4-1bb single domain antibody

Abstract

Anti-4-1BB single domain antibodies and polypeptides, e.g., bispecific antibodies and chimeric antigen receptors comprising these single domain antibodies, are provided. These antibodies, including humanized antibodies, exhibit superior activity and are suitable for use in various bispecific antibody formats. Methods of using the antibodies or polypeptides to treat and diagnose diseases, e.g., cancer, are also provided.

Description

[0001] This application is a divisional application of the invention patent application filed on October 12, 2022, with application number 202280082276.1 and invention title "4-1BB Single Domain Antibody". Background Technology

[0002] Single-domain antibodies (sdAbs), also known as nanobodies, are antibody fragments composed of a single monomeric variable antibody domain. Nanobodies produced by camels and some other animals are also called VHH fragments. Like intact antibodies, nanobodies can selectively bind to specific antigens. Single-domain antibodies have a molecular weight of only 12-15 kDa, much smaller than ordinary antibodies (150-160 kDa). Due to their small size and single-chain nature, single-domain antibodies are particularly suitable as fragments included in other proteins, such as chimeric antigen receptors (CARs) and bispecific antibodies.

[0003] 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF receptor superfamily (TNFRSF) and a co-stimulatory molecule expressed upon activation of immune cells (innate and adaptive immune cells). 4-1BB plays a crucial role in regulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and the cytolytic activity of CD8 T cells. Many other studies have shown that activating 4-1BB can enhance the immune response to eliminate tumors in mice. Therefore, 4-1BB suggests that it is a promising target molecule in cancer immunology. Summary of the Invention

[0004] Nanobodies specific to human 4-1BB protein are provided, as well as bispecific or multispecific antibodies incorporated into such nanobodies. In some embodiments, the nanobodies of this disclosure are "non-agonists" and do not activate 4-1BB signaling on their own. However, when bound to an anti-Claudin 18.2 antibody, the subsequently generated bispecific antibody exhibits potent activity in activating 4-1BB signaling in the presence of Claudin 18.2-positive cells.

[0005] 4-1BB signaling activation is achieved by agonist antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB nanobodies disclosed herein do not require such activity. In fact, it is preferable that the anti-4-1BB portion of a bispecific antibody cannot independently activate 4-1BB without binding to a tumor-associated antigen (TAA) also targeted by the bispecific antibody, as such bispecific antibodies reduce extratumor side effects.

[0006] The antibodies disclosed herein are safer than known anti-4-1BB agonist antibodies, which are typically associated with dose-limiting targeting toxicity. In tissues that do not express the corresponding TAA, such as the liver, the bispecific antibodies of this disclosure are not expected to trigger a cytotoxic immune response because they do not activate 4-1BB signaling. Conversely, in tumor tissues where TAAs are expressed and / or accessible, these bispecific antibodies can initiate an effective immune response against tumor cells. Therefore, unlike currently clinically developed anti-4-1BB antibodies with targeting / inherent toxicity, the antibodies of this disclosure can be both effective and safe in treating cancer.

[0007] Therefore, in one embodiment of this disclosure, a single-domain antibody or a polypeptide comprising said single-domain antibody is provided, wherein said single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2, and 3, wherein said CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 6, said CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 7, 39, or 40, and said CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0008] In another embodiment, a single-domain antibody or a polypeptide comprising said single-domain antibody is provided, wherein said single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2, and 3, wherein said CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 9, said CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 10, 41, or 42, and said CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 11.

[0009] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2, and 3, wherein CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 12, CDR2 comprises an amino acid sequence as shown in SEQ ID NO: 13, 43, or 44, and CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 14.

[0010] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2, and 3, wherein CDR1, 2, and 3 each comprise an amino acid sequence as shown in SEQ ID NO: 15-17.

[0011] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2, and 3, wherein CDR1, 2, and 3 each comprise an amino acid sequence as shown in SEQ ID NO: 18-20.

[0012] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2, and 3, wherein CDR1, 2, and 3 comprise the amino acid sequences of CDR1, CDR2, and 3 of any one of SEQ ID NO: 1-5.

[0013] Bispecific / multispecific antibodies incorporating one or more nanobody units are also provided. In some embodiments, the second specificity is against a tumor antigen, such as Claudin 18.2. Other exemplary tumor antigens are also disclosed herein. In some embodiments, chimeric antigen receptors (CARs) comprising nanobodies of the present disclosure are provided.

[0014] In addition, a composition comprising the antibody or polypeptide and a pharmaceutically acceptable carrier is provided. Also provided are one or more polynucleotides encoding the antibody or polypeptide, and isolated cells comprising one or more polynucleotides encoding the antibody or a fragment thereof.

[0015] Treatment methods and uses are also provided. In one embodiment, a method of treating cancer in a patient in need is provided, comprising administering an effective amount of the disclosed antibody or peptide to the patient. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, and thyroid cancer. In some embodiments, the method further includes administering a second cancer treatment agent to the patient. Attached Figure Description

[0016] Figure 1 The results of the ELISA test show the binding activity of the anti-4-1BB antibody to human 4-1BB antigen, cynomolgus monkey 4-1BB antigen and mouse 4-1BB antigen.

[0017] Figure 2 Cell-based binding to human 4-1BB protein was demonstrated.

[0018] Figure 3 Shown by Biocore The full kinetic activity of the tested anti-4-1BB antibody was measured.

[0019] Figure 4 The ELISA binding plot shows that the tested antibody does not react with human OX40 or human CD40.

[0020] Figure 5 Unlike the reference anti-4-1BB antibody Urelumab, the antibody tested did not activate 4-1BB signaling.

[0021] Figure 6 The results showed that the tested bispecific antibody activated 4-1BB signaling only in Claudin 18.2 positive CHO cells.

[0022] Figure 7 The results showed that the tested bispecific antibody could only activate 4-1BB signaling in PBMC cells in the presence of CHO cells that overexpressed Claudin 18.2.

