sequence shown in SEQ ID NO:4 or 8 by at least 80% sequence identity are predominantly (or all) present in the FR (backbone region).

[0129] In some embodiments, the anti-HHLA2 antibody described in this application is a heavy chain antibody. In some preferred embodiments, the heavy chain antibody comprises CH2 and CH3 fragments of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or the Fc region of human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some preferred embodiments, the heavy chain antibody comprises the Fc region of human IgG1 or the Fc region of human IgG4. In some preferred embodiments, the heavy chain antibody comprises the Fc region whose amino acid sequence is shown in SEQ ID NO:5.

[0130] In some embodiments, this application relates to a polynucleotide encoding the aforementioned anti-HHLA2 antibody or its antigen-binding fragment.

[0131] It is known to those skilled in the art that, due to codon degeneracy, each amino acid sequence can be encoded by multiple nucleic acid sequences (or polynucleotides). The nucleic acid sequence encoding the anti-HHLA2 antibody or its antigen-binding fragment of this disclosure can be synthesized using methods known in the art (e.g., de novo solid-phase DNA synthesis, PCR amplification). Given that specific amino acid sequences have been described in this disclosure, those skilled in the art can easily modify one or more codons of their respective coding sequences without altering the amino acid sequence of the anti-HHLA2 antibody or its antigen-binding fragment of this disclosure to prepare a variety of different nucleic acids capable of expressing proteins having the same amino acid sequence.

[0132] In some embodiments, this application relates to expression vectors comprising the aforementioned polynucleotides or expressing the aforementioned anti-HHLA2 antibody or its antigen-binding fragment. Recombinant vectors can be constructed using any vector known in the art capable of expressing the anti-HHLA2 antibody or its antigen-binding fragment. Vectors described herein include, but are not limited to, naked plasmids, phage particles, yeast plasmids, adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), retroviruses (such as lentiviruses), poxviruses, papillomaviruses, papillomaviruses (such as SV40), rod-shaped viruses, or baculoviruses. For ease of production and purification, the expression vector may also include secretory signal peptides, expression tags, etc.

[0133] In some embodiments, this application relates to host cells integrated with the aforementioned polynucleotides or expression vectors, or expressing the aforementioned anti-HHLA2 antibody or its antigen-binding fragment. The cells described herein may be prokaryotic or eukaryotic cells. In some embodiments, the cells are selected from yeast cells, mammalian cells, or any other cells suitable for expressing the relevant proteins. In some examples, the mammalian cells are, for example, Chinese hamster ovary (CHO) cells (e.g., ExpiCHO cells, GS-CHO-K1 cells, or CHO-K1 cells), CHO-S cells, 293 cells (e.g., Expi293F cells), and monkey kidney cells.

[0134] In some embodiments, this application relates to a pharmaceutical composition comprising: the above-described anti-HHLA2 antibody or its antigen-binding fragment, and optionally pharmaceutically acceptable excipients.

[0135] The excipients described herein can be any pharmaceutically acceptable excipient, such as, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc. For other pharmaceutically acceptable pharmaceutical excipients, please refer to, for example, *Handbook of Pharmaceutical Excipients* (4th Edition), by RC Luo et al., translated by Zheng Junmin, Chemical Industry Press, 2005.

[0136] In some embodiments, the pharmaceutical composition further comprises a cancer therapeutic agent, such as a chemotherapy or targeted therapy drug for cancer.

[0137] In some embodiments, the pharmaceutical composition may be in the form of a sterile aqueous solution, solution, suspension, microemulsion, liposome, or powder. In some embodiments, the pharmaceutical composition may be in the form of a unit dose for easy administration to the patient at the desired dosage.

[0138] The dosage range of the antibody or pharmaceutical composition described herein can be determined by clinicians based on experience, taking into account factors such as the route of administration (including administration time, dosing interval, and route of administration), the patient's age, weight, sex, pathological condition, diet, excretion rate, and drug sensitivity. The antibody or pharmaceutical composition described herein can be administered via routes of administration known in the art, such as parenteral routes, including but not limited to subcutaneous, intravenous, intramuscular, and intraperitoneal injections or infusions.

[0139] In some embodiments, this application relates to the use of the above-described anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition in the preparation of a medicament for treating HHLA2-related diseases. Alternatively, this application relates to an anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for treating HHLA2-related diseases. Alternatively, this application relates to a method of treating HHLA2-related diseases in a subject of need, comprising administering (e.g., a therapeutically effective amount) of the above-described anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition to the subject. Alternatively, this application relates to the use of the above-described anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for enhancing an immune response.

[0140] In some preferred embodiments, the HHLA2-related disease is cancer. In some preferred embodiments, the cancer includes, but is not limited to, solid tumors (such as lung cancer (e.g., non-small cell lung cancer), kidney cancer (e.g., renal cell carcinoma), bone cancer, skin cancer, pancreatic cancer, colorectal cancer, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, cervical cancer, glioma, glioblastoma, neuroblastoma, pancreatic ductal carcinoma, thymoma, thyroid cancer, bile duct cancer, or breast cancer) and hematologic malignancies (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma).

[0141] In some embodiments, this application relates to the use of the above-described anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition in the preparation of a medicament for enhancing an immune response. Alternatively, this application relates to an anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for enhancing an immune response. Alternatively, this application relates to a method for enhancing an immune response in a subject in need, comprising administering (e.g., a therapeutically effective amount) of the above-described anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition to the subject. Alternatively, this application relates to the above-described anti-HHLA2 antibody or its antigen-binding fragment for detecting HHLA2 protein. Alternatively, this application relates to a method for detecting HHLA2 protein in a sample, comprising contacting the above-described anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition with the sample. Alternatively, this application relates to the use of the above-described anti-HHLA2 antibody or its antigen-binding fragment for detecting HHLA2 protein.

[0142] In some embodiments, this application relates to the use of the above-described anti-HHLA2 antibody or its antigen-binding fragment in the preparation of reagents for detecting HHLA2 protein.

[0143] In some embodiments, this application relates to a method for generating the above-described anti-HHLA2 antibody or its antigen-binding fragment, comprising: (1) culturing the host cell under conditions suitable for the expression of the above-described anti-HHLA2 antibody or its antigen-binding fragment, and (2) purifying the anti-HHLA2 antibody or its antigen-binding fragment from the culture of the host cell.

[0144] In some embodiments, anti-HHLA2 antibodies or their antigen-binding fragments can be purified from the host cell culture using purification methods known in the art, such as affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc.

[0145] Example

[0146] The invention will now be described in further detail through examples, but the invention is not limited to these examples. Unless otherwise specified, the reagents, materials, or instruments involved in the following examples are commercially available.

