Anti-CTLA-4 nano antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-CTLA-4 nano antibody and a preparation method and application thereof, the nano antibody at least comprises a VHH structural domain, and the VHH structural domain comprises CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1-CDR3 are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 3. The anti-CTLA-4 nano antibody provided by the invention can be used as a candidate antibody drug for various tumors, provides support for tumor antibody immunotherapy, can be used for developing a bispecific antibody, and lays a foundation for new application of an immunotherapy technology and the like.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to an anti-CTLA-4 nanobody, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy is one of the most successful methods in cancer treatment in recent years. It works by stimulating or mobilizing the body's immune function, enhancing the anti-tumor immunity of the tumor microenvironment, thereby controlling and killing tumor cells. Currently, tumor immunotherapy mainly includes: non-specific immune stimulation, adoptive cell therapy, and immune checkpoint inhibitors. The most successful areas and research hotspots in tumor immunotherapy are mainly focused on the research and development of immune checkpoint inhibitors, such as monoclonal antibodies like CTLA-4, PD-L1, OX40, CD19, CD20, and CD80, which are the focus of a global research and development race.

[0003] Under normal circumstances, human T lymphocytes regulate immune homeostasis by expressing a series of positive (promoting T cell differentiation and proliferation) and negative (inhibiting T cell differentiation and proliferation) regulatory factors, ensuring the body completes its immune response without causing excessive immunization and self-damage. In tumor tissues, negative regulatory factors or their ligands are often overexpressed, inhibiting T cell activation and proliferation or inducing T cell apoptosis, thus leading to the formation of an immunosuppressive tumor microenvironment, allowing tumor cells to evade the body's immune surveillance and killing. In the occurrence and development of tumors, the dominant immune checkpoints become one of the causes of immune tolerance. The application of immune checkpoint inhibitors can relieve immunosuppression in cancer patients and allow T cells to exert their anti-tumor effects. Currently, the most thoroughly studied immune checkpoints include cellular T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein (PD-1), and its ligand PD-L1.

[0004] Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, was identified and discovered by Pierre Golstein and his colleagues in 1987. CTLA-4 is a member of the immunoglobulin superfamily and is a leukocyte differentiation antigen expressed by activated T cells, transmitting inhibitory signals to them. It is a transmembrane receptor on T cells, homologous to the T cell costimulatory protein CD28. Both proteins can bind to ligands CD80 (B7-1) or CD86 (B7-2), with CTLA-4 exhibiting a higher binding affinity than CD28. CTLA-4 is routinely expressed in regulatory T cells, but its expression is often upregulated in cancer patients. Current research suggests that CTLA-4 antibodies may exert their immunomodulatory function primarily through two mechanisms. One mechanism involves CTLA-4 competitively binding to B7 molecules (CD80 and CD86) via CD28, recruiting phosphatases at their cytoplasmic tails, thereby attenuating T cell signaling and reducing the immune response. CTLA-4 antibodies thus reduce negative regulation, relieve immunosuppression, and enhance T cell responses by blocking CTLA-4. The other mechanism, as recently demonstrated, involves CTLA-4 antibody drugs acting through the clearance of tumor-associated regulatory T cells (Treg cells) mediated by the Fc region of the antibody heavy chain and Fc receptors on immune cells (such as macrophages or NK killer cells).

[0005] Currently, the only approved CTLA-4 antibody on the market is BMS's Yervoy (Ipilimumab), which was approved by the FDA in 2011 for the treatment of unresectable or metastatic melanoma. There are no domestically developed and marketed CTLA-4 antibody drugs in China. This CTLA-4 antibody is an IgG1 monoclonal antibody, which is costly to treat and has a remission rate of only 11.9%-19% for melanoma, but with significant side effects. Grade 3-5 irAEs occur in 19.9%-56.3% of patients, and serious adverse immune events have been observed in 15% of patients, which can be life-threatening if not properly managed. Its toxic side effects are largely related to the strong CTLA-4 blocking effect and the strong ADCC activity caused by IgG1-FC in the antibody. Furthermore, this antibody lacks species cross-reactivity in mouse experiments, making its further development costly and difficult. CN202010979860.X discloses a mouse anti-human CTLA-4 monoclonal antibody, but its experimental data is very limited and it does not have the ability to cross-react with species. In addition, this antibody is a traditional mouse anti-CTLA-4 monoclonal antibody.

[0006] Nanobodies are typically only one-tenth the size of traditional antibodies, around 15 kDa. They contain internal disulfide bonds and have numerous hydrophilic residues on their surface, exhibiting high stability and strong resistance to heat and pH. They can even be orally absorbed without degradation. The VHH germline gene sequence of camel nanobodies is highly homologous to human VH3. The difference lies in the slightly longer CDR1 and CDR3 sequences compared to humans; the protruding CDR3 enhances the specificity and affinity for antigen binding. Furthermore, nanobodies possess numerous advantages, including low immunogenicity, high solubility, ease of screening and expression, and cost-effectiveness. Compared to traditional antibodies, nanobodies naturally lack light chains, making them more suitable for functional antibody modification. For example, combining nanobodies targeting two or more specific targets to develop bispecific or multispecific antibodies can effectively avoid problems such as heavy / light chain mismatches that occur during the development of traditional bispecific or multispecific antibodies, effectively expanding the development potential of therapeutic antibodies. Summary of the Invention

[0007] To address the shortcomings of existing anti-CTLA-4 antibodies, which are all full-length antibodies or murine antibodies, lack cross-reactivity with antigen species, and have significant side effects, this invention provides an anti-CTLA-4 nanobody, its preparation method, and its application.

[0008] Specifically, the present invention provides the following technical solutions to solve the above technical problems.

[0009] A first aspect of the present invention provides an anti-CTLA-4 nanobody, the nanobody comprising a VHH domain, the VHH domain comprising CDR1, CDR2 and CDR3; the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO:1-3, respectively.

[0010] In some embodiments, the VHH domain further comprises FR1, FR2, FR3 and FR4, wherein FR1-FR4 each comprise an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 4-7.

[0011] In some embodiments, FR1-FR4 respectively comprise amino acid sequences as shown in SEQ ID NO: 4-7.

[0012] In some embodiments, the nanobody comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0013] In some specific embodiments, the nanobody comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0014] A second aspect of the present invention provides a fusion protein comprising a nanobody as described in the first aspect of the present invention.

[0015] In some embodiments, the fusion protein is selected from one or more of the following:

[0016] (i) Monovalent or multivalent nanobodies;

[0017] (ii) Bispecific antibodies or multispecific antibodies;

[0018] (iii) Heavy chain antibodies.

[0019] In some implementations, the fusion protein includes a heavy chain constant region.

[0020] In some implementations, the heavy chain constant region is the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0021] In some embodiments, the heavy chain constant region comprises CH2-CH3 or CH1-CH2-CH3.

[0022] In some embodiments, the heavy chain constant region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

[0023] In some embodiments, the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO: 13.

[0024] A third aspect of the present invention provides an isolated nucleic acid that encodes a nanobody as described in the first aspect of the present invention or a fusion protein as described in the second aspect.

[0025] A fourth aspect of the present invention provides a recombinant vector comprising the nucleic acid as described in the third aspect of the present invention.

[0026] In some embodiments, the recombinant vector is selected from viral vectors and non-viral vectors; the non-viral vector is selected from plasmids, linear DNA fragments, and RNA.

[0027] A fifth aspect of the present invention provides a transformant comprising a nucleic acid as described in the third aspect of the present invention, or a recombinant vector as described in the fourth aspect of the present invention; and the transformant is not an animal or plant species.

[0028] In some embodiments, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0029] In some embodiments, the eukaryotic cells are yeast cells or mammalian cells.