[0023] Figure 8 The results showed that the bispecific antibody incorporated with humanized B31 antibody had NF-κB activation activity comparable to that of the chimeric B31 antibody.

[0024] Figure 9 The results showed that the bispecific antibody incorporated with humanized B31 antibody had IL-2 secretion-inducing activity comparable to that of the chimeric B31 antibody.

[0025] Figure 10 The in vivo tumor growth inhibitory activity of the tested antibody was demonstrated.

[0026] Detailed description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" is understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this document.

[0027] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence. This means that, at the time of alignment, the percentage of bases (or amino acids) is the same in the two sequences being compared. This alignment and percentage homology or sequence identity can be determined using software programs known in the art, such as Ausubel. et al Those described in Current Protocols in Molecular Biology, eds. (2007). Preferably, alignment is performed using default parameters. One alignment procedure is BLAST, using default parameters. Specifically, the procedures are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting criteria = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Bioequivalent polynucleotides are those that have the above-specified percentage of homology and encode polypeptides with the same or similar biological activities.

[0028] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a degree of homology or sequence identity with the nucleotide sequence of that nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that has a degree of homology with it or its complement. On the one hand, a homolog of a nucleic acid is capable of hybridizing with the nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide having a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0029] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any molecule containing a protein or peptide that comprises at least a portion of an immunoglobulin molecule having antigen-binding biological activity. Examples of such molecules include, but are not limited to, the complementarity-determining region (CDR), variable region, constant region, frame (FR) region, or any portion thereof of the heavy or light chain or its ligand-binding moiety, or at least a portion of the binding protein.

[0030] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to an antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror images, and dimers. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0031] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some respects, these regions are linked to short linker peptides of 10 to approximately 25 amino acids. The linker may be enriched with glycine for increased flexibility or with serine or threonine for increased solubility, and it can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite the removal of constant regions and the introduction of linkers. ScFv molecules are known in the art, as described in U.S. Patent 5,892,019.

[0032] The term "antibody" encompasses a wide range of polypeptides that can be distinguished by biochemical methods. Those skilled in the art will understand that heavy chains can be classified as γ, μ, α, δ, or ε, with further subclasses (e.g., γ1-γ4). The properties of this chain determine the "class" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and are known to have functional specificity. Given this disclosure, modified versions of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore all within the scope of this disclosure. All immunoglobulin classes are obviously within the scope of this disclosure; the following discussion generally pertains to IgG immunoglobulin molecules. For IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 Daltons. These four chains are usually connected by disulfide bonds to form a "Y"-shaped structure, in which the light chain starts from the opening of the "Y"-shaped structure and extends all the way to the variable region, surrounding the heavy chain.

[0033] The antibodies, antigen-binding peptides, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, humanized antibodies, humanized antibodies, primate-derived antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, libraries expressed by Fab, and anti-idiotype (anti-Id) antibodies (including anti-Id antibodies such as the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0034] "Specific binding" or "specificity" generally refers to an antibody binding to an epitope through its antigen-binding domain, and this binding requires a certain complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is considered to "specifically bind" to an epitope when it binds to that epitope more easily through its antigen-binding domain than to a random, unrelated epitope. This article uses the term "specificity" to define the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" can be considered more specific to a given epitope than antibody "B," or antibody "A" can be said to have a higher specificity for binding to epitope "C" than it does for binding to related epitope "D."

[0035] As used herein, the term “treatment” refers to therapeutic and preventative treatment or preventative measures aimed at preventing or slowing (reducing) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, decrease in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or reduction of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” can also refer to extended survival compared to expected survival without treatment. Those who require treatment include those who already have the condition or disorder, those who are susceptible to the condition or disorder, or those for whom prevention of the condition or disorder is desired.

[0036] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject requiring diagnosis, prognosis, or treatment, especially a mammalian subject. Mammal subjects include humans, livestock, farm animals, and zoo, sporting, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows.

[0037] As used herein, phrases such as “patients in need of treatment” or “subjects in need of treatment” include subjects, such as mammalian subjects, who will benefit from the administration of the antibodies or compositions used in this disclosure, for example, for detection, for diagnostic procedures and / or for treatment.

[0038] Single-domain anti-4-1BB antibody This disclosure provides single-chain anti-4-1BB antibodies (nanobodies) with high affinity for human 4-1BB protein. In some embodiments, the provided nanobodies do not activate 4-1BB signaling solely through this binding and are therefore referred to as "non-agonist" anti-4-1BB antibodies. Such non-agonist anti-4-1BB nanobodies are particularly suitable for the preparation of bispecific antibodies.

[0039] Activation of 4-1BB signaling is the expected mechanism of action for agonist antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB nanobodies disclosed herein do not need to possess such activity. In fact, the anti-4-1BB moiety of preferred bispecific antibodies cannot independently activate 4-1BB in the absence of CLDN18.2 binding, as such bispecific antibodies reduce off-target side effects.

[0040] Compared to known anti-4-1BB agonist antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513), which are typically associated with dose-limiting targeting toxicity, the bispecific antibodies of this disclosure are safer. In tissues that do not express CLDN18.2, the bispecific antibodies of this disclosure are not expected to trigger a cytotoxic immune response because they do not activate 4-1BB signaling. Conversely, in tumor tissues that express and / or are accessible to CLDN18.2, these bispecific antibodies can initiate an effective immune response against tumor cells. Therefore, unlike currently clinically developed anti-4-1BB antibodies with targeting / inherent toxicity, the antibodies disclosed in this invention can be both effective and safe in the treatment of cancer.

[0041] Therefore, in one embodiment of this disclosure, a single-domain antibody and a polypeptide comprising such a single-domain antibody are provided. In one embodiment of this disclosure, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody comprises CDR1, CDR2, and CDR3, each having the CDR1, CDR2, and CDR3 sequences of antibody B31 (SEQ ID NO: 1). In some embodiments, CDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, 39, or 40, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 8; in some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 6-8. In some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 6, 39, and 8. In some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 6, 40, and 8.

