Single domain 4-1BB antibody

Abstract

Single domain anti-4-1BB antibodies and polypeptides, such as bispecific antibodies and chimeric antigen receptors, comprising these single domain antibodies are provided. Methods of using the antibodies or polypeptides to treat and diagnose diseases such as cancer are also provided. The single domain antibodies of the present disclosure can be "non-agonists" that do not activate 4-1BB signaling by themselves. However, when combined with anti-claudin 18.2 antibodies, the subsequent bispecific antibodies have strong activity in activating 4-1BB signaling in the presence of claudin 18.2 positive cells.

Description

[Background technology]

[0001] background Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Nanobodies produced from camelids and certain other animals are also called VHH fragments. Like whole antibodies, nanobodies can selectively bind to specific antigens. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than common antibodies (150-160 kDa). Given their small size and single-chain nature, single domain antibodies may be particularly suitable for inclusion as fragments in other proteins such as chimeric antigen receptors (CARs) and bispecific antibodies.

[0002] 4-1BB (CD137, tumor necrosis factor receptor superfamily 9), a member of the TNF-receptor superfamily (TNFRSF), is a costimulatory molecule expressed after activation of immune cells, both innate and adaptive immune cells. 4-1BB plays an important role in regulating the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8 T cells. Many other studies have shown that activation of 4-1BB enhances immune responses to eliminate tumors in mice. Thus, 4-1BB was suggested to be a promising target molecule in cancer immunology. Summary of the Invention [Means for solving the problem]

[0003] Abstract Nanobodies specific for human 4-1BB protein and bispecific or multispecific antibodies incorporating such nanobodies are provided. In some embodiments, the nanobodies of the present disclosure are "non-agonists" that do not activate 4-1BB signaling by themselves. However, when combined with anti-claudin 18.2 antibody, the bispecific antibody then has strong activity in activating 4-1BB signaling in the presence of claudin 18.2 positive cells.

[0004] 4-1BB signaling activation is the expected mechanism for agonistic antibodies, such as utomirumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB nanobodies disclosed herein do not require such activity. In fact, the anti-4-1BB portion of the bispecific antibody is preferably unable to independently activate 4-1BB in the absence of binding to the tumor-associated antigen (TAA) that the bispecific antibody also targets, in which case such bispecific antibodies will have reduced off-tumor side effects.

[0005] Compared with known anti-4-1BB agonist antibodies that generally involve dose-limiting on-target toxicity, the antibodies of the present disclosure are much safer.In tissues where the corresponding TAA is not expressed, such as the liver, the bispecific antibodies of the present disclosure are not expected to induce cytotoxic immune responses, since they cannot activate 4-1BB signaling.In contrast, in tumor tissues where TAA is expressed and / or available, these bispecific antibodies can initiate a strong immune response to tumor cells.Therefore, unlike the anti-4-1BB antibodies currently in clinical development that suffer from on-target / intrinsic toxicity, the antibodies of the present disclosure can be simultaneously potent and safe in treating cancer.

[0006] Thus, in one embodiment of the present disclosure, a single domain antibody or a polypeptide comprising the single domain antibody is provided, having binding specificity for human 4-1BB protein and comprising complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 6, CDR2 comprises the amino acid sequence of SEQ ID NO: 7, 39 or 40, and CDR3 comprises the amino acid sequence of SEQ ID NO: 8.

[0007] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, which has binding specificity for human 4-1BB protein and comprises complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 9, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, 41 or 42, and CDR3 comprises the amino acid sequence of SEQ ID NO: 11.

[0008] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, which has binding specificity for human 4-1BB protein and comprises complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 12, CDR2 comprises the amino acid sequence of SEQ ID NO: 13, 43 or 44, and CDR3 comprises the amino acid sequence of SEQ ID NO: 14.

[0009] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, which has binding specificity for human 4-1BB protein and comprises complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 15 to 17, respectively.

[0010] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, which has binding specificity for human 4-1BB protein and comprises complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 18 to 20, respectively.

[0011] In another embodiment, a single domain antibody or a polypeptide comprising the single domain antibody is provided, which has binding specificity for human 4-1BB protein and comprises complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 of any one of SEQ ID NOs: 1 to 5.