[0030] A sixth aspect of the present invention provides a method for preparing nanobodies or fusion proteins, the method comprising culturing a transformant as described in the fifth aspect of the present invention under suitable conditions to obtain the nanobodies or fusion proteins from the culture.

[0031] A seventh aspect of the present invention provides an antibody-drug conjugate having the structure shown in Formula I below:

[0032] Ab-[(L2) n -L1-D] y Formula I;

[0033] Wherein, D is a small molecule drug with cytotoxicity, L1 and L2 are linkers connecting the drug and the antibody, respectively; n is 0 or 1; y represents the average number of D coupled to the antibody, and 0 < y ≤ 10;

[0034] The Ab is a nanobody as described in the first aspect of the present invention or a fusion protein as described in the second aspect.

[0035] In some implementations, 2≤y≤7.

[0036] In some implementations, 3≤y≤6.

[0037] In some embodiments, the small molecule drug is one or more of MMAE, MMAF, DM1, DM3, and DM4; and the linker is one or more of SPP, SIAB, SMCC, MP, VC, ala-phe, PAB, and MC-VC-PAB.

[0038] An eighth aspect of the present invention provides a chimeric antigen receptor comprising (a) an antigen-binding domain comprising a nanobody as described in the first aspect of the present invention or a fusion protein as described in the second aspect.

[0039] In some embodiments, the chimeric antigen receptor further includes one or more of: (b) a hinge domain; (c) a transmembrane domain; (d) a co-stimulatory intracellular domain; (e) a signal transduction domain; and (f) a signal peptide.

[0040] A ninth aspect of the present invention provides a genetically modified cell comprising a chimeric antigen receptor as described in an eighth aspect of the present invention.

[0041] In some implementations, the host cell of the genetically modified cell is a T cell or an NK cell.

[0042] A tenth aspect of the present invention provides a pharmaceutical composition comprising at least one of the following: a nanobody as described in the first aspect of the present invention, a fusion protein as described in the second aspect, an antibody-drug conjugate as described in the seventh aspect, a chimeric antigen receptor as described in the eighth aspect, or a genetically modified cell as described in the ninth aspect, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0043] The eleventh aspect of the present invention provides a kit comprising nanobodies as described in the first aspect of the present invention, fusion proteins as described in the second aspect, nucleic acids as described in the third aspect, recombinant vectors as described in the fourth aspect, transformants as described in the fifth aspect, antibody-drug conjugates as described in the seventh aspect, chimeric antigen receptors as described in the eighth aspect, genetically modified cells as described in the ninth aspect, or pharmaceutical compositions as described in the tenth aspect.

[0044] The twelfth aspect of the present invention provides a method for detecting CTLA-4, the method comprising contacting a sample to be tested with a nanobody as described in the first aspect of the present invention, a fusion protein as described in the second aspect of the present invention, or a kit as described in the eleventh aspect of the present invention.

[0045] In some implementations, the method is for non-diagnostic and / or therapeutic purposes.

[0046] The thirteenth aspect of the present invention provides the use of nanobodies as described in the first aspect of the present invention, fusion proteins as described in the second aspect, nucleic acids as described in the third aspect, recombinant vectors as described in the fourth aspect, transformants as described in the fifth aspect, antibody-drug conjugates as described in the seventh aspect, chimeric antigen receptors as described in the eighth aspect, genetically modified cells as described in the ninth aspect, pharmaceutical compositions as described in the tenth aspect, or kits as described in the eleventh aspect in the preparation of medicaments for treating CTLA-4-related diseases.

[0047] The fourteenth aspect of the present invention provides a method for preventing and / or treating PD-L1-related diseases, the method comprising administering to a subject in need a preventive and / or therapeutically effective amount of, as described in the first aspect of the present invention, a fusion protein as described in the second aspect, a nucleic acid as described in the third aspect, a recombinant vector as described in the fourth aspect, a transformant as described in the fifth aspect, an antibody-drug conjugate as described in the seventh aspect, a chimeric antigen receptor as described in the eighth aspect, a genetically modified cell as described in the ninth aspect, a pharmaceutical composition as described in the tenth aspect, or a kit as described in the eleventh aspect.

[0048] The fifteenth aspect of the present invention provides nanobodies as described in the first aspect of the present invention, fusion proteins as described in the second aspect, nucleic acids as described in the third aspect, recombinant vectors as described in the fourth aspect, transformants as described in the fifth aspect, antibody-drug conjugates as described in the seventh aspect, chimeric antigen receptors as described in the eighth aspect, genetically modified cells as described in the ninth aspect, pharmaceutical compositions as described in the tenth aspect, or kits as described in the eleventh aspect, for the prevention and / or treatment of PD-L1-related diseases.

[0049] In some implementations, the CTLA-4-related disease is a tumor or an autoimmune disease.

[0050] In some implementations, the tumor is a solid tumor or a hematoma.

[0051] In some implementations, the solid tumor is selected from colon cancer, lung cancer, melanoma, and gastric cancer.

[0052] In some implementations, the autoimmune disease is selected from systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antisynthetic enzyme syndrome, IgG4-related disease, autoimmune hemolytic anemia, MDA5+ dermatomyositis-related interstitial pulmonary fibrosis, idiopathic inflammatory myopathy, immune thrombocytopenic purpura, myasthenia gravis, primary membranous nephropathy, type 1 diabetes mellitus, and autoimmune lymphoproliferative syndrome.

[0053] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0054] The reagents and raw materials used in this invention are all commercially available.

[0055] The positive and progressive effects of this invention are as follows:

[0056] This invention provides an anti-CTLA-4 nanobody with a relative molecular weight of approximately 80 kDa and about 80% homology to the human gene. It exhibits extremely high affinity as determined by SPR technology. Cellular function experiments have verified that the nanobody of this invention has T-cell activation activity, and mouse tumor drug trials have demonstrated significant anti-tumor effects. The nanobody provided by this invention possesses species cross-reactivity, recognizing both monkey and mouse CTLA-4, facilitating subsequent experiments. The nanobody described in this invention has stronger tissue penetration, and by adjusting the administration method and frequency, it can overcome the shortcomings of traditional CTLA-4 antibodies, such as strong toxic side effects. This invention also performs bivalent modification on the nanobody, enabling efficient expression in eukaryotic mammalian cells. Cellular function and in vivo mouse tumor drug efficacy experiments have verified the expressed antibody, demonstrating that the nanobody can effectively stimulate immune cells and exert anti-tumor effects. The anti-CTLA-4 nanobody can serve as a candidate antibody drug for various tumors, supporting tumor antibody immunotherapy, and can also be used for the development of bispecific antibodies, laying the foundation for new applications of immunotherapy technology. Attached Figure Description

[0057] Figure 1 Purified CTLA-4-VHH1 nanobody was detected by SDS-PAGE electrophoresis. 2 μg of CTLA-4-VHH1 nanobody was spotted on 12% Sure Page, with a purity >95%. The band size indicator showed approximately 15 KD, which is consistent with the theoretical size.

[0058] Figure 2 For CTLA-4 nanobody antigen-binding affinity testing;

[0059] Figure 3 The cross-species reaction test of the CTLA-4 nanobody antigen showed that the CTLA-4-VHH1 nanobody reacted with human, monkey, and mouse CTLA-4 proteins.