[0042] Humanized antibodies, such as those provided in SEQ ID NO: 21-25, are also provided. In some embodiments, the humanized antibody comprises a reversion mutation selected from the group consisting of 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 78M, 82(82A)D, and 94A according to Kabat numbers. In some embodiments, the humanized antibody comprises reversion mutations 37Y, 44Q, 45R, and 94A. In some embodiments, the humanized antibody comprises reversion mutations 23A, 37Y, 44Q, 45R, 49A, 74N, and 94A. In some embodiments, the humanized antibody comprises reversion mutations 23A, 37Y, 44Q, 45R, 49A, 78M, 82(82A)D, and 94A. In some embodiments, the humanized antibody comprises reversion mutations 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 82(82A)D, and 94A. In some embodiments, the humanized antibody comprises reversion mutations 37Y, 44Q, 45R, and 49A.

[0043] In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 22. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 25. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in any of SEQ ID NO: 1 and 21-25.

[0044] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody comprises CDR1, CDR2, and CDR3, which have the CDR1, CDR2, and CDR3 sequences of antibody B16 (SEQ ID NO: 2), respectively. In some embodiments, CDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, 41, or 42, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; in some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 9-11. In some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 9, 41, and 11. In some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 9, 42, and 11.

[0045] Humanized antibodies, such as those provided in SEQ ID NO: 31-34, are also provided. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 37F, 44E, 45R, 47G, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 29L, 30D, 37F, 44E, 45R, 47G, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P, and 94T according to Kabat numbers.

[0046] In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 31. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 32. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 33. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 34. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO: 2 and any of SEQ ID NO: 31-34.

[0047] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody comprises CDR1, CDR2, and CDR3, each having the CDR1, CDR2, and CDR3 sequences of antibody B125 (SEQ ID NO: 3). In some embodiments, CDR1 comprises the amino acid sequence shown in SEQ ID NO: 12, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 13, 43, or 44, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; in some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 12-14. In some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 12, 43, and 14. In some embodiments, CDR1, CDR2, and CDR3 each comprise the amino acid sequences shown in SEQ ID NO: 12, 44, and 14.

[0048] Humanized antibodies, such as those provided in SEQ ID NO: 26-30, are also provided. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, 74A, 84P, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 37F, 44E, 45R, 47G, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, and 74A according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, 74A, and 94T according to Kabat numbers. In some embodiments, the humanized antibody comprises a reversion mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, 74A, 84P, and 94T according to the Kabat number.

[0049] In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 26. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with any of the sequences shown in SEQ ID NO: 3 and 26-30.

[0050] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody comprises CDR1, CDR2, and CDR3, which respectively have the CDR1, CDR2, and CDR3 sequences of antibody B164 (SEQ ID NO: 4). In some embodiments, the CDR1, CDR2, and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NO: 15-17. In some embodiments, the antibody comprises the CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0051] In another embodiment, a single-domain antibody or a polypeptide comprising the single-domain antibody is provided, wherein the single-domain antibody comprises CDR1, CDR2, and CDR3, which respectively have the CDR1, CDR2, and CDR3 sequences of antibody B210 (SEQ ID NO: 5). In some embodiments, the CDR1, CDR2, and CDR3 respectively comprise amino acid sequences as shown in SEQ ID NO: 18-20.

[0052] Humanized antibodies, such as those provided in SEQ ID NO: 26-30, are also provided. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 27L, 29L, 37F, 44E, 45R, 47G, 89D, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 37F, 44E, 45R, 47G, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 27L, 37F, 44E, 45R, 47G, and 94T according to Kabat numbers. In some embodiments, the humanized antibody contains a reversion mutation selected from the group consisting of 27L, 29L, 37F, 44E, 45R, 47G, 89D, and 94T according to Kabat numbers.

[0053] In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 35. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3, and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the amino acid sequences shown in any of SEQ ID NO: 5 and 35-38.

[0054] In some embodiments, an anti-4-1BB antibody and antigen-binding fragment that competes with any antibody disclosed herein for binding to human 4-1BB is also provided. In some embodiments, an anti-4-1BB antibody and antigen-binding fragment that binds to the same epitope as any antibody disclosed herein is also provided. In some embodiments, an anti-4-1BB antibody and antigen-binding fragment comprising CDR1, CDR2, and CDR3 of the antibody disclosed herein is also provided.

[0055] Compositions comprising the antibody or the polypeptide and a pharmaceutically acceptable carrier are also provided.

[0056] Those skilled in the art will also understand that the antibodies disclosed herein can be modified so that their amino acid sequences differ from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity, such as 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the modified antibody or fragment retains the specified CDR sequence.

[0057] In some embodiments, the antibody comprises an amino acid sequence or one or more portions that are not typically associated with the antibody. Exemplary modifications are described in more detail below. For example, the antibody of this disclosure may comprise a flexible linker sequence or may be modified to add a functional portion (e.g., PEG, drug, toxin, or label).

[0058] Bispecific and multispecific antibodies are also provided, including one, two, three or four units of the single-domain anti-4-1BB antibody disclosed herein, as well as one or more other specific antibodies (non-4-1BB). Bispecific and multispecific antibodies, as well as chimeric antigen receptors (CARs). As previously stated, the anti-4-1BB antibodies disclosed herein are particularly suitable for the preparation of bispecific and multispecific antibodies as well as chimeric antigen receptors (CARs). This is at least due to the enhanced therapeutic index and small size of these antibodies.

[0060] Therefore, in one embodiment, a bispecific antibody is provided, comprising the anti-4-1BB nanobody of this disclosure or an antigen-binding fragment thereof, and a second antibody or antigen-binding fragment having binding specificity to a non-4-1BB target antigen. In some embodiments, a third or fourth specificity is also included.