[0147] Unlike most other immune checkpoint molecules, HHLA2 possesses dual functions of immune activation and immunosuppression. Lieping Chen's research team identified the first functional receptor for HHLA2, CD28H (also known as TMIGD2, transmembrane and immunoglobulin domain containing 2). CD28H binds to HHLA2 on the cell membrane surface, acting as a co-stimulatory factor, activating T cell proliferation and cytokine secretion through an AKT phosphorylation-dependent signaling cascade. In 2021, an inhibitory receptor for HHLA2, KIR3DL3 (killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3), was discovered. In contrast to CD28H, KIR3DL3 is primarily expressed in functionally mature T cells and NK cell subtypes. Upon binding to HHLA2, KIR3DL3 recruits intracellular SHP-1 and SHP-2 (Src homology region 2 domain-containing phosphatase-1 / 2), inhibiting intracellular Vav1, ERK1 / 2, AKT, and NF-κB signaling pathways, leading to suppression of immune cell function. Under physiological conditions, CD28H and KIR3DL3 act as the stimulatory and inhibitory receptors for HHLA2, respectively, regulating the immune response of activated T cells and the cytotoxic activity of NK cells. These two receptors exhibit dynamic changes on immune cells. Simultaneously with T cell receptor (TCR) signaling, CD28H on native T cells interacts with HHLA2, co-stimulating T cell activation. However, with repeated T cell activation, the expression of the stimulatory receptor TMIGD2 gradually diminishes, allowing the expression of the inhibitory receptor KIR3DL3 to gradually become dominant. Therefore, HHLA2 plays a double-edged sword role in the regulation of the immune system.

[0148] Example 1: Construction of an alpaca immune library

[0149] Two alpacas were immunized with recombinant human HHLA2 protein (Human-HHLA2 or Human-B7-H7, purchased from Acro) and recombinant cynomolgus monkey HHLA2 protein (Cyno-HHLA2 or Cyno-B7-H7, purchased from Acro). The immunization schedule was as follows (day 0 was the day of the first immunization): On day 0, 10 mL of blood was drawn from each alpaca before immunization as a negative serum control. Then, 0.5 mg of recombinant human HHLA2 protein, 0.5 mg of recombinant cynomolgus monkey HHLA2 protein, and an equal volume of CFA (complete Freund's adjuvant, purchased from Solarbio, catalog number: F5881-10 mL) were mixed and subcutaneously injected into the alpaca. On day 14, 0.25 mg of recombinant human HHLA2 protein and 0.25 mg of recombinant cynomolgus monkey HHLA2 protein were injected into the alpaca. HLA2 recombinant protein was mixed with an equal volume of IFA (incomplete Freund's adjuvant, purchased from Solarbio, catalog number: F5506-10ML) and subcutaneously injected into alpacas. On day 21, 10 mL of blood was collected, and the titer was detected by ELISA. On day 28, 0.25 mg of human HHLA2 recombinant protein and 0.25 mg of cynomolgus monkey HHLA2 recombinant protein were mixed with an equal volume of IFA and subcutaneously injected into alpacas. On day 35, 50 mL of peripheral blood was collected, and lymphocytes were separated by centrifugation (InTrizol), and the titer was detected by ELISA. On day 42, 0.25 mg of human HHLA2 recombinant protein and 0.25 mg of cynomolgus monkey HHLA2 recombinant protein were mixed with an equal volume of IFA and subcutaneously injected into alpacas. On day 49, 50 mL of peripheral blood was collected, and lymphocytes were separated by centrifugation (InTrizol), and the titer was detected by ELISA. At this point, the immunogenicity reached the standard for establishing a library. 50 mL of peripheral blood was collected, and PBMCs were separated according to the instructions for use of lymphocyte separation solution (purchased from Haoyang Biotechnology, catalog number: LTS1077).

[0150] Total RNA was extracted from the obtained PBMCs using the Cell Total RNA Isolation Kit (purchased from Fuji Biotechnology, catalog number: RE-03111) according to its instructions. RNA purity was confirmed to be good by agarose gel electrophoresis. PrimeScript was used. TM The II1st Strand cDNA Synthesis Kit (purchased from TaKaRa, catalog number: 6210A) was used to reverse transcribe the extracted RNA into cDNA according to its instructions. The cDNA obtained was amplified by nested PCR using HSDNA Polymerase (TaKaRa, catalog number: R01A) according to its instructions to obtain the VHH gene fragment. The PCR amplification product was subjected to 1% agarose gel electrophoresis, and the VHH gene fragment (approximately 750 bp) was recovered by gel extraction using a Gel Extraction Kit (OMEGA, catalog number: D2500-01) according to its instructions. The recovered VHH gene fragment was then subjected to a second round of nested PCR amplification. The second round nested PCR amplification product was then purified by gel extraction using a Gel Extraction Kit (OMEGA, catalog number: D2500-01) according to its instructions, and used for electroporation of yeast.

[0151] EBY100 competent yeast cells were prepared by mixing purified second-round nested PCR amplification products with linearized pYDC2 vector. The resulting VHH gene fragment and vector mixture was then electroporated into the competent yeast cells. The mixture was serially diluted (10 μL) and plated onto SD-CAA plates, incubated at 30°C for 4 days, and the calculated library size was approximately 3.24 × 10⁻⁶. 8 Forty-eight single clones were randomly selected from the library plate for PCR identification, and the results showed an insertion rate of 100%. Then, 60 positive PCR products were randomly selected for further sequencing verification, confirming good diversity.

[0152] Add approximately 10 mL of SD-CAA culture medium to each of the above SD-CAA plates, scrape off the colonies, and collect the suspended cells using a pipette tip (the bacterial concentration was measured to be 2.47 × 10⁻⁶ using a cell counter). 9 Add an equal volume of 50% sterile glycerol (cells / mL) and mix well. Store as an antibody yeast display library at -80°C.

[0153] Example 2: Screening of yeast display libraries

[0154] Expression cells from the antibody-displaying yeast library obtained in Example 1 were washed with PBST buffer and incubated with negative magnetic beads at 4°C. The magnetic beads were then removed, and the suspended yeast cells were collected. The collected yeast cells were incubated with human HHLA2 recombinant protein (purchased from Acro) at 4°C, washed with PBST buffer, and then incubated with positive magnetic beads at 4°C. The resulting magnetic bead-yeast cell complex was washed with PBST buffer and then cultured overnight in SD-CAA medium. After removing the magnetic beads from the culture medium, the yeast cells were collected by centrifugation and added to SG-CAA medium for antibody expression.