[0060] Figure 4 The image shows the purified gel image of the CTLA-4-VHH-FC bivalent antibody; 1. CTLA-4-VHH1-FC, M: protein ladder; 3 μg of CTLA-4-VHH1-FC was spotted into a non-reducing 12% sure-page gel, and the size of the electrophoretic band was approximately 80 kDa, which is consistent with its theoretical size;

[0061] Figure 5 The binding affinity of CTLA-4-VHH1 to the cell surface CTLA-4 protein;

[0062] Figure 6The vertical axis represents the tumor volume changes in a C57BL / 6 mouse subcutaneous MC38 colon cancer model induced by CTLA-4 antibody. Note: The vertical axis represents tumor volume (in mm). 3 Data are expressed as Mean ± SEM (mean ± standard error); the x-axis represents the time point for tumor volume detection after the first dose (Days post treatment); BIW3: twice weekly for a total of 6 doses: *p < 0.05, **p < 0.01; mpk: 1 mg per kilogram of mouse body weight; Tumorgrowth inhibition: tumor growth inhibition; TGI: tumor inhibition rate, TGI = 41.33%.

[0063] Figure 7 A schematic diagram of the design of a bispecific nanobody.

[0064] Figure 8 SDS-PAGE assay of bispecific nanobodies and their parental monoclonal antibodies.

[0065] Figure 9 For in vitro cell function testing of BsAb-F1.

[0066] Figure 10 The inhibitory effect of different doses of BsAb-F1 on mouse tumor growth; tumor volume growth curves (Part A) and mouse body weight change curves (Part B) under different doses of BsAb-F1. (Part C) Mouse efficacy test design. Tumor weight (Part D) and tumor images (Part E) at the experimental endpoint (day 34) in different treatment groups. One mouse in group G1 had a tumor volume exceeding 3000 mm on day 28. 3 In the case of euthanasia, the tumor completely disappeared in one mouse in group G4 on day 21. All data are presented as mean ± standard error (n=6), * P < 0.05, ** P < 0.01 and > 0.001. Detailed Implementation

[0067] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0068] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0069] The term "anti-CTLA-4 nanobody" refers to an antibody that can bind to human CTLA-4 with sufficient affinity, making it usable as a diagnostic and / or therapeutic agent targeting human CTLA-4.

[0070] The terms "bispecific antibody" or "multispecific antibody," used in their broadest sense, encompass antibodies that exhibit multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies, antibody fragments covalently or non-covalently linked, etc.

[0071] The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if eight out of ten positions in two sequences match, then the two sequences have 80% identity. Typically, two sequences are compared to produce maximum identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences.

[0072] "Bispecific antibody," "bispecific antibody," or "bispecific recombinant protein" refers to an antibody that has binding specificity to two different antigens (or epitopes). It contains antigen-binding domains that are specific to different antigens (or epitopes), for example, two antigen-binding domains that are specific to different antigens (or epitopes), thereby enabling it to bind to two different binding sites and / or target molecules. The individual antigen-binding domains of a bispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or its antigen-binding fragments (e.g., Fab, Fab', F(ab')2, scFab, Fv, or scFv).

[0073] The term "variable region" refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. VH and VL each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The terms "complementarity-determining region" or "CDR" refer to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDR sequence in a specific variable region can be determined using schemes known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes, to define the regional range and combinations of CDRs. Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR (including heavy chain variable region residues), it means the numbering position according to the IMGT numbering system.

[0074] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), such as those spatially proximal due to antigen folding (i.e., tertiary folding of antigens as proteins). The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463).

[0075] The term "specific binding" refers to an antibody binding to an antigen or an epitope within that antigen with a higher affinity than it would to other antigens or epitopes. However, antibodies that specifically bind to an antigen or an epitope within an antigen may exhibit cross-reactivity with other related antigens, for example, cross-reactivity with the same antigens from other species (homologous) such as humans or monkeys, such as the cynomolgus (cyno), chimpanzee (chimp), or common marmoset (marmoset).

[0076] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen).

[0077] The term "EC" 50 The half-maximal effect concentration (WMP) is the antibody concentration that produces a 50% maximum effect and can be measured using methods known in the art.

[0078] The term "nucleic acid molecule" or "nucleic acid" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0079] The term "host cell" refers to a cell in which the vector can proliferate and whose DNA can be expressed; said cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the tested host cell. It should be understood that not all progeny are identical to the parent cell, as mutations can occur during replication; such progeny are included.

[0080] The term "transformer" refers to a host cell in which foreign DNA has been successfully introduced and stably maintained. For example, a recombinant cell formed by integrating plasmid DNA into a host cell through transformation (bacteria) or transfection (eukaryotic cells) techniques is called a transformer.

[0081] The term "antibody drug conjugate" (ADC) herein refers to a molecule formed by conjugating one or more immunoglobulin-associated molecules or fragments thereof with one or more other molecules. A conjugate typically contains at least one non-protein chemical structural part, such as a chemical linker for achieving the conjugation. In some cases, the other molecules may be the same as immunoglobulin-associated molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-associated molecules or fragments thereof. The one or more additional molecules may be the same as or different from each other. For example, the other molecules may be target-binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules.

[0082] The term "pharmaceutically acceptable" means that it can be administered to an individual or subject without producing biologically or otherwise undesirable side effects, such as serious, intolerable side effects.

[0083] The term "pharmaceuticalally acceptable carrier" refers to all substances contained in a pharmaceutical preparation other than the active pharmaceutical ingredient, generally divided into two main categories: excipients and additives. It has the same meaning as "pharmaceuticalally acceptable excipient" and can be used interchangeably.

[0084] The term “treatment” refers to a therapeutic approach or a remission measure. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition; or (4) slowing the development of the disease or one or more biological manifestations of the condition.

[0085] The term "prevention" includes the suppression of the occurrence or development of a disease or condition, or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative programs. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0086] The term "effective amount" refers to such an amount or dose of the bispecific antibody or its antigen-binding fragment or ADC molecule or composition or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0087] The term "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. The therapeutic effective amount of an antibody or ADC can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or ADC to elicit the desired response in the individual. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or ADC are less than the beneficial therapeutic effect. Relative to an untreated subject, "therapeutic effective amount" preferably inhibits measurable parameters (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors. Therapeutic effective amount will vary depending on the type of compound, disease type, disease severity, patient age, etc., but may be adjusted as appropriate by those skilled in the art.

[0088] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered in subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0089] The term "pharmaceutical composition" refers to a composition in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0090] The terms “tumor” and “cancer” are used interchangeably in this document to refer to a physiological disorder in mammals in which cell growth is not regulated, encompassing both solid and liquid tumors, as well as malignant and benign tumors, and all precancerous and cancerous cells and tissues.

[0091] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0092] Example 1: Animal Immunization

[0093] Alpaca immunization: 100 μg of CTLA-4 antigen (ARCO, CT4-H5229) was mixed with an equal volume of Freund's adjuvant and injected subcutaneously at multiple sites in the alpaca's neck and forelegs. 20 mL of venous blood was collected from the alpaca before immunization. Subsequent immunizations were administered on days 21, 35, 49, and 64, for a total of 5 immunizations. Ten days after the last immunization, 20 mL of PBMCs were collected from the alpaca for subsequent immunization library construction.

[0094] Example 2: Construction of a phage display library for CTLA-4 nanobodies

[0095] (1) PBMC isolation. Blood samples were taken from alpacas after immunization. Lymphocytes in the peripheral blood of alpacas were isolated and purified using lymphocyte separation medium (GE, 17-1440-02) and density gradient centrifugation. The cells were washed with PBS two to three times and then used for subsequent RNA extraction.

[0096] (2) Construction of CTLA-4 nanobody library.

[0097] Total RNA extraction: Take the lymphocytes isolated in Example 2 (1), add 1 ml of Trizol, pipette and mix well to fully lyse the cells, let stand at room temperature for 10 min, add 0.2 ml of chloroform, shake vigorously for 15 seconds, let stand on ice for 5-10 min, place in a refrigerated centrifuge at 4°C, centrifuge at 12,000 rpm for 10 min, collect the upper aqueous phase, add an equal volume of isopropanol, mix well, let stand at room temperature for 15 min, wait for nucleic acid precipitation, centrifuge at high speed to remove the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, centrifuge at high speed to remove the supernatant, drain the water, dissolve the RNA in nuclease-free water, and take 1 μl for concentration and purity determination.