[0061] In some embodiments, the non-4-1BB target antigen is a tumor antigen. A large number of tumor antigens are known in the art, and novel tumor antigens can be readily identified through screening. Non-limiting examples of tumor antigens include ABL, ALK, B4GALNT1, BAFF, BCL2, BRAF, BTK, CD19, CD20, CD30, CD38, CD52, CD73, Claudin 18.2, CTLA-4, EGFR, FORR1, FLT3, HDAC, HER2, IDH2, IL-1β, IL-6, IL-6R, JAK1 / 2, JAK3, KIT, LAG-3, MEK, Nectin 4, ROR1, mTOR, PARP, PD-1, PDGFR, PDGFRα, PD-L1, PI3Kδ, PIGF, PTCH, RAF, RANKL, Smoothened, VEGF, VEGFR, and VEGFR2. Other examples include Her2, EpCAM, CD33, CD47, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, GD2, GD3, GM2, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and Tenascin.

[0062] In some implementations, the bispecific antibody is specific for 4-1BB and Claudin 18.2.

[0063] A chimeric antigen receptor (CAR) comprising the nanobody of this disclosure is also provided. In the CAR, the nanobody can serve as an antigen recognition domain. Furthermore, in some embodiments, the CAR further includes an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0064] A hinge, also known as a spacer region, is a small structural domain located between the antigen recognition region and the outer membrane. A suitable hinge can enhance the flexibility of the scFv receptor head and reduce spatial confinement between the CAR and its target antigen. Example hinge sequences are based on proximal membrane regions derived from immune molecules such as IgG, CD8, and CD28.

[0065] A transmembrane domain is a structural component consisting of a hydrophobic α-helix that spans the cell membrane. It anchors the CAR to the plasma membrane and bridges the extracellular hinge and antigen recognition domain with intracellular signal transduction regions. Typically, transmembrane domains derived from proximal membrane components of intracellular domains can be used, such as the CD28 transmembrane domain.

[0066] Intracellular T cell signaling domains are located within the intracellular domains of the receptor. After an antigen binds to the external antigen recognition domain, the CAR receptor aggregates and transmits an activation signal. Then, the receptor's internal cytoplasmic terminal continues to transmit the signal within the T cell. To mimic this process, the cytoplasmic domain of CD3-zeta is often used as a major component of the CAR intracellular domain.

[0067] In addition to CD3 signaling, T cells require co-stimulatory molecules to persist after activation. In some implementations, the intracellular domain of the CAR receptor also includes one or more chimeric domains from co-stimulatory proteins, such as CD28, CD27, CD134 (OX40), and CD137 (4-1BB). Polynucleotides encoding antibodies and methods for preparing antibodies This disclosure also provides isolated polynucleotide or nucleic acid molecules encoding antibodies, variants, or derivatives of this disclosure. The polynucleotides of this disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, its variants, or derivatives, either on the same polynucleotide molecule or on different polynucleotide molecules. Alternatively, the polynucleotides of this disclosure may encode partial heavy and light chain variable regions of an antigen-binding polypeptide, its variants, or derivatives, either on the same polynucleotide molecule or on different polynucleotide molecules.

[0069] Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptide of this disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and methods described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents (6,150,584; 6,458,592; 6,420,140), the entire contents of which are incorporated herein by reference. Cancer treatment As described herein, the antibodies, bispecific antibodies, peptides, variants, or derivatives disclosed herein may be used in certain therapeutic and diagnostic approaches.

[0071] This disclosure also relates to antibody-based treatment methods involving the administration of antibodies of this disclosure to patients, such as animals, mammals, and humans, to treat one or more diseases or conditions described herein. The therapeutic compounds of this disclosure include, but are not limited to, antibodies of this disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of this disclosure (including variants and derivatives thereof as described herein).

[0072] In some embodiments, methods for treating cancer in patients in need are provided. In one embodiment, the method involves administering an effective amount of the antibody disclosed herein to the patient. In some embodiments, at least one cancer cell (e.g., stromal cells) in the patient overexpresses a tumor antigen, such as Claudin 18.2.

[0073] This disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T-cell or NK-cell therapies. Suitable cells can be used, contacted with the antibodies or CARs of this disclosure (or optionally engineered to express the antibodies or CARs of this disclosure). After such contact or modification, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any type of cancer disclosed herein. The cells (e.g., T cells or NK cells) may be, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

[0074] In some embodiments, the cells are isolated from the cancer patient themselves. In other embodiments, the cells are provided by a donor or from a cell bank. When the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0075] The antibodies or variants or derivatives thereof disclosed herein can be used to treat, prevent, diagnose, and / or predict the progression and / or metastasis of other diseases or conditions associated with increased cell survival, including but not limited to malignancies and related diseases such as leukemia (including acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia (including myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroblastic leukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, and solid tumors, including but not limited to sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and notochord. Tumors, angiosarcomas, endothelial sarcomas, lymphangiosarcomas, lymphangioendothelial sarcomas, synovial tumors, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, cerebral hemangioma, melanoma, neuroblastoma, and retinoblastoma. Diagnostic methods Overexpression of 4-1BB has been observed in certain tumor samples, and patients with 4-1BB overexpressing cells may respond to treatment with the anti-4-1BB antibody of this disclosure. Therefore, the antibody of this disclosure can also be used for diagnostic and prognostic purposes.

[0077] Preferably, the sample including cells can be obtained from a patient, who may be a cancer patient or a patient requiring diagnosis. The cells can be tumor tissue or tumor masses, blood samples, urine samples, or cells from any sample from the patient. After optional pretreatment of the sample, the sample can be incubated with an antibody of this disclosure, the incubation conditions of which should allow the antibody to interact with the 4-1BB protein that may be present in the sample. The presence of the 4-1BB protein in the sample can be detected using methods such as ELISA with anti-4-1BB antibodies.

[0078] The presence (in amount or concentration) of 4-1BB protein in a sample can be used for cancer diagnosis, as an indicator of a patient's suitability for antibody therapy, or as an indicator of whether a patient has (or has not) responded to cancer treatment. For prognostic methods, one, two, or more tests can be performed at certain stages after the initiation of cancer treatment to show progress in treatment.