[0012] Also provided are bispecific / multispecific antibodies incorporating one or more units of a nanobody. In some embodiments, the second specificity is against a tumor antigen, such as claudin 18.2. Other examples of tumor antigens are also disclosed herein. In some embodiments, chimeric antigen receptors (CARs) comprising the nanobodies of the present disclosure are provided.

[0013] Compositions comprising the antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided. Additionally, isolated cells comprising one or more polynucleotides encoding the antibody or polypeptide, one or more polynucleotides encoding the antibody or fragment thereof are also provided.

[0014] Treatment methods and uses are also provided. In one embodiment, a method of treating cancer in a patient in need thereof is provided, comprising administering to the patient an effective amount of an antibody or polypeptide of the present disclosure. 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, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer, and thyroid cancer. In some embodiments, the method further comprises administering to the patient a second cancer therapeutic agent. [Brief description of the drawings]

[0015] [Figure 1]FIG. 1 shows the ELISA results testing the binding activity of anti-4-1BB antibodies to human 4-1BB antigen, cyno4-1BB antigen and mouse 4-1BB antigen.

[0016] [Diagram 2] FIG. 2 shows cell-based binding to human 4-1BB protein.

[0017] [Diagram 3] FIG. 3 shows the complete kinetic activity of the tested anti-4-1BB antibodies as measured by Biocore.

[0018] [Figure 4] FIG. 4 depicts an ELISA binding graph showing that the antibodies tested do not react with either human OX40 or human CD40.

[0019] [Diagram 5] FIG. 5 shows that unlike the reference anti-4-1BB antibody urelumab, the tested antibodies did not activate 4-1BB signaling.

[0020] [Figure 6] FIG. 6 shows that the bispecific antibodies tested activated 4-1BB signaling only in claudin-18.2-positive CHO cells.

[0021] [Figure 7] FIG. 7 shows that the bispecific antibodies tested activated 4-1BB signaling in PBMC cells only in the presence of CHO cells overexpressing claudin 18.2.

[0022] [Figure 8] FIG. 8 shows that the bispecific antibody incorporating the humanized B31 antibody had NFκB activation activity equivalent to that of the chimeric B31 antibody.

[0023] [Figure 9]FIG. 9 shows that the bispecific antibody incorporating the humanized B31 antibody had IL-2 secretion-inducing activity equivalent to that of the chimeric B31 antibody.

[0024] [Figure 10] FIG. 10 shows the in vivo tumor growth inhibitory activity of the antibodies tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0026] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and homology or sequence identity percentage can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard; Filter=none; Strand=both; Cutoff=60; Expectation=10; Matrix=BLOSUM62; Display=50 sequences; Sort by: HIGH SCORE; Database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those that have the specified percentages of homology as described above and that encode polypeptides having the same or similar biological activity.

[0027] 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 the nucleic acid or its complementary sequence. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a degree of homology with the double-stranded nucleic acid or its complementary sequence. In one embodiment, a homolog of a nucleic acid can hybridize with the nucleic acid or its complementary sequence. Similarly, an "equivalent polypeptide" refers to a polypeptide that has a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some embodiments, an 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 embodiments, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridges) of the reference sequence.

[0028] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0029] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. 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.

[0030] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and the variable region of the light chain (V L In some embodiments, the domains are linked by a short linker peptide of 10 to about 25 amino acids. The linker can contain many amino acids, such as glycine for flexibility, as well as serine or threonine for solubility, and the V H N-terminus of V L The scFv molecule can be linked either to the C-terminus of the IgG1A or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0031] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of this disclosure and, therefore, are within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of this disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains of molecular weight approximately 23,000 daltons, and two identical heavy polypeptide chains of molecular weight 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, starting at the mouth of the "Y" and continuing through the variable region.

[0032] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0033] "Specifically binds" or "has specificity for" generally means that an antibody binds to an epitope through its antigen-binding domain, and that binding requires a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with a higher specificity than it has for related epitope "D."

[0034] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or alleviation of disease symptoms, and remission (whether partial or complete remission). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in whom a condition or disorder is to be prevented.

[0035] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, farm animals, livestock, zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

[0036] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, e.g., mammalian subjects, who will benefit from the administration of an antibody or composition of the disclosure for use, e.g., in detection, diagnostic procedures, and / or treatment. Single domain anti-4-1BB antibody

[0037] The present disclosure provides single-chain anti-4-1BB antibodies with high affinity to human 4-1BB protein. In some embodiments, the provided nanobodies cannot activate 4-1BB signaling by such binding alone, and are therefore referred to as "non-agonistic" anti-4-1BB antibodies. Such non-agonistic anti-4-1BB nanobodies are particularly suitable for preparing bispecific antibodies.