[0098] cDNA synthesis: Take 20 μg of RNA and synthesize cDNA using the SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen) kit and procedure. Store the synthesized cDNA at -20℃.

[0099] PCR amplification: Using the reverse transcription product cDNA as a template, the variable region (VHH) of the camel heavy chain antibody was amplified by Nest-PCR. Table 1 shows the names and sequences of the primers used for amplification.

[0100] Table 1. Primer information used for amplification of the alpaca VHH fragment

[0101]

[0102] The PCR reaction conditions are as follows:

[0103] Round 1:

[0104] cDNA 2 μl; 2×rTaq Mix 25 μl; CALL001 / 001-2 / 001-3 mix (10 uM) 1.5 μl; CALL002 (10 uM) 1.5 μl; Water to bring the total to 50 μl.

[0105] Reaction conditions: 95℃, 5 min; 94℃, 45 s; 56℃, 45 s; 72℃, 45 s per cycle; 72℃, 5 min; amplification 25 cycles.

[0106] Round Two:

[0107] Template 20ng; 2×rTaq Mix 25μl; VHH For (10uM) 1.5μl; VHH Back / VHH back-2 / VHH back-3 mix (10uM) 1.5μl; Water make up to 50μl.

[0108] Reaction conditions: 95℃, 5 min; 94℃, 30 s; 56℃, 45 s; 72℃, 35 s per cycle; 72℃, 10 min; amplification 17 cycles.

[0109] After the PCR reaction, the target fragment was recovered using the QIAgen gel purification kit (Qiagen) or PCR purification kit (Qiagen) and their operating procedures. The recovered product was then tested for concentration and purity using Nanodrop 2000 and stored at -20°C.

[0110] (3) Construction of phage display library.

[0111] Enzyme digestion and ligation: The VHH fragment and pMECs obtained in (2) were double-digested with restriction endonucleases NotI and PstI (NEB), respectively.

[0112] Vector digestion system:

[0113] pMECS vector 30 μg; PstI 10 μl; NotI 20 μl; Cutsmart (buffer) 30 μl; add H2O to 300 μl; digest overnight at 37 degrees Celsius.

[0114] VHH fragment digestion system:

[0115] VHH fragment 10 μg; PstI 5 μl; NotI 10 μl; 10×Cutsmart buffer 20 μl; add H2O to 200 μl; digest overnight at 37 degrees Celsius.

[0116] The enzyme digestion products of the VHH fragment and pMECs vector were recovered by agarose gel electrophoresis and agarose gel recovery kit (QIAGEN, 20051), and then the enzyme digestion products were ligated.

[0117] The connection system is as follows:

[0118] 1.5 μg pMECs vector; 0.5 μg VHH fragment; 1 μl T4 ligase (NEB); 2 μl 10×T4 ligation buffer; add H2O to 20 μl; ligate overnight at 16 °C.

[0119] Transformation: Mix 1 μl of the ligation product with 30 μl of TG1 supercompetent cells, incubate on ice for 5 min, transfer the mixture to an electroporation cuvette, and electroporate at 1.5 kV. After electroporation, add 1 ml of SOC medium, pipette and mix well, then transfer to a 2 ml centrifuge tube. Incubate at 37°C for 1 h for recovery, and then perform serial dilutions of 10⁻⁶. 2 10 3 and 10 4 The diluted bacterial culture was spread onto plates and incubated overnight at 37°C. The colony count was calculated the following day, reaching approximately 10. 6 One clone / plate. Repeat the transformation using the same method described above until the number of clones in the library reaches 10. 7 The transformed bacterial culture, which is the antibody phage library, is then added with an equal volume of 50% glycerol and stored at -20°C.

[0120] (4) Detection of diversity of nanobodies in phage display library. Sixty clones from (3) were randomly selected and sent for sequencing by 3730. The antibody VHH region sequence was obtained and compared. It was found that only 3 pairs of clone sequences were repeated. Among the 60 clones, 57 clones had different antibody VHH region sequences, indicating that the antibody library has good diversity.

[0121] (5) The obtained library was amplified, and a phage strain carrying the CTLA-4 nanobody was rescued using helper phage. The phage library preserved in (3) was inoculated into 100 mL of culture medium and cultured to the logarithmic growth phase. 20 μL of helper phage (pfu=2×10⁻⁶) was added. 12 At room temperature, let stand for 30 min, centrifuge at low speed, resuspend the precipitate in culture medium, inoculate into 300 mL of culture medium, and incubate overnight. The next day, centrifuge at 3,000 g for 30 min, collect the supernatant, add PEG solution to precipitate the phage, let stand on ice for 30 min, centrifuge at 2,200 g for 30 min to precipitate the phage library carrying PD-L1 nanoantibodies, and resuspend the precipitate in an appropriate amount of PBS to a titer of 2 × 10⁻⁶. 12 pfu / mL.

[0122] Example 3: Obtaining high-affinity CTLA-4 nanobodies using phage display technology

[0123] (1) Washing of the affinity CTLA-4 nanobody phage library.

[0124] Coat an ELISA plate with 100 ng of antigen and incubate overnight at 4°C. The next day, add 2 × 10⁻⁶ CTLA-4 nanoparticle antibody phages obtained above. 11Incubate at room temperature for 2 hours; wash 10 times with PBST, add 100 μL of triethylamine elution buffer, incubate at room temperature for 10-30 minutes, and collect the phages, which are the CTLA-4 nanobody phage library obtained by affinity washing; take 10 μL to infect TG1 cells, spread on a plate, and use the remaining screened phage library for further amplification and rescue.

[0125] (2) Amplification and rescue of the phage library after screening. The amplification and rescue method is the same as in Example 2 (5). The phage library obtained after the first round of screening is stored at 4°C and the screening is repeated for 2-3 rounds.

[0126] (4) Selection of CTLA-4 high affinity nanobody monoclonal antibodies.

[0127] As above, 100 ng of CTLA-4 antigen was used to coat the ELISA plate as the experimental group. At the same time, an equal number of wells were left uncoated as negative controls. Incubate overnight at 4℃; randomly select 32 single clones from the plates of the 4th round of screening and add them to 1 ml of culture medium, incubate at 37℃ until the logarithmic phase, add 10 μL of 1M IPTG for induction overnight; the next day, centrifuge to collect bacterial sediment, lyse, centrifuge at 5,000g for 15 min, and collect the supernatant; at the same time, add 2% BSA to the ELISA plate and block at room temperature for 1 h; add the supernatant of one single clone to each well of the experimental group and the negative control group, incubate at room temperature for 2 h; wash 10 times with PBST, add anti-HA-HRP tag antibody, incubate at room temperature for 1 h; wash 5 times with PBST, add substrate chromogenic agent, react for 10-20 min, add stop agent, and read the absorbance value on a microplate reader (BioTek, EPOCH); when the ratio of absorbance value to control well is greater than 2.1, it is judged as a positive clone. The test results are shown in Table 2. In this case, 10 CTLA-4 positive clones were screened. These 10 positive clones were sent to Guangzhou Liuhe Biotechnology for 3730 sequencing, which detected 5 VHH antibody sequences, named CTLA-4-VHH1 ~ CTLA-4-VHH5. The antibody nucleic acid sequences were analyzed using IMGT / V-QUEST for the frame region (FR1~FR4) and variable region (CDR1~CDR3). The five sequences are shown below:

[0128] CTLA-4-VHH1

[0129] Amino acid sequence (SEQ ID NO: 8)

[0130] QVQLQESGGGLVQAGGSLRLSCEVSGDITNFNAMGWGRQAPGNKRELVATIGQTEGSGYADSVKGRFTISTDSARNTVYLQMNSLKPEDTAVYYCYAFRDGANQLWGQGTQVTVSS

[0131] Among them,

[0132] CDR1: GDITNFNA (SEQ ID NO: 1)

[0133] CDR2: IGQTEGS (SEQ ID NO: 2)

[0134] CDR3: YAFRDGANQL (SEQ ID NO: 3)

[0135] FR1: QVQLQESGGGLVQAGGSLRLSCEVS (SEQ ID NO: 4)

[0136] FR2: MGWGRQAPGNKRELVAT (SEQ ID NO: 5)

[0137] FR3: GYADSVKGRFTISTDSARNTVYLQMNSLKPEDTAVYYC (SEQ ID NO: 6)

[0138] FR4: WGQGTQVTVSS (SEQ ID NO: 7)

[0139] Amino acid sequence of CTLA-4-VHH2 (SEQ ID NO: 9)

[0140] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSSIYSDGRNTYYADSVKGRFTISRDNAKNTLYLQMNSLKPDDTAVYYCAAGFPSIGLLPYEYDYWGQGTQVTVSS

[0141] Amino acid sequence of CTLA-4-VHH3 (SEQ ID NO: 10)

[0142] QVQLQESGGGLVQPGGSLTLSCAASGFTLTSVDINWVRQAPGKGLEWVSGINPGGHTTYQIDSVKGRFAISRDNAKNTVYLQMNSLKPEDTARYYCTEGVGAFWGQGTQVTVSS

[0143] CTLA-4-VHH4 amino acid sequence (SEQ ID NO: 11)

[0144] QVQLQESGGGLVQAGGSMKLSCTASGFPFARTIIAWFRQAPGKEREALSCVNADDDTPYYKDFVKGRFTISRDAAKKTVSLQMNSLTPEDTAVYYCAAADVPRCLPLYFGNEDIYEAWGQGTQVTVSS

[0145] CTLA-4-VHH5 amino acid sequence (SEQ ID NO: 12)

[0146] QVQLQESGGGLVQPGGSLRLSCAASGFSLDNYAIGWFRQAPGKEREGVSCITSSDGTTYYPESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADAKEDQSSTTAWAMCNNPGRIYGMDYWGKGTLVTVSS

[0147] Table 2. CTLA-4 nanobody ELISA screening

[0148]

[0149] Example 4: Induction, expression and purification of CTLA-4 nanobodies

[0150] (1) Induction of CTLA-4 nanobody expression.

[0151] Single clones of CTLA-4-VHH1 were picked from the plate and inoculated into 10 ml of ampicillin-containing medium. The culture was incubated overnight at 37°C and 220 rpm. The next day, 2 ml of the overnight culture was inoculated into 200 ml of ampicillin-containing medium and cultured at 37°C and 220 rpm until the logarithmic growth phase (OD600 0.6-0.8). IPTG was then added to induce nanobody expression overnight. The following day, the bacterial pellet was collected, and the cells were lysed using a hypotonic method. The supernatant was collected by high-speed centrifugation for subsequent protein purification.

[0152] (2) Purification of CTLA-4 nanobody.

[0153] Purified CTLA-4 nanobody was obtained using an affinity purification column (His-Trap 1 ml, GE). The Ni column was first washed with ultrapure water, then with equilibration buffer and affinity A. The resulting supernatant was added to the Ni column at a flow rate of 1 ml / min. Impurities were washed away with 30 column volumes of affinity A1 buffer (30 mM imidazole) until the OD280 was below 0.0001. The target protein was then eluted with 10 column volumes of elution buffer (150 mM imidazole). The purified target protein was detected by vertical electrophoresis on 12% SDS-PAGE using a Bio-rad, Mini PROTEAN Tetra System. The expression and purification of the CTLA-4 nanobody are shown in the figure below. Figure 1 As shown in the figure, the CTLA-4 nanobody band is approximately 15 kDa in size and has a purity >90%, indicating that the nanobody was successfully and correctly expressed.

[0154] Example 5: Assay of CTLA-4 nanobody antigen binding affinity

[0155] Coat an ELISA plate with 100 ng of CTLA-4 antigen and incubate overnight at 4°C. Wash three times with PBST (0.05%). Add 200 μl of 2% BSA to each well and incubate at room temperature for 2 h. Wash three times with PBST. Serially dilute the purified CTLA-4 nanoantibodies obtained in Example 4. The starting concentration is 4.5 μg / ml (300 nM), and each well is serially diluted 3-fold for a total of 7-8 concentration gradients. Add 100 μl of each concentration gradient to three wells and incubate at room temperature for 1 h. Wash six times with PBST, add 100 μl / well of 2000-3000 times diluted antiHA and HRP (abcam), and incubate at room temperature for 1 h. Wash six times with PBST, add 100 μl of TMB chromogenic solution (abcam), develop for 10 min, and then add an equal volume of TMB stop buffer (abcam) to stop the OD450 reading.

[0156] ELISA test results are as follows ( Figure 2 The results showed that the CTLA-4-VHH1 nanobody had strong affinity binding activity to the CTLA-4 antigen, with a half-maximal effective concentration (EC50) of 1.676 nM, indicating strong affinity binding activity. The other four VHH groups showed weak affinity, so only CTLA-4-VHH1 was selected for subsequent studies.

[0157] Example 6: Cross-species reaction of CTLA-4 nanobody antigens

[0158] 100 ng of human CTLA-4, mouse CTLA-4, and cynomolgus CTLA-4 (purchased from Beijing Baipusaisi) were added to each well of an ELISA plate and incubated overnight at 4°C. The plates were washed three times with 0.05% PBST. 200 μl of 2% BSA was added to each well and incubated at room temperature for 2 hours. The plates were washed three times with PBST. The purified CTLA-4 nanobodies from Example 4 were diluted to a concentration of 1.5 μg / ml and then added to the wells of ELISA plates coated with human CTLA-4, mouse CTLA-4, and cynomolgus CTLA-4, respectively, at 100 μl per well. The plates were incubated at room temperature for 1 hour, with PBST added as a blank control. The plates were washed six times with PBST, and 100 μl / well of anti-HA and HRP (abcam) diluted 2000-3000 times were added and incubated at room temperature for 1 hour. Wash 6 times with PBST, add 100 μl of TMB chromogenic solution (abcam), incubate for 10 min, then add an equal volume of TMB stop buffer (abcam) to stop the incubation. Read the OD450 value using a microplate reader (BioTek, EPOCH). The results are as follows: Figure 3 It can be seen that the CTLA-4-VHH1 nanobody reacts with human, monkey, and mouse CTLA-4 proteins.

[0159] The aforementioned cross-reactivity of antigens is beneficial for the direct in vivo validation experiments of CTLA-4-VHH1 on different animal species (without the need for humanization of the CTLA-4 gene, saving experimental validation costs).

[0160] Example 7: Bivalent modification and eukaryotic expression of CTLA-4 nanobodies

[0161] The CTLA-4-VHH1 nanobody was synthesized by fusing the human IgG4 Fc gene sequence (the corresponding amino acid sequence is SEQ ID NO: 13). The synthesized gene sequence was subcloned into the expression vector pcMV3 (SinoBiological, CV011) using seamless cloning PCR technology (specific procedures refer to the HieffClone® plus One Step Cloning Kit instructions). The recombinant single-domain antibody VHH-FC fusion protein particle was transfected into HEK293T cells (ATCC, CRL-11268) for expression. The recombinant plasmid was diluted with PBS, and PEI (polyethylenimine) solution required for transformation was added. After mixing, the mixture was added to the HEK293T cell suspension and incubated at 37°C, 5% CO2, and ≥80% relative humidity at 150 rpm. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by protein A affinity chromatography to obtain the target CTLA-4-VHH1-FC fusion protein. Figure 4 The purity is greater than 95%, and the expression level is high, about 150 mg / L. The amino acid sequence is shown in SEQ ID NO: 12. The molecular weight of the obtained bivalent antibody is about 80 KD.