[0079] Composition This disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0080] In one specific implementation, the term "pharmaceutical acceptable" means a substance approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more specifically, in humans. Furthermore, "pharmaceutical acceptable carriers" are typically non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of formulation adjuvant.

[0081] The term "carrier" refers to a diluent, adjuvant, excipient, or vessel that is administered with a therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as glucose and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tonic agents such as sodium chloride or glucose are also considered. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in a purified form, and an appropriate amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the route of administration. Parent formulations can be packaged in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0082] In one embodiment, the composition is formulated according to standard procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the components are provided individually or mixed together in unit doses, for example as dry lyophilized powders or anhydrous concentrates, in sealed containers (e.g., ampoules or sachets indicating the amount of active agent). If the composition is administered via infusion, an infusion bottle containing sterile pharmaceutical-grade water or saline may be used. When the composition is administered by injection, ampoules of sterile injectable water or saline may be provided so that the components can be mixed prior to administration. Detailed Implementation

[0083] Example 1: Generation of VHH Antibody against Human 4-1BB This example describes the generation of a single-domain (VHH) antibody against human 4-1BB protein.

[0084] Immunization: To generate VHH antibodies against human 4-1BB, two alpacas were immunized with human 4-1BB protein. After two rounds of immunization, the antibody titers in the serum of the immunized alpacas were evaluated using ELISA.

[0085] Immune library construction: Phage libraries were constructed using a phagemid vector consisting of a VHH gene fragment amplified from PBMCs of 4-1BB immunized alpacas. Antibodies were used to display the VHH fragment from the library as phages. Three immune libraries were generated from PBMCs of different alpacas in different immunization rounds. Each library was larger than 1 × 10⁻⁶. 9 The sequence diversity analysis is as follows. Forty-eight clones were selected from each library and further sequenced. The CDR sequences of the three libraries showed sufficient diversity.

[0086] Phage screening and clone selection: The 4-1BB protein is used as an antigen for phage library screening.

[0087] For human 4-1BB phage libraries, solution panning or solid-phase panning is used. The bound phages were eluted with Gly-HCl. The resulting phage was designated as output 1. The bound phages were incubated with SS320 cells and plated on 2YT plates for the next round of selection. A total of three rounds of selection were performed. After three rounds of selection, ELISA showed enrichment of 4-1BB conjugates in phages output 1, output 2, and output 3.

[0088] Single clones were selected from output 2 and output 3 phages. These clones were subjected to antigen-binding ELISA. Clones exhibiting good binding efficacy were selected for subsequent sequencing.

[0089] Five candidate sequences were cloned into the pcDNA 3.4 vector and expressed in 293F cells. Monoclonal antibodies were purified from the culture supernatant using protein G. The purified antibodies were assessed for binding to the 4-1BB-His protein by ELISA.

[0090] The amino acid sequences of the single variable domains B31, B16, B125, B164, and B210 are listed in Table 1 below. CDR sequences are summarized in Tables 1A-1E. Some CDR sequences include dipeptides, such as NG and DG, which may be susceptible to post-translational modifications. Certain "risk-free" versions of these sequences are also provided in the table, which are expected to retain antibody activity while reducing the risk of such post-translational modifications.

[0091] Table 1. Variable Region Sequence

[0092] Table 1A. CDR Sequence of B31

[0093] Table 1B. CDR Sequences of B16 Table 1. CDR Sequence of B125 Table 1. CDR Sequences of D.B164

[0096] Table 1E. CDR Sequence of B210

[0097] Example 2: Binding activity with 4-1BB antigen This embodiment tested the binding activity of the antibody to the 4-1BB protein. 2.1 ELISA combined with 4-1BB To assess the binding activity of clones B31, B16, B125, B164, and B210, chimeric monoclonal antibodies from these clones, as well as Urelumab (an anti-4-1BB agonist antibody used as a control), were tested by ELISA.

[0099] In summary, microplates were coated with PBS solution containing 0.5 μg / ml human 4-1BB-His protein, 100 μl / well, incubated overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Triple dilutions of B31, B16, B125, B164, and B210 antibodies (starting from 3 μg / ml) were added to each well, and the plates were incubated at 37°C for 1 h. Plates were washed with PBS / Tween and then incubated with peroxidase-conjugated anti-human IgG (H&L) (GOAT) antibody at 37°C for 30 min. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. Figure 1 As shown in Table 2, all of these clones bind to human 4-1BB with high affinity.

[0100] Microplates were coated with PBS solution containing 0.5 μg / ml cynomolgus monkey 4-1BB-His protein, 100 μl / well, and incubated overnight at 4°C, followed by blocking with 150 μl / well of 1% BSA. Triple dilutions of antibodies against B31, B16, B125, B164, and B210 (starting from 15 μg / ml) were added to each well, and the plates were incubated at 37°C for 1 h. Plates were washed with PBS / Tween and then incubated with peroxidase-conjugated anti-human IgG (H&L) (GOAT) antibody at 37°C for 30 min. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. Figure 1 As shown in Table 2, all of these clones bind with high affinity to cynomolgus monkey 4-1BB.

