Anti-4-1BB antibody and use thereof

Abstract

An anti-4-1BB antibody and a use thereof. The antibody or an antigen-binding fragment comprises heavy chains CDR1, CDR2 and CDR3, wherein the CDR1 has an amino acid sequence as shown in SEQ ID NO: 1, 10, 19, 28 or 37; the CDR2 has an amino acid sequence as shown in SEQ ID NO: 2, 11, 20, 29 or 38; and the CDR3 has an amino acid sequence as shown in SEQ ID NO: 3, 12, 21, 30 or 39. Compared with commercially available drugs, the antibody or antigen-binding fragment has better efficacy and higher safety.

Description

Anti-4-1BB antibodies and their applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to anti-4-1BB antibodies and their applications. Background Technology

[0002] 4-1BB, also known as CD137 or TNFRSF9, is a type I transmembrane protein whose extracellular region consists of four CRD regions. 4-1BB molecules are mainly expressed in activated T cells, NK cells, regulatory T cells, dendritic cells, monocytes, neutrophils, and eosinophils. Endothelial cells of tumor vessels have also been reported to express 4-1BB.

[0003] Anti-4-1BB antibodies exert their biological functions by binding to 4-1BB on the surface of T cells, activating downstream signaling pathways. The agonist 4-1BB antibody Urelumab (BMS-663513), a human IgG4 antibody developed by Bristol-Myers Squibb, has shown good clinical efficacy, but some patients experienced dose-related hepatotoxicity. Another weak 4-1BB agonist antibody, Utomilumab (PF-05082566), a human IgG2 antibody developed by Pfizer, has shown comparable efficacy to anti-PD-1 antibody therapy when used in combination with the anti-PD-1 antibody Pembrolizumab, although it did not cause any dose-limiting toxicities.

[0004] Therefore, there is a lack of more effective and safer 4-1BB agonists for the treatment of patients with a variety of diseases, including cancer, that are suitable for treatment with 4-1BB agonists. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems of limited efficacy and poor safety in existing technologies. The antibody or antigen-binding fragment provided by the embodiments of this invention can effectively bind to 4-1BB with high specificity and has lower immunogenicity, thus improving both efficacy and safety compared to commercially available drugs.

[0006] Therefore, in a first aspect of the invention, an antibody or antigen-binding fragment is provided. In some embodiments, the antibody or antigen-binding fragment comprises heavy chains CDR1, CDR2, and CDR3, wherein (1) CDR1 has the amino acid sequence shown in SEQ ID NO: 1, 10, 19, 28, or 37; and (2) CDR2 has the amino acid sequence shown in SEQ ID NO: 2, 11, 20, 29, or 38; and (3) CDR3 has the amino acid sequence shown in SEQ ID NO: 3, 12, 21, 30, or 39.

[0007] In some embodiments, the antibody or antigen-binding fragment comprises heavy chain CDR1, CDR2, and CDR3, wherein,

[0008] (1) The CDR1 has the amino acid sequence shown in SEQ ID NO:1, the CDR2 has the amino acid sequence shown in SEQ ID NO:2, and the CDR3 has the amino acid sequence shown in SEQ ID NO:3; or

[0009] (2) The CDR1 has the amino acid sequence shown in SEQ ID NO:10, the CDR2 has the amino acid sequence shown in SEQ ID NO:11, and the CDR3 has the amino acid sequence shown in SEQ ID NO:12; or

[0010] (3) The CDR1 has the amino acid sequence shown in SEQ ID NO:19, the CDR2 has the amino acid sequence shown in SEQ ID NO:20, and the CDR3 has the amino acid sequence shown in SEQ ID NO:21; or

[0011] (4) The CDR1 has the amino acid sequence shown in SEQ ID NO:28, the CDR2 has the amino acid sequence shown in SEQ ID NO:29, and the CDR3 has the amino acid sequence shown in SEQ ID NO:30; or

[0012] (5) The CDR1 has the amino acid sequence shown in SEQ ID NO:37, the CDR2 has the amino acid sequence shown in SEQ ID NO:38, and the CDR3 has the amino acid sequence shown in SEQ ID NO:39.

[0013] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain framework region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof.

[0014] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region comprising an amino acid sequence shown in any one of SEQ ID NO: 46-50, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identity with it.

[0015] In some implementations, CDR1, CDR2, and CDR3 are defined by any one or a combination of systems such as Kabat, Chothia, AbM, Contact, or IMGT. The specific sequences of CDR1, CDR2, and CDR3 defined by the Kabat, Chothia, and IMGT systems are shown in Table 1.

[0016] In some implementations, CDR1, CDR2, and CDR3 are defined by the IMGT system. The specific sequence is shown in Table 1.

[0017] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof.

[0018] In some implementations, the heavy chain constant region is derived from a human IgG antibody or a mutant thereof.