[0162] IgG4 Fc sequence (SEQ ID NO: 13)

[0163] PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0164] IgG4 hinge region sequence (SEQ ID NO: 14)

[0165] EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP

[0166] Example 8: CTLA-4 Nanobody Affinity Assay

[0167] The affinity of CTLA-4 nanobodies was analyzed using the SPR method. An NTA chip (GE, BR-1005-32) was used. Following the chip's instruction manual, the antigen human CTLA-4-his was first captured at a flow rate of 10 μl / min for 30 s. The antibody CTLA-4-VHH1-FC was diluted with system running buffer HBSP (0.005%) to create suitable concentration gradients. In this example, the gradient was 2-fold, starting from approximately 1.5 μg / ml (20 nM), resulting in six concentration gradients. Each injection lasted 120 s, followed by dissociation for 240 s, and then regeneration with 350 mM EDTA. This cycle was repeated until all concentration gradients were injected. After the program ran, the built-in analysis program of the Biacore T200 (GE) instrument was used for fitting analysis to obtain the nanobody affinity constants (Table 3).

[0168] Table 3. Detection of binding affinity constants of human and mouse CTLA-4 nanobodies.

[0169]

[0170] The data in Table 2 above show that the CTLA-4-VHH1-FC bivalent nanobody can bind to both human and mouse CTLA-4 simultaneously, with high affinity (above nanomolar). This antibody's ability to recognize mouse CTLA-4 allows for direct tumor efficacy experiments in wild-type mice. While the affinity of CTLA-4-VHH1-FC for human CTLA-4 is slightly weaker than that of Yervoy, both are at the 0.1 nanomolar level. Recent studies suggest that the mechanism of action of CTLA-4-like antibodies may rely more on the clearance of tumor microenvironment regulatory T cells (Tregs) mediated by the constant region Fc of the antibody heavy chain and Fc receptors of immune cells (such as macrophages or NK killer cells) to exert immunotherapeutic effects. Furthermore, CTLA-4-VHH1-FC, with its stable nanobody characteristics and small molecular weight, may be more conducive to penetrating tumor tissue and reaching the site of action.

[0171] Example 9: Cellular Binding Affinity Test of CTLA-4 Bivalent Nanobody

[0172] GS-J1 (GenScript, M00612) cells were seeded in 384-well plates (white plate, flat bottom) at a density of 40,000 cells / well / 20 μL of culture medium containing 5 μg / ml PHA (Promega, G7940). Then, 20 μL of GS-C1 / CD80 (GenScript, M00613) cells (20,000 cells / well), 20 μL of 2 μg / mL CTLA-4 solution (Genscript, Z03373), and 20 μL of serially diluted CTLA-4-VHH1 sample solution were added to each well. The plates were incubated at 37°C for 24 hours. Transfer the supernatant from the assay plate to a new culture dish. Detect the concentration of IL-2 in the supernatant using a human IL-2 assay kit (Cisbio, 62HIL02PET). Detect the value using Phera Star, with excitation light at 334 nm and simultaneous readings of two absorption wavelengths at 660 nm and 620 nm.

[0173] See results Figure 6 CTLA-4-VHH1-Fc can block the immunosuppression of effector cells by CTLA-4 and effectively stimulate effector cells to secrete the cytokine IL-2, with a half-maximal effective concentration (IC50) of 5.592 μg / ml, which is comparable to the positive control Yervoy (2.159 μg / ml). Since the mechanism of action of CTLA monoclonal antibodies may rely more on the clearance of Tregs mediated by the antibody's Fc fragment, the CTLA-4 blocking effect of this patented antibody is weaker than that of Yervoy, thus reducing adverse reactions caused by excessive immunostimulation from strong CTLA-4 blocking antibodies.

[0174] Example 10: Inhibitory effect of CTLA-4 bivalent nanobody on mouse tumors

[0175] Mouse wild-type colon cancer cells MC38 were resuscitated and cultured. MC38 cells in the logarithmic growth phase were collected, and cell viability was determined to be above 95%. The culture medium was removed, and the cells were washed twice with PBS before being inoculated into the right forelimb of ordinary C57BL / 6 wild-type mice (derived from Jicui Yaokang, not CTLA-4 humanized mice). The inoculation volume was 1×102 6 / 100 μL / mouse, on day 7 post-inoculation, the average tumor volume reached 94 mm. 3Mice were selected and divided into two groups (Group 1 - IgG4 negative control group, Group 2 - CTLA-4-VHH1-FC test group), with 6 mice in each group. Day 0 was defined as the day of grouping. Drug administration began on Day 0 and continued twice weekly for a total of 6 weeks. The IgG group received a dose of 1.5 mpk (1.5 mg per kilogram of mouse weight) per administration; the CTLA-4-VHH1-FC group received a dose of 0.8 mpk per mouse. Tumor volume inhibition in the mice was observed.

[0176] Test results as follows Figure 6 As shown, compared with the IgG4 negative control group, CTLA-4-VHH1-FC significantly inhibited tumor volume starting from day 7 after administration. The final tumor volume inhibition efficiency TGItv = 41.33%, demonstrating a significant tumor-inhibiting effect.

[0177] Example 11: Bispecific antibody modification of CTLA-4 nanobodies

[0178] 1) Molecular design

[0179] The CTLA-4 nanobody (VHH1) and PD-L1 target nanobody (SEQ ID NO: 15) of this invention were combined to design an IgG-like bispecific antibody. Figure 7 There are three main types. The first is a symmetrical configuration, in which two target nanobodies are respectively linked to the N-terminus and C-terminus of the Fc (BsAb-F1); the second is a symmetrical "Y"-shaped structure, in which two target nanobodies are tandemly linked to the N-terminus of the Fc (BsAb-F2); the third is an asymmetrical "Y"-shaped structure, in which two target nanobodies are bivalently linked to both ends of the Fc and specifically combined using the Knob-in-hole technique (Spiess et al., 2013; Ridgway et al., 1996) to form a bispecific antibody (BsAb-F3).