[0101] Microplates were coated with PBS solution containing 0.5 μg / ml mouse 4-1BB-His protein, 100 μl / well, and incubated overnight at 4°C, followed by blocking with 150 μl / well of 1% BSA. Triple dilutions of antibodies against B31, B16, B125, B164, and B210 (starting from 15 μg / ml) were added to each well, and the plates were incubated at 37°C for 1 h. Plates were washed with PBS / Tween and then incubated with peroxidase-conjugated anti-human IgG (H&L) (GOAT) antibody at 37°C for 30 min. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. Figure 1 As shown in Table 2, only B16, B125, and B210 bind to mouse 4-1BB. Table 2. Cross-species activities of B31, B16, B125, B164, and B210

[0103] --: No combination 2.2 Cellular binding of 4-1BB To evaluate the 4-1BB binding properties, the binding of the anti-CLDN18.2-4-1BB bispecific antibody to HEK293 expressing 4-1BB was analyzed by FACS. A total of 1×10⁻⁶ cells were used per well. 5 HEK293-4-1BB cells were incubated with serially diluted antibody (4-fold dilution starting at 100 nM) in FACS buffer at 4°C for 30 min. After washing with FACS buffer, PE-conjugated anti-human IgG antibody was added to each well and incubated at 4°C for 30 min. Following washing, the MFI of PE was evaluated using a MACSQuant analyzer 16. Figure 2 As shown, the tested anti-CLDN18.2-4-1BB bispecific antibody exhibited concentration-dependent binding ability to 4-1BB. 2.3 Protein Kinetics of 4-1BB Biacore tested the binding of B31, B16, B125, B164, and B210 antibodies to recombinant 4-1BB protein (human 4-1BB-his tag) using a capture method. Monoclonal antibodies (mAbs) of B31, B16, B125, B164, and B210 were captured using a Protein A chip. Serial dilutions of the human 4-1BB-his-tagged protein were injected onto the captured antibodies at a flow rate of 30 μl / min for 30 s. Antigen dissociation was allowed for 360 s. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software. Results are shown in [Table data would be inserted here]. Figure 3 And in Table 3 below.

[0105] Table 3: Total Dynamics Measured by Biacore 2.4 Cross-binding activity with OX40 and CD40 To evaluate the cross-binding activity of clones B31, B16, B125, B164, and B210 with human CD40 and human OX40, chimeric monoclonal antibodies from these clones were tested by ELISA.

[0107] In summary, microplates were coated with PBS solution containing 0.5 μg / ml human CD40 or human OX40 protein, 100 μl / well, incubated overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Five-fold dilutions of B31, B16, B125, B164, and B210 antibodies (starting from 3 μg / ml) were added to each well, and the plates were incubated at 37°C for 1 h. The plates were washed with PBS / Tween and then incubated with peroxidase-conjugated anti-human IgG (H&L) (GOAT) antibody at 37°C for 30 min. After washing, the plates were developed with TMB substrate and analyzed spectrophotometer at OD 450 nm. Figure 4 As shown, none of these clones cross-bind with human CD40 or human OX40.

[0108] Example 3: Functional activity of 4-1BB nanobodies This embodiment tested the functional activity of the antibody and showed that, unlike Urelumab, the antibody of the present invention does not activate 4-1BB signaling.

[0109] Functional characterization of 4-1BB monoclonal antibodies based on cell lines To evaluate the ability of 4-1BB monoclonal antibodies to activate the 4-1BB signaling pathway, a commercial 4-1BB NF-κB luciferase reporter system was used. In this assay, HEK-4-1BB NF-κB was used as the reporter cell line. The HEK-4-1BB NF-κB cell line was genetically engineered to stably express 4-1BB and luciferase downstream of the response element (Genomeditech, cat#GM-C04832). Antibody binding to the 4-1BB receptor induced luciferase expression. Briefly, reporter cells were cultured at 2.5 × 10⁶ cells per well. 4 Cells were cultured at a density of 100 μC in white 96-well plates. Antibodies were serially diluted and added to the white 96-well plates, with final concentrations of B31 ranging from 150 nM to 0.0000768 nM, and B16, B125, B164, and B210 molecules ranging from 100 nM to 0.000381 nM. After incubation at 37°C for 6 h, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0110] like Figure 5 As shown, the Urelumab monoclonal antibody dose-dependently activates 4-1BB signaling, while the B31, B16, B125, B164 and B210 antibodies do not promote 4-1BB induction under the same experimental conditions.

[0111] Example 4: Functional activity of anti-Claudin 18.2 / 4-1BB bispecific antibody In this embodiment, a bispecific antibody incorporating anti-4-1BB nanobodies and specific for Claudin 18.2 was generated and tested.

[0112] 4.1 Functional characterization of Claudin 18.2-4-1BB bispecific antibody based on cell line To evaluate the ability of the generated anti-Claudin 18.2 / 4-1BB bispecific antibody to activate the 4-1BB signaling pathway, a commercial 4-1BB NF-κB luciferase reporter system was used. In this assay, H_TNFRSF9 (4-1BB) NFkB-reporter Jurkat (Genomeditech, cat#GM-C09468) cells were used as effector cells, and CHO-K1 cells expressing or not expressing Claudin 18.2 were used as target cells. The H_TNFRSF9 (4-1BB) NFkB-Reporter Jurkat cell line was genetically modified to stably express 4-1BB and luciferase downstream of the response element (Genomeditech, cat#GM-C04832). Antibody binding to the 4-1BB receptor induced luciferase expression. Briefly, effector cells were cultured at 2.5 × 10⁶ cells per well. 4 The density of cells is 2.5 × 10⁻⁶. 4 Target cells (E / T ratio = 1:1) were co-cultured in white 96-well plates. Antibody was serially diluted 4-fold and added to the white 96-well plates, with final concentrations ranging from 100 nM to 0.000381 nM. After incubation at 37°C for 6 h, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0113] like Figure 6 As shown, the Urelumab monoclonal antibody can dose-dependently enhance 4-1BB signaling in CHO-K1 and CHO-Claudin18.2 overexpressing cells. The activities of the anti-Claudin18.2 / 4-1BB bispecific antibodies B31-BiAb, B16-BiAb, B125-BiAb, B164-BiAb, and B210-BiAb depend on Claudin 18.2 expression on cells. 4.2 Activity of bispecific antibodies in promoting immune responses in human peripheral blood mononuclear cells (PBMCs) To investigate the ability of Claudin 18.2-4-1BB bispecific antibody to stimulate the response of human PBMCs, the release of IL-2 cytokines was detected using the LANCEUltra TR-FRET assay kit. Human PBMCs stimulated with 0.5 μg / ml human anti-CD3 antibody were used as effector cells. CHO-K1 cells expressing Claudin 18.2 were used as target cells. In the presence of human anti-CD3 antibody, human PBMCs (1×10⁻⁶ cells / mL) were stimulated with 0.5 μg / ml human anti-CD3 antibody. 5 ) with CHO-K1-Claudin 18.2 or parental CHO-K1 cells (2.5 × 10⁻⁶) 4(E / T ratio = 4:1) Co-culture. Five-fold serially diluted bispecific antibody was added to the culture medium, starting at a final concentration of 100 nM. After 48 h, the secretion level of IL-2 in the culture medium was measured using the IL-2 (human) LANCE Ultra TR-FRET assay kit (PerkinElmer). Figure 7 As shown, the bispecific antibody can only activate the PBMC response in the presence of Claudin 18.2 overexpressing cells.