[0019] In some embodiments, the heavy chain constant region is derived from human IgG1 or IgG4 antibodies or mutants thereof; the heavy chain constant region may have one or more amino acid substitutions, deletions or additions compared to the natural sequence from which it is derived (e.g., up to 20, up to 15, up to 10, up to 5, up to 3 or up to 1 substitution, deletion or addition).

[0020] In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:51 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identity with it.

[0021] In some implementations, the antibody is a monoclonal antibody.

[0022] In some embodiments, the antibody is a natural intact antibody, a nanobody, a single-chain antibody, a single-chain Fv-Fc antibody, a nanobody-Fc antibody, an scFv dimer, or a dimer monoclonal antibody of an scFv-Fc antibody.

[0023] In some implementations, the antibody is a nanobody.

[0024] In some embodiments, the antibody is a nanobody-Fc antibody, wherein the C-terminus of the nanobody is linked to the N-terminus of the Fc region.

[0025] More specifically, the Fc region includes a hinge region, a CH2 region, and a CH3 region. The C-terminus of the nanobody is connected to the N-terminus of the hinge region, the C-terminus of the hinge region is connected to the N-terminus of the CH2 region, and the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region. Those skilled in the art will understand that the nanobody-Fc antibody may contain a linker peptide. The type of linker peptide is not particularly limited, as long as it does not affect the efficacy and safety of the nanobody-Fc antibody; for example, flexible linker peptides, rigid linker peptides, etc.

[0026] In some implementations, the Fc region is the human wild-type IgG1 Fc region or a variant thereof.

[0027] In some embodiments, the antigen-binding fragment is selected from: Fab, Fab'-SH, Fv, and (Fab')2 fragments.

[0028] In some implementations, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0029] In a second aspect, the invention provides an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody or antigen-binding fragment as described in the first aspect.

[0030] In a third aspect, the present invention provides a nucleic acid construct. In some embodiments, the nucleic acid construct comprises the isolated nucleic acid molecule described in the second aspect. Further, the nucleic acid construct comprises one or more control sequences operatively linked to the nucleic acid molecule, the one or more control sequences directing the production of the antibody or antigen-binding fragment thereof described in the present invention in a suitable host cell, the one or more control sequences being selected from: promoters, enhancers, stop signals, signal peptides, leader sequences, transcription terminators, and any group thereof.

[0031] In a fourth aspect, the present invention provides an expression vector. In some embodiments, the expression vector carries the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, or expresses the antibody or antigen-binding fragment described in the first aspect.

[0032] In some embodiments, the expression vector is selected from plasmids, granules, viruses, mini-chromosomes, and artificial chromosomes.

[0033] In some implementations, the expression vector is a eukaryotic expression vector.

[0034] In some embodiments, the eukaryotic expression vector is selected from pCRII, pCR3 and pcDNA3.4, pB SII, pET 15, pGEX, pEGFP-N1, pETL, pDSR-α, and pFastBacDual.

[0035] In a fifth aspect, the present invention provides a recombinant cell. In some embodiments, the recombinant cell carries the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or expresses the antibody or antigen-binding fragment described in the first aspect. According to embodiments of the present invention, the recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under suitable conditions, and the antibody or its antigen-binding fragment can effectively bind to 4-1BB. Furthermore, the antibody or antigen-binding fragment has a good effect in preventing and / or treating 4-1BB-mediated diseases.

[0036] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma fissicerum, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, including HEK-293 cells, HELA cells, BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0037] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody or its antigen-binding fragment described in this application. Those skilled in the art will readily understand that suitable conditions for antibody or antigen-binding fragment expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody or its antigen-binding fragment according to the specific environment of their laboratory.

[0038] In a sixth aspect, the present invention provides a composition. In some embodiments, the composition comprises the antibody or antigen-binding fragment described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect.

[0039] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof of the present invention can also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit 4-1BB activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with upstream or downstream signal transduction of 4-1BB.

[0040] In a seventh aspect, the present invention provides a medicament. In some embodiments, the medicament comprises the antibody or antigen-binding fragment described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect.

[0041] In some implementations, the drug also includes a pharmaceutically acceptable carrier.

[0042] In an eighth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect in the preparation of a medicament for the prevention, relief, and / or treatment of cancer.

[0043] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, melanoma, pancreatic cancer, multiple myeloma, and acute myeloid leukemia.

[0044] In a ninth aspect, the present invention provides a kit. In some embodiments, the kit comprises the antibody or antigen-binding fragment described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cells described in the fifth aspect.

[0045] In some implementations, the kit may also include reagents commonly used for detecting 4-1BB, such as coating solutions.

[0046] In some implementations, the kit may also include instructions for use.

[0047] In a tenth aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect in the preparation of a kit for detecting 4-1BB.

[0048] In an eleventh aspect of the invention, the invention provides for the use of the antibody or antigen-binding fragment of the first aspect, the isolated nucleic acid molecule of the second aspect, the nucleic acid construct of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, or the composition of the sixth aspect or the pharmaceutical composition of the seventh aspect in the prevention, relief and / or treatment of cancer.