[0180] Amino acid sequence of PD-L1 nanobody (SEQ ID NO: 15)

[0181] QVQLQESGGGLVQTGGSLRLSCVSSGRERTAMGWFRQAPGKERELVGSITSTGGDTYIADAMQGRFTISRDNAKNTVYLQMNNLRPEDTAVYYCAARNGMMALATHRALYEYWGQGTQVTVSS

[0182] CDR1: SSGRERTA (SEQ ID NO: 16)

[0183] CDR2: ITSTGGDT (SEQ ID NO: 17)

[0184] CDR3: AARNGMMALATHRALYEY (SEQ ID NO: 18)

[0185] Amino acid sequence of BsAb-F1 (SEQ ID NO: 19)

[0186] QVQLQESGGGLVQTGGSLRLSCVSSGRERTAMGWFRQAPGKERELVGSITSTGGDTYIADAMQGRFTISRDNAKNTVYLQMNNLRPEDTAVYYCAARNGMMALATHRALYEYWGQGTQVTVSSPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGKEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPQVQLQESGGGLVQAGGSLRLSCEVSGDITNFNAMGWGRQAPGNKRELVATIGQTEGSGYADSVKGRFTISTDSARNTVYLQMNSLKPEDTAVYYCYAFRDGANQLWGQGTQVTVSS

[0187] Amino acid sequence of BsAb-F2 (SEQ ID NO: 20)

[0188] QVQLQESGGGLVQTGGSLRLSCVSSGRERTAMGWFRQAPGKERELVGSITSTGGDTYIADAMQGRFTISRDNAKNTVYLQMNNLRPEDTAVYYCAARNGMMALATHRALYEYWGQGTQVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPQVQLQESGGGLVQAGGSLRLSCEVSGDITNFNAMGWGRQAPGNKRELVATIGQTEGSGYADSVKGRFTISTDSARNTVYLQMNSLKPEDTAVYYCYAFRDGANQLWGQGTQVTVSSPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0189] Amino acid sequence of the heavy chain 1 of BsAb-F3 (SEQ ID NO: 21)

[0190] QVQLQESGGGLVQTGGSLRLSCVSSGRERTAMGWFRQAPGKERELVGSITSTGGDTYIADAMQGRFTISRDNAKNTVYLQMNNLRPEDTAVYYCAARNGMMALATHRALYEYWGQGTQVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPQVQLQESGGGLVQTGGSLRLSCVSSGRERTAMGWFRQAPGKERELVGSITSTGGDTYIADAMQGRFTISRDNAKNTVYLQMNNLRPEDTAVYYCAARNGMMALATHRALYEYWGQGTQVTVSSPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0191] Amino acid sequence of the BsAb-F3 heavy chain 2 (SEQ ID NO: 22)

[0192] QVQLQESGGGLVQAGGSLRLSCEVSGDITNFNAMGWGRQAPGNKRELVATIGQTEGSGYADSVKGRFTISTDSARNTVYLQMNSLKPEDTAVYYCYAFRDGANQLWGQGTQVTVSSEPKIPQ PQPKPQPQPQPQPKPQPKPEPECTCPKCPQVQLQESGGGLVQAGGSLRLSCEVSGDITNFNAMGWGRQAPGNKRELVATIGQTEGSGYADSVKGRFTISTDSARNTVYLQMNSLKPEDTAVYY CYAFRDGANQLWGQGTQVTVSSPPCPSCPAPEFLGGPSSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0193] Example 12: Expression and purification of bispecific nanobodies

[0194] The designed bispecific nanobody gene sequence was synthesized by Beijing Yiqiao Biotechnology Co., Ltd., and the synthesized gene was directly subcloned into a customized eukaryotic expression vector (SinoBiological, CV011). The recombinant plasmid was transfected into HEK293T cells (ATCC, CRL-11268) for expression. The recombinant plasmid was diluted with PBS, and PEI (polyethylenimine) solution required for transformation was added. After mixing, the mixture was added to the HEK293T cell suspension and incubated at 37°C, 8% CO2, and ≥80% relative humidity at 150 rpm. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by protein A affinity chromatography (purchased from GE) to obtain the target antibody.

[0195] The results show that ( Figure 8The expression yields of the three bispecific nanobodies, from highest to lowest, were: BsAb-F1 (300 mg / L, 2.68 μmol / L), BsAb-F2 (~210 mg / L, 1.88 μmol / L), and BsAb-F3 (~207 mg / L, 1.85 μmol / L); protein purity was 90% or higher, and the target band was approximately 115 KD. The parental monoclonal antibodies VHH15-8-FC and VHH1-FC showed similar expression yields (molar yields) of approximately 80 KD.

[0196] Example 13: Detection of Bispecific Nanobody EC50

[0197] Add 100 ng of PD-L1, his tag or CTLA-4, his tag (Beijing Baipusaisi) to each well of the ELISA plate and incubate overnight at 4°C. The next day, wash three times with PBST, add 200 μl of 2% BSA to each well, and incubate at room temperature for 2 hours. Wash three times with PBST, then add a series of serially diluted bispecific nanobody and its parent monoclonal antibody, Tecentriq and Yervoy as positive controls. The specific dilution concentrations are as follows: starting concentration 5 nM, followed by 5-fold serial dilutions, for a total of 8 concentration gradients, with each gradient repeated in 3 wells, 100 μl per well, and incubate at room temperature for 1 hour. Wash 5-6 times with PBST, add 100 μl of diluted anti-human IgG, HRP (A80-304P, Bethyl) to each well, and incubate at room temperature for 1 hour. Wash 5-6 times with PBST, add 100 μl of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 10 minutes. Add an equal volume of TMB stop solution. The buffer is used to terminate color development, and OD450 readings and statistical analysis are performed.

[0198] ELISA results showed (Table 4): BsAb-F1 had a higher affinity for PD-L1 and CTLA-4 than its parental nanomonoantibodies VHH15-8-FC and VHH1-FC; and its EC50 binding to PD-L1 and CTLA-4 was similar (ratio close to 1), indicating that there was little or no mutual interference between the VHH binding arms of the two targets or between the VHH binding arm and the Fc domain. However, the EC50 binding to CTLA-4 of BsAb-F2 and BsAb-F3 was lower than that of their parental monoclonal antibodies, and also lower than that of PD-L1. This may be because the CTLA-4-VHH arm in the two antibodies is sterically hindered by the PD-L1-VHH arm in series with it, or by another CTLA-4-VHH arm in series with it.

[0199] Table 4. EC50 of bispecific nanobody binding to PD-L1 and CTLA-4

[0200]

[0201] Note: "n / a" means not applicable.

[0202] Example 14: Detection of KD Affinity for Bispecific Nanobodies

[0203] The antigen was diluted to 2 μg / ml, and then the parameters were set on the Biacore T200 (GE) instrument to inject the antigen at a flow rate of 10 μL / min for 30-60 s, so that the NTA chip could capture about 60 RU of His-tagged antigen. Next, the antibody to be tested was diluted 2-fold with 0.05% HBS-T injection buffer to concentrations of 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 nM, respectively, for 120 s for each concentration, followed by 240 s for dissociation. 3.5M EDTA was then injected at 30 μL / min for 60 s, followed by regeneration. Nickel chloride was then injected at 10 μL / min for 60 s. Steps 1-4 were then repeated until all concentration gradients were completed. After the program was completed, the instrument's built-in analysis program was used for fitting analysis.

[0204] The results are shown in Table 5. The dissociation constants (KD) of the three bispecific nanobodies bound to the PD-L1 target were all in the pmol level. Among them, BsAb-F1 and BsAb-F2 showed similar affinities to Tecentriq, almost exceeding the instrument's detection limit, at 1 pmol, 6 pmol, and 3 pmol, respectively. The KD values ​​of the three bispecific nanobodies bound to PD-L1 were all higher than those of the parent monoclonal antibody VHH15-8-FC (87 pmol), indicating that the affinity of the PD-L1 target nanobodies in the three configurations was not affected by steric hindrance. However, the affinity of the three bispecific nanobodies for CTLA-4 was weaker than that of the positive antibody Yervoy. Furthermore, except for BsAb-F1, the affinity of the other two bispecific nanobodies for CTLA-4 was lower than that of their parent monoclonal antibody VHH1-FC, consistent with the ELISA results. These results further demonstrate that the two target binding arms of BsAb-F1 are less affected by steric hindrance, exhibiting a more significant structural advantage. In addition, BsAb-F1 also exhibits strong binding activity to mouse-derived CTLA-4 (KD: 0.776 nM, Table 5).

[0205] In summary, among the three molecular configurations, BsAb-F1 exhibits higher expression yield and greater affinity for binding to both target antigens, demonstrating a clear structural advantage.