[0155] Then, recombinant human HHLA2 protein (purchased from Acro) and cynomolgus monkey HHLA2 protein (purchased from Acro) were sorted by flow cytometry. Cells were plated (SD-CAA medium) and cultured at 37°C for 48 hours; single clones were selected for sequencing. Simultaneously, single clones were incubated with dyes SA-PE and SA-650, as well as Anti-HA-488, Human-HHLA2, and Cyno-HHLA2, and identified by flow cytometry. Positive clones binding both recombinant human and cynomolgus monkey HHLA2 proteins were obtained. Human HHLA2 / KIR3DL3 competitive sorting was also performed. Figure 2A Screening was performed, and high-affinity binding monoclonal clones were selected from the positive monoclonal clones for antibody sequence analysis. Figure 2B The results show the determination of the interaction between exemplary test clones M1F1-55 and M1F1-92 and biotinylated human HHLA2 (Bio-Human-B7-H7) and biotinylated cynomolgus monkey HHLA2 (Bio-Cyno-B7-H7), respectively.

[0156] Example 3 Recombinant Expression and Screening of VHH-Fc Fusion Protein

[0157] Based on the monoclonal antibody sequence screened in Example 2, the corresponding VHH gene was synthesized by Tianlin Biotechnology Wuxi Co., Ltd. The VHH gene fragment was ligated with the hIgG1 Fc fragment (SEQ ID NO: 5) and cloned into the pcDNA3.4 vector. The resulting recombinant vector was then transformed into competent E. coli DH5α cells, and the resulting recombinant E. coli was amplified to obtain a plasmid. The plasmid was added to OPM-293-CD05-Medium medium (Shanghai Aopumai Biotechnology Co., Ltd.) according to the instructions using PEImax transfection reagent (Shanghai Qifa Experimental Reagent Co., Ltd.). After mixing, the resulting mixture was added to HEK293 cells for transient transfection. After culturing at 37°C for 5 days, the supernatant was collected by centrifugation and purified by Protein A column affinity chromatography. The purity of the obtained fusion protein was confirmed to be >95% by SDS-PAGE gel electrophoresis and SEC-HPLC. Simultaneously, the endotoxin level was confirmed to be <1 EU / mg using the Limulus Amebocyte Lysate (LAL) assay.

[0158] The purified VHH-Fc fusion proteins were divided into two batches and detected by the following ELISA assay, along with biotinylated human HHLA2 (Biotinylated Human B7-H7 / HHLA2 Protein, His, Avitag). TMThe abbreviation "Bio-Human-B7-H7" (purchased from Acro) and biotinylated cynomolgus monkey HHLA2 (Biotinylated Cynomolgus B7-H7 / HHLA2 Protein, His, Avitag) TM The study combined [Bio-Cyno-B7-H7, purchased from Acro], with Isotype (batch number: 400-101-001) purchased from Apak Biotech as a negative control and B7-H7 PC (Harbour) (purchased from Harbour BioMed, also referred to as "B7-H7 PC" in this article) as a positive control:

[0159] (1) Bio-Human-B7-H7 and Bio-Cyno-B7-H7 in 50mM NaHCO3 (pH=9.6) at a concentration of 2μg / mL were coated into 96-well microplates at 100μL / well and incubated overnight at 4℃.

[0160] (2) Wash the plate three times with PBST buffer, add 300 μL / well of 5% milk for blocking, and incubate at 37°C for 1 h;

[0161] (3) Wash the plate once with PBST buffer. Dilute the antibody sample to be tested (i.e., the VHH-Fc fusion protein to be tested) and the control with 5% milk at a volume ratio of 1:5, starting from 100 nM, for a total of 7 dilutions (100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM). At the same time, set up a blank well without antibody. Add 100 μL of the diluted antibody sample to be tested to the well of the 96-well plate and incubate at 37°C for 1 h.

[0162] (4) Wash the plate 5 times with PBST buffer and add 5% milk diluted secondary antibody (Anti-Human IgGFc, HRP, 1:10K, purchased from Jackson);

[0163] (5) Wash the 96-well plate incubated with secondary antibody 5 times with PBST buffer, add 100 μL / well of TMB single-component colorimetric solution, incubate at 37°C for 7 min, then add 50 μL / well of 1M HCl to terminate the reaction, and read the OD450.

[0164] (6) Import the data into Graphpad Prism for curve fitting (the first batch of fusion protein ELISA test results are in...). Figure 3A and Figure 3B The results of the second batch of fusion protein ELISA tests are shown in [the image / data]. Figure 4A and Figure 4B(as shown in the image).

[0165] The results of the two batches of ELISA tests are shown in Tables 1 and 2, respectively.

[0166] Table 1. Results of the first batch of fusion protein ELISA detection

[0167] Test sample Bio-Human-B7-H7(EC50,nM) Bio-Cyno-B7-H7 (EC50, nM) B7-H7 PC (Harbour) 0.2247 0.5831 M1F1-55 0.3318 0.3064 M1F1-182 0.2675 0.2679 M1F1-62 0.2448 0.2416 M1F1-92 0.2909 0.3049 Isotype Do not combine Do not combine

[0168] Table 2. ELISA results of the second batch of fusion proteins

[0169] Test sample Bio-Human-B7-H7(EC50,nM) B7-H7 PC (Harbour) 0.1739 M1F1-21 0.1443 M1F1-72 0.1689 M1F1-75 0.1449 M1F1-201 13.19 M1F1-369 0.5102 M1F1-373 0.5234 Isotype Do not combine Test sample Bio-Cyno-B7-H7 (EC50, nM) B7-H7 PC (Harbour) 0.5962 M1F1-55 0.1924 M1F1-182 0.193 M1F1-62 0.04596 M1F1-92 0.1258 M1F1-21 0.09055 M1F1-72 0.06743 M1F1-75 0.3325 M1F1-201 158.9 M1F1-369 Do not combine M1F1-373 Do not combine Isotype Do not combine

[0170] Then, the binding effect of the above fusion protein on HHLA2-expressing cell lines (HCC827 cells (purchased from Procell) and / or ASPC-1-OE-B7H7 cells (provided by Apak Biotechnology); with 293T cells (purchased from Procell) as a control) was determined in two batches according to the following method:

[0171] (1) Wash the cells (HCC827 cells, ASPC-1-OE-B7H7 cells or 293T cells) used for the assay twice with PBS buffer, resuspend the cells with PBS buffer, and aliquot the cell suspension into PCR plates, 50 μL per well.

[0172] (2) Dilute the fusion protein to be tested and the control (B7-H7 PC (Harbour) and Isotype) with PBS buffer. The initial concentration is 400 nM, and it is diluted 5 times to a total of 7 gradients (400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, 0.128 nM, 0.0256 nM). Add 50 μL of the dilution buffer to the PCR plate at 50 μL / well. At the same time, set up one blank well without the fusion protein to be tested. Incubate at 4°C for 1 hour.

[0173] (3) After incubation, wash the cells three times with PBS buffer, add 100 μL of fluorescent secondary antibody (647 Anti-Human IgG Fc, purchased from Jackson) to each well, and incubate at 4°C for 1 hour;

[0174] (4) After incubation, wash the cells once with PBS buffer, transfer the cells to a 96-well cell plate, and analyze them by flow cytometry.

[0175] (5) Export the analysis data and analyze it using FlowJo software to obtain the MFI value;

[0176] (6) Import the data into Graphpad Prism for curve fitting.