[0049] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, melanoma, pancreatic cancer, multiple myeloma, and acute myeloid leukemia.

[0050] In a twelfth aspect of the invention, a method for preventing, alleviating, and / or treating cancer is provided. In some embodiments, the method includes administering to a subject at least one of: (1) an antibody or antigen-binding fragment as described in the first aspect; (2) an isolated nucleic acid molecule as described in the second aspect; (3) a nucleic acid construct as described in the third aspect; (4) an expression vector as described in the fourth aspect; (5) recombinant cells as described in the fifth aspect; (6) a composition as described in the sixth aspect; and (7) a drug as described in the seventh aspect.

[0051] Typically, the antibody or its antigen-binding fragment is administered in an effective amount, that is, an amount sufficient to achieve the desired therapeutic and / or preventive effect, for example, an amount that causes prevention or relief of symptoms associated with the disease being treated, such as diseases associated with 4-1BB. The effective amount of the composition administered to the subject will depend on the type and severity of the disease, as well as on individual characteristics such as general health status, age, sex, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, factors that a person skilled in the art will be able to determine the appropriate dosage based on.

[0052] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, melanoma, pancreatic cancer, multiple myeloma, and acute myeloid leukemia.

[0053] According to some embodiments of the present invention, the administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0054] In a thirteenth aspect of the invention, the invention provides for use in diagnosing whether a subject has cancer in at least one of the following: (1) the antibody or antigen-binding fragment of the first aspect; (2) the isolated nucleic acid molecule of the second aspect; (3) the nucleic acid construct of the third aspect; (4) the expression vector of the fourth aspect; (5) the recombinant cell of the fifth aspect; (6) the composition of the sixth aspect; and (7) the drug of the seventh aspect.

[0055] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, melanoma, pancreatic cancer, multiple myeloma, and acute myeloid leukemia.

[0056] In a fourteenth aspect of the invention, a method for diagnosing whether a subject has cancer is provided. In some embodiments, the method includes detecting 4-1BB in a test sample using at least one of the following: (1) the antibody or antigen-binding fragment described in the first aspect; (2) the isolated nucleic acid molecule described in the second aspect; (3) the nucleic acid construct described in the third aspect; (4) the expression vector described in the fourth aspect; and (5) the recombinant cells described in the fifth aspect. As previously stated, the antibody or antigen-binding fragment of the embodiments of the present invention can effectively bind to 4-1BB. Therefore, the antibody or antigen-binding fragment can be used to detect 4-1BB, which mediates a variety of diseases. Thus, the level of 4-1BB contained in a biological sample from the subject can be used to determine whether the subject has a 4-1BB-mediated disease. Furthermore, the above-mentioned substances can also be used to monitor the content of 4-1BB in the test sample of the subject. That is, the above-mentioned substances can also be used for disease staging of subjects with 4-1BB-mediated diseases, and can also be used for prognostic assessment of 4-1BB-mediated diseases, such as cancer.

[0057] According to specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:

[0058] According to a specific embodiment of the present invention, the requirement that the 4-1BB content in the test sample is not lower than the minimum standard for cancer incidence indicates that the test sample originates from a patient with a 4-1BB-mediated disease. The minimum standard value can be determined through comparative analysis and verification of the 4-1BB content in test samples from a large number of individuals with the 4-1BB-mediated disease and a large number of healthy individuals.

[0059] According to a specific embodiment of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, melanoma, pancreatic cancer, multiple myeloma, and acute myeloid leukemia.

[0060] According to a specific embodiment of the present invention, the sample to be tested includes at least one of the following: tissue, cells, blood, serum, sweat, feces, and urine.

[0061] The technical effects of this invention include at least the following:

[0062] 1. The antibody or antigen-binding fragment of the present invention can bind highly specifically to human 4-1BB protein, while cross-binding cynomolgus monkey 4-1BB protein, and can block the binding of 4-1BB ligand to 4-1BB in the natural state;

[0063] 2. The antibody or antigen-binding fragment of the present invention can be a small molecular weight nanobody, which requires external cross-linking to exert its activation effect and has excellent activity and safety.

[0064] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0065] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0066] Figure 1 shows the FACS results of the candidate combined with Human 4-1BB in Embodiment 1 of the present invention;

[0067] Figure 2A shows the ELISA results of the candidate anti-4-1BB nanobody binding to different species of 4-1BB in Embodiment 3 of the present invention;

[0068] Figure 2B shows the ELISA results of the candidate anti-4-1BB nanobody binding to TNFRSF family members in Example 3 of the present invention;

[0069] Figure 3 shows the ELISA results of the binding of the candidate anti-4-1BB nanobody to the soluble 4-1BB antigen in Example 4 of the present invention;

[0070] Figure 4A shows the FACS results of the candidate anti-4-1BB nanobody binding to HEK293 cells overexpressing Human 4-1BB in Example 5 of the present invention;

[0071] Figure 4B shows the FACS results of the candidate anti-4-1BB nanobody binding to HEK293 cells overexpressing Cynomolgus 4-1BB in Example 5 of the present invention;

[0072] Figure 5 shows the detection results of the candidate anti-4-1BB nanobody blocking the binding of 4-1BB to its ligand in Example 6 of the present invention;

[0073] Figure 6A shows the activation results of the 4-1BB signaling pathway in the presence of FcγRIIb in the candidate anti-4-1BB nanobody in Example 7 of the present invention.