[0115] Example 5. Humanization of B31 / B125 / B16 / B210 VHH antibodies Humanized monoclonal antibodies were prepared using the variable region genes of B31 / B125 / B16 / B210. In the first step of this process, the amino acid sequence of the VHH of B31 / B125 / B16 / B210 was compared with available human Ig gene sequence databases to find the best-matching human Ig gene sequence overall. Then, humanized variable domain sequences of B31 / B125 / B16 / B210 were designed, with CDRH1, H2, and H3 located on the frame sequence of the VH gene.

[0116] The amino acid and nucleotide sequences of some humanized antibodies are listed in Table 4 below. Table 4A. Humanized antibody sequence of B31 (underlined CDR; bold / italic indicates reversion mutation)

[0118] Table 4B. Humanized antibody sequence of B125 (underlined CDR; bold / italic indicates reversion mutation)

[0119] Table 4C. Humanized antibody sequences for B16 (underlined CDRs; bold / italicized text indicates reversion mutations)

[0120] Table 4D. Humanized antibody sequence of B210 (underlined CDR; bold / italic indicates reversion mutation)

[0121] B31 reversion mutations include 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 78M, 82(82A)D, and 94A. More specifically, VHH-v2 includes reversion mutations 37Y, 44Q, 45R, and 94A; VHH-v3 includes reversion mutations 23A, 37Y, 44Q, 45R, 49A, 74N, and 94A; VHH-v4 includes reversion mutations 23A, 37Y, 44Q, 45R, 49A, 78M, 82(82A)D, and 94A; VHH-v5 includes reversion mutations 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 82(82A)D, and 94A; and VHH-v6 includes reversion mutations 37Y, 44Q, 45R, and 49A.

[0122] B16 reversion mutations include 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P, and 94T. More specifically, VHH-v2 includes reversion mutations 37F, 44E, 45R, 47G, and 94T; VHH-v3 includes reversion mutations 29L, 30D, 37F, 44E, 45R, 47G, and 94T; and VHH-v4 includes reversion mutations 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P, and 94T.

[0123] The reversion mutations for B125 include 37F, 43K, 44E, 45R, 47G, 74A, 84P, and 94T. More specifically, VHH-v2 includes reversion mutations 37F, 44E, 45R, 47G, and 94T; VHH-v3 includes reversion mutations 37F, 43K, 44E, 45R, 47G, and 74A; VHH-v4 includes reversion mutations 37F, 43K, 44E, 45R, 47G, 74A, and 94T; and VHH-v5 includes reversion mutations 37F, 43K, 44E, 45R, 47G, 74A, 84P, and 94T.

[0124] B210 reversion mutations include 27L, 29L, 37F, 44E, 45R, 47G, 89D, and 94T. More specifically, VHH-v2 includes reversion mutations 37F, 44E, 45R, 47G, and 94T; VHH-v3 includes reversion mutations 27L, 37F, 44E, 45R, 47G, and 94T; VHH-v4 includes reversion mutations 27L, 29L, 37F, 44E, 45R, 47G, and 94T; and VHH-v5 includes reversion mutations 27L, 29L, 37F, 44E, 45R, 47G, 89D, and 94T.

[0125] The humanized VHH gene was cloned into the pcDNA3.4 vector and transfected into 293F cells for further analysis.

[0126] Example 6: Antigen-binding properties of humanized antibodies Biacore measures the pharmacokinetic affinity of humanized antibodies. Biacore tested the binding of humanized antibodies to recombinant human 4-1BB protein (human 4-1BB-histag) using a capture method. B31-V2, B31-V3, B31-V4, B31-V5, and B31-V6 were captured using a Protein A chip. Serial dilutions of the human 4-1BB-histag protein were injected onto the captured antibodies at a flow rate of 10 μl / min for 30 s. Antigen dissociation was allowed for 360 s. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software, and the results are shown in Table 5 below. All humanized antibodies showed strong binding. Table 5. Total Kinetics as Measured by Biacore

[0128] Example 7: Functional activity of humanized bispecific antibodies Anti-Claudin 18.2 / 4-1BB bispecific antibodies were prepared using humanized 4-1BB nanobodies and tested in this example.

[0129] 7.1 Functional characterization of Claudin 18.2-4-1BB bispecific antibody based on cell line To evaluate the ability of the Claudin 18.2-4-1BB humanized bispecific antibody to activate the 4-1BB signaling pathway, a commercial 4-1BB NF-κB luciferase reporter system was used. In this assay, H_TNFRSF9 (4-1BB) NFkB-Reporter Jurkat (Genomeditech, cat#GM-C09468) cells were used as effector cells, and CHO-K1 cells expressing or not expressing Claudin18.2 were used as target cells. The H_TNFRSF9 (4-1BB) NFkB-Reporter Jurkat cell line was genetically engineered to stably express 4-1BB and luciferase downstream of the response element (Genomeditech, cat#GM-C04832). Antibody binding to the 4-1BB receptor induced luciferase expression. Briefly, effector cells were cultured at 2.5 × 10⁶ cells per well. 4 The density of cells is 2.5 × 10⁻⁶. 4Target cells (E / T ratio = 1:1) were co-cultured in white 96-well plates. Antibody was serially diluted 3-fold and added to the white 96-well plates, with final concentrations ranging from 100 nM to 0.005 nM. After incubation at 37°C for 6 h, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0130] like Figure 8 As shown, the activity of the anti-Claudin 18.2 / 4-1BB humanized bispecific antibody is comparable to that of the B31 chimeric antibody.