[0177] The results of the first batch of measurements were in Figures 5A-5C The results are shown in Table 3 (for HCC827 cells) and Table 4 (for ASPC-1-OE-B7H7 cells); the results of the second batch of assays are... Figure 6A and Figure 6B And as shown in Table 5 below.

[0178] Table 3. First batch of results on the binding effect of the fusion protein to HCC827 cells.

[0179] Test sample HCC827 (EC50, nM) B7-H7 PC (Harbour) 0.1996 M1F1-55 0.3813 M1F1-182 0.2088 M1F1-62 0.04147 M1F1-92 0.4644 Isotype Do not combine

[0180] Table 4. First batch of results on the binding effect of the fusion protein to ASPC-1-OE-B7H7 cells.

[0181] Test sample ASPC-1-OE-B7H7(EC50,nM) B7-H7 PC (Harbour) 1.778 M1F1-55 0.5388 M1F1-182 1.629 M1F1-62 5.025 M1F1-92 3.263 Isotype Do not combine

[0182] Table 5. Results of the second batch of assays on the binding efficacy of the fusion protein to ASPC-1-OE-B7H7 cells.

[0183] Test sample ASPC-1-OE-B7H7(EC50,nM) B7-H7 PC (Harbour) 1.854 M1F1-21 31.1 M1F1-72 1.687 M1F1-75 247.2 M1F1-201 2.048 M1F1-369 2.07 M1F1-373 4.18 Isotype Do not combine

[0184] from Figure 5C and Figure 6B It can be seen that the fusion proteins from clones M1F1-72 and M1F1-182 bind nonspecifically to 293T cells; therefore, these two clones are excluded.

[0185] The KIR3DL3 binding inhibition of the fusion protein was detected using ASPC-1-OE-B7H7 cells as follows. The results of the inhibition detection are shown in [Figure 1]. Figure 7A and Figure 7B And Tables 6 and 7:

[0186] (1) Wash ASPC-1-OE-B7H7 cells twice with PBS buffer, resuspend the cells with PBS buffer, and aliquot the cell suspension into PCR plates, 50 μL per well;

[0187] (2) Dilute the fusion protein to be tested and the control B7-H7 PC (Harbour) with PBS buffer, starting at a concentration of 1000 nM, and dilute 5 times to 7 concentration points (1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM). Add 50 μL of the dilution buffer to the PCR plate at 50 μL / well. At the same time, set up one blank well (Control) without adding the fusion protein. Incubate at 4°C for 1 hour.

[0188] (3) After incubation, wash the cells three times with PBS buffer, add 50 μL of Bio HumanKIR3DL3-FC Avi (purchased from Acro) to each well, with a concentration of 2 μg / mL, and incubate at 4°C for 1 hour.

[0189] (4) After incubation, wash the cells three times with PBS buffer, add 100 μL of fluorescent secondary antibody (SA-650; purchased from Thermo Fisher Scientific) to each well, and incubate at 4°C for 1 hour;

[0190] (5) After incubation, wash the cells once with PBS buffer, transfer the cells to a 96-well cell plate, and analyze them using a flow cytometer.

[0191] (6) Export the analysis data and analyze it using FlowJo software to obtain the MFI value;

[0192] (7) Import the data into Graphpad Prism for curve fitting.

[0193] Table 6. First batch of detection results combining blocking

[0194]

[0195] Table 7. Detection results of the second batch of combined blocking assays.

[0196] Test sample IC50(nM) B7-H7 PC (Harbour) 3.341 M1F1-21 Non-blocking M1F1-72 Non-blocking M1F1-75 Non-blocking M1F1-201 3.947 M1F1-369 2.197 M1F1-373 4.481

[0197] In summary, two antibodies (derived from clones M1F1-55 and M1F1-92, named M1F1-55-VHH and M1F1-92-VHH, respectively) with high affinity for HHLA2-expressing cells and good KIR3DL3 binding blocking activity were screened for further experiments. Their corresponding antibody sequence information is as follows:

[0198]

[0199] Example 4 Affinity determination of VHH antibody

[0200] The affinity of the VHH antibody obtained in Example 3 for human HHLA2 recombinant protein (Human-B7-H7) and cynomolgus monkey HHLA2 recombinant protein (Cyno-B7-H7) was determined using biofilm thin-layer interferometry (BLI).

[0201] The selected antibodies M1F1-55-VHH and M1F1-92-VHH were both diluted to 2 μg / mL using PBST buffer (PBS + 0.05% Tween-20, supplemented with 0.1% BSA). The antigens Bio-Human-B7-H7 and Bio-Cyno-B7-H7 (both purchased from Acro) were diluted to 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM, respectively, using PBST buffer. Fortebio was used. The K2 system (purchased from Sartorius) was used for detection. After pre-wetting the SA (Streptavidin) sensor with 200 μL of PBST buffer, the diluted antigen was loaded and solidified. It was then immersed in the diluted anti-antibody solution for binding and transferred to the PBST buffer for dissociation. Affinity and kinetic parameters were then analyzed using a 1:1 Langmuir binding model, as shown in the table below.

[0202] Table 8. Binding of VHH antibodies to human HHLA2 and cynomolgus monkey HHLA2.

[0203]

[0204] Table 9. Binding signal values ​​of VHH antibody with human HHLA2 and cynomolgus monkey HHLA2

[0205]

[0206] It can be seen that the VHH antibody obtained in Example 3 exhibits good affinity for HHLA and can specifically bind to HHLA.

[0207] Example 5: SPR binding and blocking assay of VHH antibody

[0208] 5.1 SPR Combination Test

[0209] Select the CM5 chip and fully equilibrate the channels with run buffer (1×PBS). Activate the chip surface with an EDC / NHS mixture, setting one channel as a blank control (run buffer) and the other channel for ligand coupling. Dilute the ligand B7-H7 (HHLA2) His protein (purchased from Acro) with 10 mM acetate buffer (pH 4.0–5.0).

[0210] The M1F1-92-VHH antibody was prepared using running buffer and serially diluted 3-fold starting from 100 nM. Antibody association and dissociation were recorded, and the results were fitted using a 1:1 Langmuir model to obtain k. a k d and K DThe values ​​are 1.339 × 10⁻⁶. 5 (1 / Ms), 1.469×10 -4 (1 / s) and 1.097 nM.

[0211] 5.2 SPR blocking test

[0212] This experiment aims to evaluate whether the M1F1-92-VHH antibody can block the interaction between human B7-H7 and its receptor KIR3DL3, and to calculate the inhibition rate (IC50). 50 ).