[0074] Figure 6B shows the activation status of the 4-1BB signaling pathway in the absence of FcγRIIb in the candidate anti-4-1BB nanobody in Example 7 of the present invention. Detailed Implementation

[0075] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0076] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, and immunology, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0077] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0078] In this document, the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof will not be considered restrictive terms, but will be interpreted as meaning “but not limited to” or “not limited to.”

[0079] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0080] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0081] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., double Fab), as long as they exhibit the desired antigen-binding activity.

[0082] The term "complementarity-determining region" or "CDR" refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. Nanobodies contain three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), or the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). For a given nanobodies, those skilled in the art will readily identify the CDRs as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefrance et al., Dev. Comparat. Immunol. 27:55-77, 2003). Specifically, the CDR sequences of each antibody in this application in different numbering systems are shown in Table 1.

[0083] Table 1:

[0084] The term "frame region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3, and FR4.

[0085] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0086] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0087] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

[0088] The term "natural antibody" refers to a naturally occurring immunoglobulin molecule with a different structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain. The light chains of an antibody, based on the amino acid sequence of their constant domains, can be classified into one of two types, called kappa (κ) and lamuda (λ).

[0089] The terms "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to molecules other than intact antibodies that contain a portion of the intact antibody and bind to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to: double Fab; Fv; Fab; Fab, Fab'-SH; F(ab')2; single-chain antibody molecules (e.g., scFv, ScFab); and multispecific antibodies formed from antibody fragments.

[0090] The Fab fragment is an antigen-binding fragment produced by papain digestion of an antibody and consists of the intact L chain, a variable region domain (VH) of the H chain, and a first constant domain (CH1) of a heavy chain. Papain digestion of an antibody produces two identical Fab fragments. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, possessing divalent antigen-binding activity and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment in that it has residues added to the carboxyl terminus of the CH1 domain, these residues containing one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the designation used herein for Fab' fragments in which the cysteine ​​residues of the constant domain have free thiol groups. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with hinge cysteine ​​residues in between. Other chemical couplings of antibody fragments are also known.

[0091] "Fv" consists of a tight, non-covalently associated dimer of a heavy-chain variable region domain and a light-chain variable region domain. The folding of these two domains generates six hypervariable rings (three rings each from the H and L chains), which contribute amino acid residues to achieve antigen binding, thus giving the antibody antigen-binding specificity. However, even a single variable domain (or half of Fv, containing only three antigen-specific CDRs) can recognize and bind antigens, although its affinity is often lower than that of the intact binding site.

[0092] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes both the native sequence Fc region and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly designing the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies may include antibody populations with all Lys447 residues removed, antibody populations with Lys447 residues not removed, and antibody populations having mixtures containing and without Lys447 residues.

[0093] Light chains (LCs) derived from any vertebrate can be classified into two distinct types based on the amino acid sequence of their constant domain, referred to as κ and λ, respectively. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their heavy chain constant domain (CH). Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, with heavy chains referred to as α, δ, γ, ε, and μ, respectively. The γ and α classes are further subdivided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0094] The term "human antibody" refers to an antibody whose amino acid sequence corresponds to that of antibodies produced by humans or human cells, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0095] The term "humanized" antibody refers to a chimeric antibody containing amino acid residues from a non-human HVR and amino acid residues from a human FR. In some respects, a humanized antibody will substantially contain at least one of all, typically two, variable domains, where all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody. In which all or substantially all of the FRs of the humanized antibody correspond to certain aspects of the FRs of the human antibody, any FR of the humanized antibody may contain one or more amino acid residues from the non-human FR (e.g., one or more vernier position residues of the FR). A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. An antibody in a "humanized form," such as a non-human antibody, refers to an antibody that has been humanized.

[0096] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding to that antigen to screen libraries of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887, 1993; Clarkson et al., Nature 352:624-628, 1991.

[0097] The term "hypervariant region" or "HVR" refers to the individual regions (complementarity-determining regions or CDRs) within the variable domains of an antibody that are hypervariable in sequence. Typically, an antibody contains six CDRs: three in the VH domain (H-CDR1, H-CDR2, H-CDR3) and three in the VL domain (L-CDR1, L-CDR2, L-CDR3).

[0098] The term "single-chain Fv," also abbreviated as "sFv" or "scFv," refers to an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired antigen-binding structure.