[0131] 7.2 Activity of humanized bispecific antibodies in promoting immune responses in human peripheral blood mononuclear cells (PBMCs) To investigate the ability of human PBMCs to respond to Claudin 18.2-4-1BB humanized bispecific antibody, the release of IL-2 cytokines was detected using the LANCE Ultra TR-FRET assay kit. Human PBMCs stimulated with 0.5 μg / ml human anti-CD3 antibody were used as effector cells. CHO-K1 cells expressing Claudin 18.2 were used as target cells. Human PBMCs (1×10⁻⁶ cells / mL) were stimulated with human anti-CD3 antibody in the presence of human anti-CD3 antibody. 5 ) with CHO-K1-Claudin 18.2 or parental CHO-K1 cells (2.5 × 10⁻⁶) 4 (E / T ratio = 4:1) Co-culture. Add 5-fold serially diluted humanized bispecific antibody to the culture medium, starting at a final concentration of 100 nM. After 48 h, measure the IL-2 secretion level in the culture medium using the IL-2 (human) LANCE Ultra TR-FRET assay kit (PerkinElmer). Figure 9 As shown, the IL-2 secretion induced by the humanized bispecific antibody is comparable to that induced by the B31 chimeric antibody.

[0132] Example 8: Anti-Claudin 18.2 / 4-1BB bispecific antibody inhibits tumor growth In this embodiment, humanized mice expressing the human 4-1BB extracellular domain were used to test the activity of the bispecific antibody in inhibiting tumor growth.

[0133] Mouse colon adenocarcinoma cells (MC38) were modified to express human CLDN18.2. Humanized mice (h4-1BB) were subcutaneously implanted with MC38-hCLDN18.2 cells. Mice were intraperitoneally injected three times weekly with the following antibodies: PBS control (1 mg / kg), Urelumab (0.85 mg / kg), B31 anti-CLDN18.2 / 4-1BB bispecific antibody (1 mg / kg), B16 anti-CLDN18.2 / 4-1BB bispecific antibody (1 mg / kg), B125 anti-CLDN18.2 / 4-1BB bispecific antibody (1 mg / kg), B164 anti-CLDN18.2 / 4-1BB bispecific antibody (1 mg / kg), and B210 anti-CLDN18.2 / 4-1BB bispecific antibody (1 mg / kg). Tumor volume was monitored twice weekly using calipers during the experiment. Figure 10 As shown, Urelumab, B31, B16, B125, B164, and B210 can all inhibit tumor growth (62.4%-96.9%) together with TG1.

[0134] This disclosure is not limited to the specific embodiments described, which are intended as a single illustration of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0135] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication or patent application is specifically and individually indicated to be incorporated herein by reference.

Claims

1. A single-domain antibody or a polypeptide comprising said single-domain antibody, characterized in that, The single-domain antibody has binding specificity to human 4-1BB protein and includes complementarity-determining regions 1 (CDR1), 2 (CDR2), and 3 (CDR3), wherein: (a) The CDR1 contains the amino acid sequence shown in SEQ ID NO: 6, the CDR2 contains the amino acid sequence shown in SEQ ID NO: 7, 39 or 40, and the CDR3 contains the amino acid sequence shown in SEQ ID NO: 8; (b) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, 41 or 42, and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; or (c) The CDR1 contains the amino acid sequence shown in SEQ ID NO: 12, the CDR2 contains the amino acid sequence shown in SEQ ID NO: 13, 43 or 44, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

14.

2. The antibody or polypeptide according to claim 1, characterized in that, The CDR1 contains the amino acid sequence shown in SEQ ID NO: 6, the CDR2 contains the amino acid sequence shown in SEQ ID NO: 7, 39 or 40, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

8.

3. The antibody or polypeptide according to claim 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 21-25.

4. The antibody or polypeptide according to claim 1, characterized in that, The CDR1 contains the amino acid sequence shown in SEQ ID NO: 9, the CDR2 contains the amino acid sequence shown in SEQ ID NO: 10, 41 or 42, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

11.

5. The antibody or polypeptide according to claim 4, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 31-34.

6. The antibody or polypeptide according to claim 1, characterized in that, The CDR1 contains the amino acid sequence shown in SEQ ID NO: 12, the CDR2 contains the amino acid sequence shown in SEQ ID NO: 13, 43 or 44, and the CDR3 contains the amino acid sequence shown in SEQ ID NO:

14.

7. The antibody or polypeptide according to claim 6, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 26-30.

8. The antibody or polypeptide according to any one of claims 1-7, characterized in that, The polypeptide is a chimeric antigen receptor (CAR) or a bispecific antibody that has binding specificity to antigens different from 4-1BB.

9. A bispecific antibody comprising the antibody of any one of claims 1-7 and a second antibody or antigen-binding fragment having binding specificity to a non-4-1BB target antigen.

10. The bispecific antibody according to claim 9, characterized in that, The target antigens were selected from the group consisting of Claudin18.2, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and Tenascin.

11. A polynucleotide encoding an antibody or polypeptide as described in any one of claims 1-10.

12. A cell comprising the polynucleotide as described in claim 11.

13. A composition comprising an antibody or polypeptide as described in any one of claims 1-10 and a pharmaceutically acceptable carrier.

14. A method of treating cancer in a patient in need, comprising administering to the patient an effective amount of an antibody or polypeptide as described in any one of claims 1-10.

15. Use of the antibody or polypeptide according to any one of claims 1-10 in the preparation of a medicament for treating cancer.

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