[0213] The CM5 chip was processed as described above, with B7-H7 immobilized in the working channel and a blank control channel set up. The M1F1-92-VHH antibody was prepared using running buffer and serially diluted 4-fold starting at 1024 nM. The M1F1-92-VHH antibody was added, and the run was completed for 120 s. KIR3DL3 was then added, and the binding response (RU) was recorded. Using the "KIR3DL3 binding response without nanobody (0 nM)" as 100%, the inhibition rate was calculated using the following formula:

[0214]

[0215] The inhibition rate-concentration data were imported into GraphPad Prism, and the IC50 was obtained by fitting the inhibition curve. 50 The IC50 of the M1F1-92-VHH antibody was determined. 50 It is 3.08 nM.

[0216] Example 6: In vitro tumor cell killing experiment using VHH antibody

[0217] In this experiment, HHLA2-overexpressing AsPC-1 cells were used as target cells, NK cells were used as effector cells, and PanC-1 cells (PanC-1-B7H7-OE cells) without HHLA2 expression were used as control target cells. A "target cell culture alone (without NK cells)" control was set up, and the number of 7-AAD+ cells was recorded.

[0218] The in vitro antitumor activity of M1F1-55-VHH antibody (hereinafter referred to as "55 Nanobody") and M1F1-92-VHH antibody (hereinafter referred to as "92 Nanobody") was determined according to the following procedure (with Isotype (Apak Biotech, batch number: 400-101-001) as the control protein).

[0219] Target cells were collected and their concentration adjusted. The cells were then resuspended in RPMI-1640 medium supplemented with 1% FBS, and CFSE (Thermo Fisher Scientific) was added. The cells were incubated at 37°C for 15 min. The incubated cell culture was washed twice with RPMI-1640 medium supplemented with 2% FBS to remove free dye. The cells were then resuspended in RPMI-1640 medium supplemented with 10% FBS to obtain CFSE-labeled target cells. The target cells were then cultured at 2 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 cells / well and cultured overnight. NK cells (E:T ratio 1:1) were then added to each well and incubated at 37°C and 5% CO2. After incubation, cell samples were collected, and 7-AAD working solution (BioLegend) was added and incubated on ice in the dark for 20 min. Flow cytometry and gated analysis were then performed using a CytoFlex S (Beckman) analyzer.

[0220] The kill rate is calculated and corrected according to the following formula:

[0221] NK-mediated cytotoxicity (%) = (7-AAD+ cells / CFSE+ cells) × 100%

[0222] Corrected lethality (%) = Test group lethality (%) - Spontaneous mortality (%).

[0223] In addition, HPAF-II cell line and SW1116 cell line were used as target cells, and NK cells were used as effector cells. The in vitro antitumor activity of M1F1-55-VHH antibody and M1F1-92-VHH antibody was determined according to the above experimental procedure.

[0224] The result is Figure 8A (PanC-1 cell line) and Figure 8B As shown in the (AsPC-1 cell line), the M1F1-55-VHH and M1F1-92-VHH antibodies of this disclosure exhibit significant pro-killing functions against HHLA2-overexpressing tumor cell lines, but show no pro-killing function against the PanC-1 cell line without HHLA2 expression (i.e., no non-specific binding to this cell line). Furthermore, as... Figure 8C and Figure 8D As shown, the M1F1-55-VHH and M1F1-92-VHH antibodies disclosed herein exhibit significant cytotoxic effects on both HPAF-II and SW1116 cell lines.

[0225] Example 7: In vivo tumor-killing experiment of VHH antibody

[0226] Six- to eight-week-old female Hu-IL15 NCG mice were acclimatized for one week, and then orthotopic pancreatic injection (io) was performed under tribromoethanol anesthesia at a dose of 2 × 10⁻⁶ mg / L. 5 AsPC-1-EGFP-Luc2 cells were inoculated into mice at a density of 10 cells / mouse (approximately 0.05 mL volume with matrix gel, 1:1 ratio) to establish an AsPC-1-NCG mouse model. On day 1 post-modeling, 7 × 10⁻⁶ cells were injected into the tail vein of the mice. 6 Human PBMCs were injected at a density of 1 cell / mouse (0.2 mL) to construct mice with a reconstructed human PBMC immune system. Peripheral blood was collected weekly after injection of human PBMCs, and mCD45, hCD45, hCD3, and hCD56 were detected by flow cytometry to assess the reconstruction of the human PBMC immune system.

[0227] Mice were randomly assigned to three groups: an Isotype control group, an M1F1-92-VHH antibody treatment group (5 mg / kg), and an M1F1-55-VHH antibody treatment group (5 mg / kg), with 5 mice in each group (n=5). All mice were administered the drug intraperitoneally (ip) at a dose of 10 μL / g, twice weekly (BIW) for 1 week. The mean bioluminescence intensity in the region of interest (ROI) was approximately 130,000 p / s / cm², as measured using an in vivo imaging system. 2 Dosing was initiated at 10:00 AM ( / sr). Changes in tumor burden were assessed after dosing using bioluminescence intensity measured by an in vivo imaging system, and the results were reported. Figure 9 As shown in the image.

[0228] After drug administration, mice in each group were euthanized by CO2 overdose. After dissection, tumor lesions were removed and weighed, and the results were statistically analyzed using one-way ANOVA.

[0229] The results showed that, compared with the control, the M1F1-55-VHH antibody and M1F1-92-VHH antibody disclosed herein exhibited significant antitumor activity in tumor-bearing animals.

[0230] Example 8: Determination of the effect of VHH antibody on promoting the secretion of NK cell inflammatory factors (IFN-γ and TNF-α)

[0231] In this embodiment, the secretion of IFN-γ and TNF-α after co-incubation of NK cells with tumor cells (e.g., PanC-1-B7H7-OE) is used as an indicator to evaluate the killing effect, thereby reflecting the antibody's role in promoting the immune killing effect of NK cells on tumor cells.

[0232] In this measurement, a total of 5 groups were set up:

[0233] Blank control group: PanC-1-B7H7-OE cells and NK cells were co-incubated without any drugs;

[0234] Isotype control group: Isotype was added during the co-incubation of the two cell types;

[0235] Nanobody group No. 92: M1F1-92-VHH antibody was added during the co-incubation of the two cell types;

[0236] PanC-1-B7H7-OE cell group: PanC-1-B7H7-OE cells were cultured alone without co-incubation with NK cells;

[0237] NK cell group: NK cells were cultured alone and not co-incubated with tumor cells.

[0238] NK cells and PanC-1-B7H7-DE cells were incubated separately or co-incubated in DMEM medium supplemented with 10% FBS and 1% P / S at 37°C, followed by centrifugation at 13000 rpm for 5 min to collect the supernatant. 25 μL of supernatant was added to each well of a V-bottom 96-well plate and mixed with magnetic beads. Then, LEGENDPlex was added to the mixture. TM 25 μL of human CD8 / NK Panel Standard V02 (Cat. No.: 741189) and 25 μL of assay buffer and standard (final volume 75 μL per well) were added. The mixture was incubated at 800 rpm for 120 min with shaking at room temperature. The mixture was then centrifuged at 350–500 g for 5 min to precipitate the beads, and the liquid was discarded. 200 μL of LEGEND Plex solution was added to each well. TM Soak in 1× wash buffer of Human CD8 / NK Panel Standard V02 (Cat.No.: 741189) for 1 min, then centrifuge at 350-500g for 5 min and discard the supernatant.