[0206] Table 5. KD values ​​of bispecific nanobody binding to PD-L1 and CTLA-4

[0207]

[0208] Notes: Ligand - ligand; Analyte - analyte; kon - binding constant; koff - dissociation constant; KD - binding-dissociation equilibrium constant, KD = koff / kon; Chi2 - deviation between measured and fitted values, the smaller the value, the smaller the deviation.

[0209] Example 15: In vitro cell function detection of bispecific nanobodies

[0210] 10,000 stable CHO-K1 / aAPC / PD-L1 cell lines (GenScript, M00613) were seeded into each well of a 384-well plate and incubated overnight at 37°C with 5% CO2. The next day, the culture supernatant was removed, and 20 μL of serially diluted BsAb-F1 and Tecentriq series samples (5-fold serial dilution) were added to each well. Then, 40,000 Jurkat / NFAT-Luc / PD-1 effector cells (GenScript, M00612) were added to each well and incubated at 37°C for 6 h. 30 μL of Luciferase assay reagent (Genscript, L00877C-100) was added to each well, and the assay plate signal was read using a ThermoFisher (Varioskan lux) microplate reader to analyze the blocking effect of BsAb-F1 on PDL1.

[0211] In addition, 40,000 Jurkat / CD28 (GenScript, M00611) cells were added to each well of a 384-well plate (white plate, flat bottom), using a culture medium containing 5 μg / mL PHA (Promega, G7940). Then, 20,000 CHO-K1 / CD80 (GenScript, M00614) cells, 20 μL of 2 μg / mL CTLA-4 solution (Genscript, Z03373), and 20 μL of serially diluted BsAb-F1 sample solution were added to each well. The plate was incubated at 37°C for 24 h. The supernatant was then collected, and the IL-2 content in the supernatant was detected using an IL-2 detection kit (Cisbio, 62HIL02PET).

[0212] Test results ( Figure 9 The study found that BsAb-F1 had a similar blocking effect to Tecentriq (0.3880 μg / mL vs. 0.3115 μg / mL), exhibiting a strong blocking effect; its blocking effect on CTLA-4 was weaker than that of Yervoy, which is consistent with the expectation of a strong PD-L1 binding arm and a weak CTLA-4 binding arm, both of which can stimulate lymphocytes to secrete IL-2.

[0213] Example 16: Pharmacodynamic Detection of Bispecific Nanobody in Mice

[0214] Six- to seven-week-old MC38-tumor-bearing C57BL / 6-PDL1 humanized mice (source: Jiangsu Jicui Biotechnology) were randomly divided into four groups (G1, G2, G3, G4), with six mice in each group. The day of grouping was defined as D0. On days 0, 3, 7, 10, 14, 17, 21, and 24, mice were intraperitoneally injected with IgG4 3 mpk (G1), BsAb-F1 0.3 mpk (G2), BsAb-F1 1 mpk (G3), and BsAb-F1 3 mpk (G4). Tumor size was observed and mouse weight was recorded on days 0, 3, 7, 10, 14, 17, 21, 24, 28, and 34. Tumor volume was calculated as: tumor volume (mm). 3 = 0.5 × (Tumor long diameter × Tumor short diameter) 2 At the end of the experiment (day 34), the mice were euthanized, the tumors were isolated, photographed, and weighed for recording.

[0215] result( Figure 10 It was found that treatment with all three doses of BsAb-F1 significantly inhibited tumor growth, with a tumor volume inhibition rate (TGI). tv The rates were 37.8%, 71.03%, and 94.75%, respectively, showing a dose-dependent effect. Figure 10 (A); meanwhile, there was no significant difference in growth weight between the mice and the IgG4 control group (A). Figure 10 No other significant adverse reactions were observed during the experiment, indicating that our bispecific antibody has minimal side effects. At the experimental endpoint, mouse tumors were isolated and weighed. It was found that at doses of 1 mg / kg and 3 mg / kg, the tumor weight in mice treated with BsAb-F1 was significantly lower than that in the control group (P < 0.01). Figure 10 D&E).

Claims

1. A Nanobody against CTLA-4, characterized in that, The nanobody comprises a VHH domain comprising CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1-CDR3 are respectively as shown in SEQ ID NOs: 1-3.

2. The Nanobody of claim 1, wherein The VHH domain further comprises FR1, FR2, FR3 and FR4, which respectively comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequences as shown in SEQ ID NOs: 4-7; or, which respectively comprise the amino acid sequences as shown in SEQ ID NOs: 4-7.

3. The Nanobody according to claim 1 or 2, characterized in that, The nanobody comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 8; or, the nanobody comprises the amino acid sequence as shown in SEQ ID NO:

8.

4. A fusion protein, characterized in that, The fusion protein comprises the nanobody as described in any one of claims 1-3; Preferably, the fusion protein is selected from one or more of the following: (i) a monovalent or multivalent nanobody; (ii) a bispecific antibody or a multispecific antibody; (iii) a heavy chain antibody; More preferably, the fusion protein comprises a heavy chain constant region, preferably a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4; the heavy chain constant region preferably comprises CH2-CH3 or CH1-CH2-CH3.

5. The fusion protein of claim 4, wherein, The heavy chain constant region comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence as shown in SEQ ID NO: 13; Preferably, the heavy chain constant region comprises the amino acid sequence as shown in SEQ ID NO:

13.

6. An isolated nucleic acid, comprising, The nucleic acid encodes the nanobody as described in any one of claims 1-3 or the fusion protein as described in claim 4 or 5.

7. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid as described in claim 6; Preferably, the recombinant vector is selected from the group consisting of: a viral vector and a non-viral vector; the non-viral vector is selected from the group consisting of: a plasmid, a linear DNA fragment and an RNA.

8. A transformant characterized in that, The transformant comprises the nucleic acid as described in claim 6 or the recombinant vector as described in claim 7; and the transformant is not an animal and a plant variety; Preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; More preferably, the eukaryotic cell is a yeast cell or a mammalian cell.

9. A method of preparing a Nanobody or fusion protein, characterized in that, The method comprises culturing the transformant as described in claim 8 under suitable conditions to obtain the nanobody or fusion protein from the culture.

10. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the nanobody as described in any one of claims 1-3 or the fusion protein as described in claim 4 or 5, and at least one of a pharmaceutically acceptable carrier, excipient or diluent.

11. A kit characterized in that, The kit comprises the nanobody as described in any one of claims 1-3, the fusion protein as described in claim 4 or 5, the nucleic acid as described in claim 6, the recombinant vector as described in claim 7, the transformant as described in claim 8 or the pharmaceutical composition as described in claim 10.

12. A method of detecting CTLA-4, characterized by, The method comprises contacting a Nanobody according to any one of claims 1 to 3, a fusion protein according to claim 4 or 5, or a kit according to claim 11 with a sample to be tested; Preferably, the method is for non-diagnostic and / or therapeutic purposes.

13. Use of a Nanobody according to any one of claims 1 to 3, a fusion protein according to claim 4 or 5, a nucleic acid according to claim 6, a recombinant vector according to claim 7, a transformant according to claim 8, a pharmaceutical composition according to claim 10, or a kit according to claim 11 for the manufacture of a medicament for the treatment of a CTLA-4 related disease; Preferably, the CTLA-4 related disease is a tumor or an autoimmune disease; More preferably, the tumor is a solid tumor, preferably selected from the group consisting of colon cancer, lung cancer, melanoma and gastric cancer, or a hematological tumor, and the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, anti-synthetase syndrome, IgG4-related disease, autoimmune hemolytic anemia, MDA5+ dermatomyositis interstitial lung fibrosis, idiopathic inflammatory myopathy, immune thrombocytopenia, myasthenia gravis, primary membranous nephropathy, type 1 diabetes and autoimmune lymphoproliferative syndrome.

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