[0099] The terms “single-domain antibody,” “nanobody,” and “VHH antibody” are used interchangeably. Originally described as the antigen-binding immunoglobulin (variable) domain of “heavy chain antibody” (i.e., “antibody lacking light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)), it is a single-domain antibody fragment derived from unique antibodies in camel-dwelling animals (such as camels, alpacas, etc.) and sharks. Unlike traditional antibodies, nanobodies consist only of the variable region (VHH) of heavy chain antibodies.

[0100] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of monoclonal antibody preparations, such variants are typically presented in small quantities), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0101] The term "bispecific" antibody refers to an artificial antibody or antigen-binding fragment derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0102] The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies with multi-epitope specificity. In one aspect, a multispecific antibody binds to two different targets (e.g., a bispecific antibody). Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH / VL unit has multi-epitope specificity; antibodies having two or more VL and VH domains, wherein each VH / VL unit binds to a different epitope; antibodies having two or more single variable domains, wherein each single variable domain binds to a different epitope; full-length antibodies; antibody fragments such as Fab, Fv, dsFv, scFv, bisomatic antibodies, bispecific bisomatic antibodies and trisomatic antibodies, and covalently or non-covalently linked antibody fragments.

[0103] The term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "proliferative disorder," "cell proliferation disorder," "proliferative lesion," and "tumor" are not mutually exclusive in this document.

[0104] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. Cancer includes both solid tumors and non-solid tumors. Solid tumors include, but are not limited to, colorectal cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, or prostate cancer, or their metastatic forms. Cancer may be 4-1BB positive.

[0105] The term "treatment" (and its grammatical variations, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a treated individual, and can be for prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some aspects, the antibodies of the present invention (e.g., the anti-4-1BB antibody of the present invention) are used to delay the development of disease or slow its progression.

[0106] "Isolated" proteins or peptides are those that have been separated from components of their natural environment. In some aspects, proteins or peptides are purified to a purity greater than 95% or 99% by means of, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain said nucleic acid molecules, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations.

[0107] The term "construct" as used in this application refers to a genetic vector containing a specific nucleic acid sequence and capable of transferring the target nucleic acid sequence into a host cell to obtain recombinant cells. The form of the construct is not particularly limited. According to embodiments of the present invention, it can be at least one of plasmids, bacteriophages, artificial chromosomes, cosmids, viruses, and viral vectors, preferably viruses or viral vectors, and those skilled in the art can choose as needed.

[0108] The term "expression vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site.

[0109] The term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA, with no particular limitation on their length. The term "nucleic acid molecule" as used in this application actually includes any one or both complementary double strands.

[0110] The term "identity" or "sequence identity" refers to the matching of sequences between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). 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 six out of ten positions in two sequences match, then the two sequences are 60% identical. Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using methods conveniently, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0111] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 1 9th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delay agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delay agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0112] The term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease associated with 4-1BB.

[0113] The term "administration" means a method of administering a dose of a compound (e.g., the anti-4-1BB antibody of the present invention) to a subject. The compositions used in the methods described herein can be administered, for example, intramuscularly, intravenously, subcutaneously, percutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, intraperitoneally, subcutaneously, subconjunctivally, intracysticly, intraarticularly, intraarticularly, intraarticularly, intraarticularly, intraprostatically, intrapleurally, intravaginally, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by direct perfusion of target cells via local perfusion, via catheter, by perfusion, in the form of an emulsion or a lipid composition. The method of administration can vary depending on a variety of factors (e.g., the compound or composition to be administered and the severity of the condition, disease, or ailment to be treated).

[0114] Other nucleotide or amino acid sequences in this invention are shown in Table 2. In the amino acid sequences of various 4-1BB proteins (Human, Cynomolgus, Mouse, Rat), the bolded parts are the extracellular regions of each protein.

[0115] Table 2

[0116] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0117] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0118] Example 1: Preparation of 4-1BB antibody

[0119] Phage-displayed nanobodies (VHHs) were screened using phage library technology and enzyme-linked immunosorbent assay (ELISA) to generate monoclonal antibodies that highly bind to Human 4-1BB (SEQ ID NO:54) at the molecular level. Specifically, the extracellular region encoding the Human 4-1BB protein was designed using the NCBI database, constructed into a mammalian eukaryotic expression system, and expressed to obtain the extracellular region of the Human 4-1BB protein (the bolded portion in SEQ ID NO:54), which was then used for nanobodies screening. Phages from the recombinant human nanolibrary were panned using the Human 4-1BB protein. After three rounds of panning, single clones were selected for initial screening using ELISA, and DNA sequencing was used to determine the unique antibody sequence contained within. The sequenced unique antibody sequence was further detected using flow cytometry (FACS), with an antibody-free solvent serving as a negative control. The ELISA results are shown in Table 3 below. It can be seen that five single clones exhibited strong binding to the Human 4-1BB protein at the phage level. As shown in Figure 1, the peaks of phages 1A9, 1F9, 61A3, and 81C6 are shifted to the right compared to the negative control (NC), indicating that the above phages bind to the overexpressing cell line 293-H4-1BB.