[0239] Add 25 μL of M1F1-92-VHH antibody to each well of the 92 nanobody group and 25 μL of Isotype to each well of the Isotype control group. Incubate the 96-well plate at room temperature with shaking at 800 rpm for 1 h. Then add 25 μL of LEGENDPlex antibody to each well. TMHuman CD8 / NK Panel Standard V02 (Cat. No.: 741189) SA-PE was incubated at 800 rpm for 30 min at room temperature with shaking. After centrifugation and discarding the supernatant, 200 μL of 1× wash buffer was added and the mixture was washed once. Then, 200–300 μL of 1× wash buffer was added to each well to resuspend the magnetic beads. Flow cytometry analysis was performed on the supernatant to determine the secretion of NK cell inflammatory factors IFN-γ and TNF-α. Results were presented in... Figure 10A and Figure 10B As shown in the image.

[0240] The results showed that the M1F1-92-VHH antibody disclosed herein could significantly enhance the immune response.

[0241] Simultaneously, changes in the secretion of other cytokines in the supernatant were measured, and the results were... Figures 10C-10G As shown in the figure, the antibody disclosed herein does not induce an increase in the secretion of other cytokines, suggesting that it will not cause an adverse immune response after administration.

[0242] Example 9: Construction of Humanized VHH Antibody

[0243] In this embodiment, the M1F1-92-VHH antibody (also known as "NB690-92") is humanized through the following process.

[0244] 9.1 Humanization Design of Nanobody (Structure-Guided CDR Grafting)

[0245] The amino acid sequences of parental VHH were annotated with CDR1-CDR3 and framework regions (FR1–FR4) using the IMGT and / or Kabat numbering system. Humanization employed a structure-guided CDR grafting strategy: first, receptor frameworks with high homology to the parental VHH framework regions and good druggable characteristics were screened from the human immunoglobulin VH germline sequence library. Then, three-dimensional structural models of the parental VHH and its humanized variants were performed to assess the compatibility of the CDR conformation with the receptor framework and to identify key framework sites (including support sites such as the Vernier zone) that may affect the CDR spatial conformation and antigen binding. After CDR grafting, based on structural analysis and conservation principles, back-mutations were performed on necessary framework sites to reduce the risk of affinity loss and maintain folding stability. This resulted in a set of humanized candidate sequences corresponding to each parental VHH.

[0246] 9.2 Screening of Humanized Candidate Sequences

[0247] The humanized candidate sequences obtained above were comprehensively ranked, and the screening indicators included: (1) improvement in humanization degree / “humanness” (similarity with human VH germline framework); (2) structural rationality (CDR conformation retention, retention of key support sites of framework-(near)CDR); (3) physicochemical property prediction (charge distribution, surface hydrophobic patches and other features related to aggregation tendency). Finally, the top 5 humanized sequences with the highest comprehensive scores were selected for subsequent drugability prediction and verification.

[0248] 9.3 Gene synthesis, HEK293 transient transexpression and protein purification

[0249] The top 5 humanized VHH antibodies obtained from screening were optimized for mammalian expression (codon optimization), and a secretion signal peptide was added to the construction to achieve secretory expression. A His tag for purification and detection was also designed. HEK293 cells were used for transient transfection expression. After culture, the cell culture supernatant was collected, clarified by centrifugation / filtration, and purified by affinity chromatography. Humanized VHH antibodies NB690-92hz1, NB690-92hz2, NB690-92hz3, NB690-92hz4, and NB690-92hz5 were obtained.

[0250] The sequence information of the humanized antibodies obtained through the above screening (whose CDR1 to CDR3 sequences are shown in SEQ ID NOs:1-3) is shown in the table below:

[0251]

[0252] Example 10: ELISA results of humanized VHH antibody

[0253] 96-well culture plates were coated with biotinylated human HHLA2 (Bio-Human-B7-H7, purchased from Acro) and biotinylated cynomolgus monkey HHLA2 (Bio-Cyno-B7-H7, purchased from Acro) at a concentration of 1 μg / mL and incubated overnight at 4°C.

[0254] The 96-well plate was washed three times with PBST at 300 μL / well, then blocked with 5% milk and incubated at 37°C for 1 h. The plate was then washed once with PBST. The humanized VHH antibody obtained in Example 9, along with negative and positive controls, was serially diluted 3-fold from 100 nM with 5% milk. 100 μL of the diluted antibody or control was added to each well of the 96-well plate, and the plate was incubated at 37°C for 1 h. Isotype (batch number: 400-101-001) purchased from Apak Biotech was used as the negative control, and B7-H7 PC (Harbour) (purchased from Harbour BioMed) was used as the positive control.

[0255] The 96-well plates were washed five times with PBST, and then the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted with PBST containing 3% BSA (containing 0.05% Tween-20) blocking buffer was added. After incubation, the 96-well plates were washed five times with PBST, and 100 μL / well of TMB single-component chromogenic solution was added to each well. The plates were incubated at 37°C for 7 min. The reaction was then terminated by adding 50 μL / well of 1 MHCl, and the data were read at OD450. The data were imported into Graphpad Prism for curve fitting. The results are shown in Tables 10 and 11.

[0256] Table 10. ELISA results based on human HHLA2.

[0257] Test sample <![CDATA[EC 50 (nM)]]> B7-H7 PC (Harbour) 0.3384 NB690-92 10.41 NB690-92hz1 2.597 NB690-92hz2 3.218 NB690-92hz3 1.447 NB690-92hz4 3.278 NB690-92hz5 4.933 Isotype ～

[0258] Table 11 ELISA results based on cynomolgus monkey-derived HHLA2

[0259] Sample to be tested <![CDATA[EC 50 (nM)]]> B7-H7 PC (Harbour) 2.732 NB690-92 0.5044 NB690-92hz1 0.8881 NB690-92hz2 0.1122 NB690-92hz3 0.5974 NB690-92hz4 0.5417 NB690-92hz5 0.7456 Isotype ～

[0260] Example 11: Sandwich ELISA results of humanized VHH antibody

[0261] Pre-coat 96-well plates with Anti-His tag, Rabbit antibody (Proteintech) at a concentration of 5 μg / mL (100 μL / well) in 50 mM NaHCO3 (pH = 9.6) and incubate overnight at 4°C. Wash the 96-well plates three times with 300 μL / well of PBST, then block the plates with 5% milk and incubate at 37°C for 2 h. Wash the 96-well plates once with PBST, add Bio-Human-B7-H7 (Acro) at a concentration of 2 μg / mL (100 μL / well), and incubate at 37°C for 1 h.