[0120] Table 3: ELISA results of candidate substance binding to Human 4-1BB

[0121] Example 2: Antibody Expression, Purification, and Detection of Antibody Binding Properties

[0122] Building upon Example 1, this example employs molecular cloning to construct a series of antibody expression vectors, which are then recombinantly expressed in the 293F expression system. Specifically, the nucleotide sequences encoding the heavy chains of a series of monoclonal antibodies are obtained by extracting plasmids from panning phage culture. The obtained VHH sequences are double-digested and inserted into the same restriction enzyme sites (SFII) of the modified pcdna3.4 eukaryotic expression vector, constructing expression vectors for antibodies numbered 1A9, 1F9, 61A3, 81C6, and 71A9. Then, a series of validated expression vectors are extracted using a plasmid extraction kit (purchased from Invitrogen) according to its instructions. After linearization with restriction endonucleases, the vectors are purified and recovered, and stored at -20°C.

[0123] After resuscitating and culturing 293F host cells (purchased from Gibco) in OPM-CD Trans293 medium (purchased from Shanghai OPMA), when the cell density was approximately 3*10⁻⁶, 6Cells were collected at a concentration of [number] cells / mL and transfected using PEI reagent. On day 2 of culture, feed and glucose were added to bring the culture concentration to 8 g / L. On day 6 of culture, the cell culture medium was collected.

[0124] A series of antibodies were analyzed at the translational level. The collected cell culture media were purified using a Protein A chromatography column, and the absorption peaks were collected for mass spectrometry analysis. Mass spectrometry analysis showed that the molecular weight of the chimeric antibody VHH-Fc was approximately 80 kDa, consistent with the theoretical molecular weight, indicating a dimer form. Simultaneously, the collected samples were analyzed by 10% SDS-PAGE electrophoresis after reduction and non-reduction. The reduced SDS-PAGE electrophoresis pattern showed approximately 40 kDa, while the non-reduced SDS-PAGE electrophoresis pattern showed a single band at approximately 80 kDa, consistent with the theoretical band size. The purified samples were dialyzed overnight at 4°C using 0.02 M PBS buffer (pH 7.4).

[0125] Example 3: Detection of species cross-species and specificity of anti-4-1BB antibodies

[0126] This embodiment uses ELISA to detect the binding characteristics of the anti-4-1BB nanobody obtained in Example 2 to different species of soluble 4-1BB proteins (SEQ ID NO: 54-57) and their selective binding to other structurally similar TNFRSF family members (CD40, OX40, GITR proteins, SEQ ID NO: 58-60). Specifically, the ELISA plate was coated overnight with an antigen concentration of 1 μg / mL, and then the non-specific binding sites were blocked with 3% BSA at 37°C for 2 h. The plate was then incubated with an antibody concentration of 10 μg / mL for 1 h. The bound nanobodies were detected using HRP-goat anti-human antibody, followed by washing, addition of TMB substrate (Yunqiao Biotechnology), and analysis at 450 nm. The ELISA results are shown in Figure 2A. The obtained antibodies all exhibited human-monkey cross-activity and did not bind to mouse or rat 4-1BB proteins. Furthermore, Figure 2B shows that the obtained antibody selectively binds to 4-1BB of the TNFRSF family, but does not bind to OX40, CD40 and GITR proteins of the same family and with similar structures.

[0127] Example 4: Binding characteristics of anti-4-1BB antibody to soluble 4-1BB antigen

[0128] This embodiment confirms the binding of the antibodies obtained in Example 2 to the soluble 4-1BB antigen using ELISA. Specifically, ELISA plates were coated with 1 μg / mL 4-1BB and incubated overnight at 4°C. Non-specific binding sites were then blocked with 3% BSA at 37°C for 2 hours. The plates were incubated with 100 μL of nanobodies of various concentrations for 1 hour. The bound nanobodies were detected using HRP-goat anti-human antibody. After washing, TMB substrate (Yunqiao Biotechnology) was added, and analysis was performed at 450 nm. The ELISA results are shown in Figure 3 and Table 4, demonstrating that all antibodies bound to the Human 4-1BB protein, with binding EC50 between 0.5-20 nM, indicating high binding activity.

[0129] Table 4: Human 4-1BB protein binding EC50 (nM)

[0130] Example 5: Binding of anti-4-1BB nanobody to overexpressing cell lines

[0131] This embodiment evaluates the 4-1BB binding characteristics of the nanobodies obtained in Example 2. The binding of the obtained antibodies to HEK293 cells (purchased from Jimon Biotechnology, denoted as HEK293-human 4-1BB) overexpressing Human 4-1BB or CHO-K1 cells (self-constructed, i.e., CHO-K1-Cynomolgus 4-1BB) overexpressing Cynomolgus 4-1BB (SEQ ID NO:55) was analyzed by FACS. Specifically, 1×10⁻⁶ nanoparticles were added to each well. 5 HEK293-human 4-1BB cells or CHO-K1-Cynomolgus 4-1BB cells and serially diluted antibodies were incubated in PBS buffer at 4°C for 1 h. After washing with PBS buffer, PE-conjugated anti-human IgG antibody was added to each well and incubated at 4°C for 30 min. After washing, the mean fluorescence intensity (MFI) of phycoerythrin (PE) was evaluated by CytoFlex (Bechman). As shown in Figures 4A, 4B and Table 5, the tested anti-4-1BB nanobodies exhibited concentration-dependent binding ability to human or cynomolgus 4-1BB.

[0132] Table 5: EC50 (nM) of binding between human 4-1BB cells and cynomolgus monkey 4-1BB cells

[0133] Example 6: Blocking the interaction between 4-1BB / 4-1BB ligands using anti-4-1BB nanobodies

[0134] This example investigated the ability of the various anti-4-1BB nanobodies obtained in Example 2 to block the binding of 4-1BB to its ligand. Specifically, HEK293 cells overexpressing Human 4-1BB were incubated with biotinylated human 4-1BB ligand (0.3 μg / mL, purchased from ACRO, 41L-H52D4) at 4°C for 1 hour in the presence of serially diluted anti-4-1BB nanobodies, with Urelumab used as a reference antibody. Then, the cells were incubated with PE-conjugated streptavidin at 4°C for 30 min. Binding was measured using a CytoFlex (Bechman) flow cytometer. The results are shown in Figure 5. Except for 71A9, all tested nanobodies blocked the binding of the 4-1BB ligand to 4-1BB in a concentration-dependent manner, reducing toxicity; 71A9 and the positive control Urelumab did not block the binding of 4-1BB to the ligand 4-1BBL.

[0135] Example 7 Functional characteristics of anti-4-1BB nanobody in HEK293-NF-κB-luciferase-4-1BB reporter gene assay

[0136] To test whether the obtained antibody could activate the 4-1BB signaling pathway without a cross-linking agent, this embodiment used the HEK293 4-1BB / NF-κB luciferase reporter gene cell line (purchased from Jiman Biotechnology) as material to detect the ability of the candidate nanobody to bind 4-1BB and thereby activate the expression of the downstream NF-κB luciferase reporter gene. The specific implementation method is as follows: The candidate molecule and control antibody Urelumab were serially diluted using culture medium (10 μg / mL for the first well, 5-fold serial dilutions). 20 μL of the diluted candidate antibody and control antibody were added to each well of a 384-well plate; the density of the HEK293 4-1BB / NF-κB luciferase reporter gene cell line was adjusted to 5 × 10⁻⁶ cells / well. 5 Cells / mL were added at a rate of 10 μL per well to a 384-well cell culture plate containing antibody, resulting in a cell density of 5 × 10⁶ cells / mL. 5 CHO-K1 (CHO-K1-CD32b) cells or CHO-K1 cells (purchased from Lonza) overexpressing wild-type CD32b (FcγRIIb) were constructed at a density of 1 / mL and used as the experimental and control groups, respectively. These cells were incubated at 37°C for 8 hours. After incubation, 30 μL of Bright-Lite luciferase substrate (purchased from Vazyme, DD1204-03) was added to each well, and the cells were shaken and incubated for 5 minutes before detecting the fluorescence value of the 384-well plate.

[0137] The results are shown in Figures 6A and 6B. In the presence of Fc crosslinking, the tested 4-1BB nanobodies induced 4-1BB-mediated NF-κB activity. In the absence of crosslinking agents, Urelumab showed relatively strong activation of downstream NF-κB luciferase reporter gene signaling activity of 4-1BB. However, 1A9, 1F9, and 81C6 of the obtained antibodies showed weak activation ability, while 61A3 and 71A9 showed no activation ability. Therefore, it can be expected that the obtained antibodies are weakly agonistic 4-1BB molecules with limited self-activation of 4-1BB. They require Fc or TAA-mediated crosslinking to make the activation more specific and have fewer toxic side effects than Urelumab.

[0138] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0139] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen binding fragment, characterized in that, The antibody or antigen-binding fragment comprises heavy chains CDR1, CDR2, and CDR3, wherein... (1) The CDR1 has the amino acid sequence shown in SEQ ID NO: 1, 10, 19, 28 or 37; and (2) The CDR2 has the amino acid sequence shown in SEQ ID NO: 2, 11, 20, 29 or 38; and (3) The CDR3 has the amino acid sequence shown in SEQ ID NO:3, 12, 21, 30 or 39.