[0262] The 96-well plate was washed three times with PBST. The humanized VHH antibody constructed in Example 9 was serially diluted 3-fold from 100 nM with 5% milk (containing 4 μg / mL ligand Bio Human KIR3DL3-FC Avi (purchased from Acro)). 100 μL of the diluted antibody was added to each well of the 96-well plate, and the plates were incubated at 37°C for 1 h. Isotype (batch number: 400-101-001) purchased from Apak Biotech was used as a negative control, and B7-H7 PC (Harbour) (purchased from Harbour BioMed) was used as a positive control.

[0263] The 96-well plate was washed five times with PBST, and then the secondary antibody (SA, HRP, 1:10K) diluted with PBST containing 3% BSA (containing 0.05% Tween-20) blocking buffer was added. After incubation, the 96-well plate was washed five times with PBST, and 100 μL / well of TMB single-component chromogenic solution was added to each well. The plate was incubated at 37°C for 7 min. The reaction was then terminated by adding 50 μL / well of 1M HCl, and the data were read at OD450. The data were imported into Graphpad Prism for curve fitting. The results are shown in Table 12.

[0264] Table 12 Results of sandwich ELISA assay

[0265] Sample to be tested <![CDATA[IC 50 (nM)]]> B7-H7 PC (Harbour) 0.9399 NB690-92 2.223 NB690-92hz1 2.851 NB690-92hz2 2.53 NB690-92hz3 4.064 NB690-92hz4 2.05 NB690-92hz5 4.653 Isotype ～

[0266] Example 12: FACS assay results of humanized VHH antibody

[0267] ASPC-1, ASPC-1-OE-B7H7, or HEK293 cells were washed twice with PBS, the supernatant was discarded, and the cells were resuspended in PBS. The resuspended cells were seeded into 96-well plates at 50 μL / well.

[0268] The test antibody was serially diluted 3-fold from 100 nM to 11 concentrations using PBS. 50 μL of the diluted antibody was added to each well of a 96-well plate and incubated at 4°C for 1 hour. Isotype (batch number: 400-101-001) purchased from Apak Biotech was used as a negative control, and B7-H7PC (Harbour) (purchased from Harbour BioMed) was used as a positive control. After incubation, the cells were washed three times with PBS, and 100 μL of fluorescent secondary antibody (647 Anti-Human IgG Fc, purchased from Jackson) was added to each well. The plates were then incubated at 4°C for 1 hour.

[0269] The cells were then washed once with PBS buffer and transferred to a 96-well cell plate for flow cytometry analysis. The data were exported and analyzed using FlowJo software to obtain the MFI values. The data were then imported into GraphpadPrism for curve fitting. The humanized antibody disclosed herein did not specifically bind to HEK293 cells. The FACS assay results for ASPC-1-OE-B7H7 and ASPC-1 cells are shown in Tables 13 and 14.

[0270] Table 13 FACS assay results of ASPC-1-OE-B7H7 cells

[0271] Sample to be tested <![CDATA[EC 50 (nM)]]> B7-H7 PC (Harbour) 0.4889 NB690-92 0.8674 NB690-92hz1 1.192 NB690-92hz2 0.7821 NB690-92hz3 1.111 NB690-92hz4 0.8967 NB690-92hz5 1.424 Isotype ～

[0272] Table 14 FACS assay results of ASPC-1 cells

[0273] Sample to be tested <![CDATA[EC 50 (nM)]]> B7-H7 PC (Harbour) 0.174 NB690-92 0.8379 NB690-92hz1 0.4 NB690-92hz2 0.515 NB690-92hz3 0.8307 NB690-92hz4 0.688 NB690-92hz5 1.04 Isotype ～

[0274] Example 13: Blocking detection results of humanized VHH antibody (FACS)

[0275] ASPC-1-OE-B7H7 cells were washed twice with PBS, the supernatant was discarded, and the cells were resuspended in PBS. The resuspended cells were seeded into 96-well plates at a rate of 50 μL / well.

[0276] The test antibody was serially diluted 3-fold from 1000 nM to seven concentrations using PBS. 50 μL of the diluted antibody was added to each well of a 96-well plate and incubated at 4°C for 1 hour. B7-H7 PC (Harbour) (purchased from Harbour BioMed) was used as a positive control. After incubation, the cells were washed three times with PBS, and 50 μL of the 2 μg / mL ligand protein BioHumanKIR3DL3-FC Avi (purchased from Acro) was added to each well. The plates were then incubated at 4°C for 1 hour.

[0277] After incubation, cells were washed three times with PBS, and 100 μL of fluorescent secondary antibody (SA-650, Thermo Fisher Scientific) was added to each well. Four wells without secondary antibody were designated as blank wells. Cells were incubated at 4°C for 1 hour. Then, the cells were washed once with PBS buffer and transferred to a 96-well cell plate for flow cytometry analysis. The data were exported and analyzed using FlowJo software to obtain MFI values. The data were then imported into Graphpad Prism for curve fitting. The results are shown in Table 15.

[0278] Table 15. Blocking detection results of humanized VHH antibodies

[0279] Sample to be tested <![CDATA[IC 50 (nM)]]> B7-H7 PC (Harbour) 0.5096 NB690-92 5.208 NB690-92hz1 7.473 NB690-92hz2 6.927 NB690-92hz3 9.35 NB690-92hz4 3.877 NB690-92hz5 10.29

[0280] Example 14: BLI detection results of humanized VHH antibody

[0281] Biomembrane interference (BLI) technique was used to analyze the binding kinetics between antigens and antibodies.

[0282] Using FortéBio The K2 system was used for detection. Experiments were conducted at 25℃ and 1000 rpm with shaking. The kinetic buffer was PBST (PBS + 0.05% Tween-20) supplemented with 0.1% BSA to reduce non-specific adsorption. Using an SA (Streptavidin) sensor, the sensor was pre-wetted in the kinetic buffer for ≥10 min. Subsequently, biotinylated antigen Bio-Human-B7-H7 (human HHLA2, purchased from Acro) or Bio-Cyno-B7-H7 (cynomolgus monkey HHLA2, purchased from Acro) was loaded onto the SA sensor at 5-10 μg / mL to a stable loading level. After establishing a baseline in the buffer, the sensor was immersed in humanized VHH antibody solutions of different concentrations for association, and then transferred to the kinetic buffer for dissociation. The humanized VHH antibody was prepared using PBST and serially diluted (100, 50, 25, 12.5, 6.25 nM). A reference sensor without antigen loading was set up and dual reference subtraction was performed. Octet Data Analysis software was used to globally fit the sensing curves, and k was calculated using a 1:1 Langmuir combined model. a k d and K D Each concentration was tested at least twice. The obtained affinity and kinetic parameters are shown in Tables 16 and 17. Table 16 shows the binding of the humanized antibody to Bio-Human-B7-H7.

[0283] Test sample <![CDATA[K D (M)]]> <![CDATA[k a (1 / Ms)]]> <![CDATA[k dis (1 / s)]]> <![CDATA[Full R 2 ]]> B7-H7 PC (Harbour) 1.38E-08 9.68E+04 1.34E-03 0.9958 NB690-92 6.60E-09 7.65E+04 5.04E-04 0.9978 NB690-92hz1 2.27E-09 9.19E+04 2.09E-04 0.9976 NB690-92hz2 5.13E-09 1.03E+05 5.26E-04 0.9948 NB690-92hz3 3.97E-09 7.85E+04 3.11E-04 0.9973 NB690-92hz4 6.23E-09 8.00E+04 4.98E-04 0.9981 NB690-92hz5 4.48E-09 9.28E+04 4.16E-04 0.9971

[0284] Table 17 Binding of humanized proteins to Bio-Cyno-B7-H7

[0285] Test sample <![CDATA[K D (M)]]> <![CDATA[k a (1 / Ms)]]> <![CDATA[k dis (1 / s)]]> <![CDATA[Full R 2 ]]> B7-H7 PC (Harbour) 2.18E-08 2.40E+05 5.23E-03 0.7438 NB690-92 4.33E-09 5.56E+04 2.40E-04 0.9926 NB690-92hz1 3.63E-09 4.95E+04 1.80E-04 0.994 NB690-92hz2 1.03E-08 3.67E+04 3.78E-04 0.9754 NB690-92hz3 5.90E-09 5.23E+04 3.08E-04 0.9941 NB690-92hz4 4.59E-09 5.97E+04 2.74E-04 0.995 NB690-92hz5 7.50E-09 4.85E+04 3.64E-04 0.9962

[0286] Those skilled in the art will recognize that the scope of this application is not limited to the various specific implementations and embodiments described above, but rather that various modifications, substitutions, or recombinations can be made without departing from the spirit of this application, all of which fall within the protection scope of this application.

Claims

1. An anti-HHLA2 antibody or its antigen-binding fragment, comprising: (1) Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1; (2) Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2 or 6; and (3) Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3 or 7.

2. The anti-HHLA2 antibody or its antigen-binding fragment as described in claim 1, wherein, The anti-HHLA2 antibody or its antigen-binding fragment comprises: FR1 comprises the amino acid sequence shown in SEQ ID NO:9 or 13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; FR2 comprises the amino acid sequence shown in SEQ ID NO:17, 10 or 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; FR3 comprises the amino acid sequence shown in SEQ ID NO: 18, 19, 20, 11 or 15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and FR4 comprises an amino acid sequence shown in SEQ ID NO:21, 22, 12 or 16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

3. The anti-HHLA2 antibody or its antigen-binding fragment as described in claim 1 or 2, wherein, (1) The anti-HHLA2 antibody or its antigen-binding fragment comprises: FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1; FR2 comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2; FR3 comprises the amino acid sequence shown in SEQ ID NO:18, 20 or 11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and FR4 comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (2) The anti-HHLA2 antibody or its antigen-binding fragment comprises: FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1; FR2 comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2; FR3 comprises the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and FR4 comprises the amino acid sequence shown in SEQ ID NO:22, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (3) The anti-HHLA2 antibody or its antigen-binding fragment comprises: FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1; FR2 comprises the amino acid sequence shown in SEQ ID NO:17, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2; FR3 comprises the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and FR4 comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; (4) The anti-HHLA2 antibody or its antigen-binding fragment comprises: FR1 comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1; FR2 comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:2; FR3 comprises the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:3; and FR4 comprises the amino acid sequence shown in SEQ ID NO:12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or (5) The anti-HHLA2 antibody or its antigen-binding fragment comprises: FR1 comprises the amino acid sequence shown in SEQ ID NO:13, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR1, comprising the amino acid sequence shown in SEQ ID NO:1; FR2 comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR2, comprising the amino acid sequence shown in SEQ ID NO:6; FR3 comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Heavy chain CDR3, comprising the amino acid sequence shown in SEQ ID NO:7; and FR4 comprises the amino acid sequence shown in SEQ ID NO:16, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

4. The anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-3, wherein, The anti-HHLA2 antibody or its antigen-binding fragment contains VHH, wherein the VHH comprises an amino acid sequence shown in SEQ ID NO: 23, 24, 25, 26, 27, 4 or 8 or an amino acid sequence having at least 80% sequence identity with it; Preferably, the amino acids that differ from those shown in the amino acid sequence with at least 80% sequence identity are mainly present or all present in the FR region compared to the amino acid sequence shown in SEQ ID NO:4 or 8.

5. The anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-4, wherein, The anti-HHLA2 antibody is a nanobody or a heavy chain antibody.

6. The anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-5, wherein, The anti-HHLA2 antibody is a nanobody, and the nanobody contains the amino acid sequence shown in SEQ ID NO: 23, 24, 25, 26, 27, 4 or 8.

7. The anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-6, wherein, The anti-HHLA2 antibody is a heavy chain antibody, and the heavy chain antibody contains the CH2 and CH3 fragments of human IgG.

8. The anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-7, wherein, The heavy chain antibody comprises the Fc region of human IgG1 or the Fc region of human IgG4; preferably, the heavy chain antibody comprises the Fc region whose amino acid sequence is shown in SEQ ID NO:

5.

9. A polynucleotide encoding an anti-HHLA2 antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

10. An expression vector comprising the polynucleotide of claim 9, or expressing the anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-8.

11. A host cell integrated with the polynucleotide of claim 9 or the expression vector of claim 10, or expressing the anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-8.

12. A pharmaceutical composition comprising: The anti-HHLA2 antibody or its antigen-binding fragment as described in any one of claims 1-8, and optionally a pharmaceutically acceptable excipient.

13. The pharmaceutical composition of claim 12, wherein, The pharmaceutical composition further includes a cancer therapeutic agent.

14. The anti-HHLA2 antibody or antigen-binding fragment thereof as described in any one of claims 1-8, the polynucleotide as described in claim 9, or the pharmaceutical composition as described in claim 12 or 13, for use in treating HHLA2-related diseases, enhancing immune responses, or detecting HHLA2 protein.

15. The anti-HHLA2 antibody or its antigen-binding fragment, polynucleotide, or pharmaceutical composition for the said use as described in claim 14, wherein, The HHLA2-related diseases mentioned are cancers; Preferably, the cancer is a solid tumor or a hematologic malignancy; Preferably, the cancer is lung cancer, kidney cancer, bone cancer, skin cancer, pancreatic cancer, colorectal cancer, head and neck cancer, liver cancer, ovarian cancer, prostate cancer, uterine cancer, cervical cancer, glioma, glioblastoma, neuroblastoma, pancreatic ductal carcinoma, thymoma, thyroid cancer, bile duct cancer, breast cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, or lymphoma.

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