2. The antibody or antigen-binding fragment of claim 1, wherein, The antibody or antigen-binding fragment comprises heavy chains CDR1, CDR2, and CDR3, wherein... (1) The CDR1 has the amino acid sequence shown in SEQ ID NO:1, the CDR2 has the amino acid sequence shown in SEQ ID NO:2, and the CDR3 has the amino acid sequence shown in SEQ ID NO:3; or (2) The CDR1 has the amino acid sequence shown in SEQ ID NO:10, the CDR2 has the amino acid sequence shown in SEQ ID NO:11, and the CDR3 has the amino acid sequence shown in SEQ ID NO:12; or (3) The CDR1 has the amino acid sequence shown in SEQ ID NO:19, the CDR2 has the amino acid sequence shown in SEQ ID NO:20, and the CDR3 has the amino acid sequence shown in SEQ ID NO:21; or (4) The CDR1 has the amino acid sequence shown in SEQ ID NO:28, the CDR2 has the amino acid sequence shown in SEQ ID NO:29, and the CDR3 has the amino acid sequence shown in SEQ ID NO:30; or (5) The CDR1 has the amino acid sequence shown in SEQ ID NO:37, the CDR2 has the amino acid sequence shown in SEQ ID NO:38, and the CDR3 has the amino acid sequence shown in SEQ ID NO:

39.

3. The antibody or antigen binding fragment of claim 1 or 2, characterized in that, The antibody or antigen-binding fragment includes a heavy chain framework region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof. Optionally, the antibody or antigen-binding fragment includes a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence shown in any one of SEQ ID NO: 46-50, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identity with it.

4. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein, The antibody or antigen-binding fragment includes a heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof. Optionally, the heavy chain constant region is derived from a human IgG antibody or a mutant thereof; Optionally, the heavy chain constant region is derived from human IgG1 or IgG4 antibodies or mutants thereof; Optionally, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:51 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identity with it.

5. The antibody or antigen-binding fragment of any one of claims 1-4, wherein, The antibody is a monoclonal antibody; Optionally, the antibody is a natural intact antibody, a nanobody, a single-chain antibody, a single-chain Fv-Fc antibody, an scFv dimer, or a dimer monoclonal antibody of an scFv-Fc antibody; Optionally, the antibody is a nanobody; Optionally, the antigen-binding fragment is selected from: Fab, Fab'-SH, Fv, and (Fab')2 fragments; Optionally, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

6. An isolated nucleic acid molecule, comprising, The nucleic acid molecule comprises a nucleotide sequence encoding an antibody or antigen-binding fragment as described in any one of claims 1 to 5.

7. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the isolated nucleic acid molecule as described in claim 6.

8. An expression vector, characterized by, The expression vector carries the isolated nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, or expresses the antibody or antigen-binding fragment of any one of claims 1 to 5.

9. A recombinant cell, characterized in that, The recombinant cells carry the isolated nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, the expression vector of claim 8, or express the antibody or antigen-binding fragment of any one of claims 1 to 5.

10. A composition characterized in that, It comprises the antibody or antigen-binding fragment according to any one of claims 1 to 5, the isolated nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7, the expression vector according to claim 8, or the recombinant cell according to claim 9.

11. A medicament, characterized by comprising: It comprises the antibody or antigen-binding fragment according to any one of claims 1 to 5, the isolated nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 9, or the composition according to claim 10.

12. Use of the antibody or antigen-binding fragment of any one of claims 1 to 5, the isolated nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, the expression vector of claim 8, the recombinant cell of claim 9, or the composition of claim 10 in the preparation of a medicament for the prevention, relief, and / or treatment of cancer.

13. A kit characterized in that, It comprises the antibody or antigen-binding fragment according to any one of claims 1 to 5, the isolated nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7, the expression vector according to claim 8, or the recombinant cell according to claim 9.

14. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 5, the isolated nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7, the expression vector according to claim 8, or the recombinant cell according to claim 9 in the preparation of a kit for detecting 4-1BB.

15. Use of the antibody or antigen-binding fragment of any one of claims 1 to 5, the isolated nucleic acid molecule of claim 6, the nucleic acid construct of claim 7, the expression vector of claim 8, the recombinant cell of claim 9, the composition of claim 10, or the medicament of claim 11 in the prevention, relief, and / or treatment of cancer.

16. A method of preventing, alleviating and / or treating cancer, characterized in that, This includes administering at least one of the following to the subject: (1) The antibody or antigen-binding fragment according to any one of claims 1 to 5; (2) The isolated nucleic acid molecule as described in claim 6; (3) The nucleic acid construct according to claim 7; (4) The expression vector according to claim 8; (5) The recombinant cells according to claim 9; (6) The composition of claim 10; and (7) The medicine according to claim 11.

17. The use of at least one of the following in diagnosing whether a subject has cancer: (1) The antibody or antigen-binding fragment according to any one of claims 1 to 5; (2) The isolated nucleic acid molecule as described in claim 6; (3) The nucleic acid construct according to claim 7; (4) The expression vector according to claim 8; and (5) The recombinant cells according to claim 9.

18. A method of diagnosing whether a subject has cancer, comprising, This includes detecting 4-1BB in the sample using at least one of the following: (1) The antibody or antigen-binding fragment according to any one of claims 1 to 5; (2) The isolated nucleic acid molecule as described in claim 6; (3) The nucleic acid construct according to claim 7; (4) The expression vector according to claim 8; and (5) The recombinant cells according to claim 9.

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