[0038] 4-1BB signaling activation is the expected mechanism for agonistic antibodies, such as utomirumab (PF-05082566) and urelumab (BMS-663513). However, the anti-4-1BB nanobody disclosed herein does not need to have such activity. In fact, it is preferred that the anti-4-1BB part of the bispecific antibody cannot independently activate 4-1BB in the absence of CLDN18.2 binding, in which case such a bispecific antibody will have reduced off-target side effects.

[0039] Compared with known anti-4-1BB agonist antibodies, such as utomirumab (PF-05082566) and urelumab (BMS-663513), which are generally associated with dose-limiting on-target toxicity, the bispecific antibodies of the present disclosure are much safer. In tissues where CLDN18.2 is not expressed, the bispecific antibodies of the present disclosure are not expected to induce cytotoxic immune responses, since they cannot activate 4-1BB signaling. In tumor tissues where CLDN18.2 is expressed and / or available, in contrast, these bispecific antibodies can initiate a strong immune response to tumor cells. Thus, unlike anti-4-1BB antibodies currently in clinical development that suffer from on-target / intrinsic toxicity, the antibodies of the present disclosure can be simultaneously potent and safe in treating cancer.

[0040] Thus, in one embodiment of the present disclosure, single domain antibodies and polypeptides comprising such single domain antibodies are provided. In one embodiment of the present disclosure, single domain antibodies comprising CDR1, CDR2 and CDR3 having the CDR1, CDR2 and CDR3 sequences, respectively, of antibody B31 (SEQ ID NO: 1), or polypeptides comprising the single domain antibodies, are provided. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 6, CDR2 comprises the amino acid sequence of SEQ ID NO: 7, 39 or 40, and CDR3 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 6-8, respectively. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 6, 39 and 8, respectively. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 6, 40 and 8, respectively.

[0041] Also provided are humanized antibodies, such as those provided in SEQ ID NOs: 21-25 for antibody B31. In some embodiments, the humanized antibody comprises back mutations selected from the group consisting of 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 78M, 82(82A)D, and 94A according to Kabat numbering. In some embodiments, the humanized antibody comprises back mutations 37Y, 44Q, 45R, and 94A. In some embodiments, the humanized antibody comprises back mutations 23A, 37Y, 44Q, 45R, 49A, 74N, and 94A. In some embodiments, the humanized antibody comprises back mutations 23A, 37Y, 44Q, 45R, 49A, 78M, 82(82A)D, and 94A. In some embodiments, the humanized antibody comprises the back mutations 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 82(82A)D, and 94A. In some embodiments, the humanized antibody comprises the back mutations 37Y, 44Q, 45R, and 49A.

[0042] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:21. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:22. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:23. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:24. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:25. In some embodiments, the antibody comprises the set forth CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs:1 and 21-25.

[0043] In another embodiment, a single domain antibody is provided that comprises a CDR1, CDR2 and CDR3 having the CDR1, CDR2 and CDR3 sequences, respectively, of antibody B16 (SEQ ID NO: 2), or a polypeptide comprising the single domain antibody. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 9, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, 41 or 42, and CDR3 comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequence of SEQ ID NO: 9-11, respectively. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequence of SEQ ID NO: 9, 41 and 11, respectively. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequence of SEQ ID NO: 9, 42 and 11, respectively.

[0044] Also provided are humanized antibodies, such as those provided in SEQ ID NOs: 31-34 for antibody B16. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 29L, 30D, 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 29L, 30D, 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering.

[0045] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:31. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:32. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:33. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:34. In some embodiments, the antibody comprises the set forth CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs:2 and 31-34.

[0046] In another embodiment, a single domain antibody is provided that comprises a CDR1, CDR2 and CDR3 having the CDR1, CDR2 and CDR3 sequences, respectively, of antibody B125 (SEQ ID NO: 3), or a polypeptide comprising the single domain antibody. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 12, CDR2 comprises the amino acid sequence of SEQ ID NO: 13, 43 or 44, and CDR3 comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 12-14, respectively. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 12, 43 and 14, respectively. In some embodiments, CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NO: 12, 44 and 14, respectively.

[0047] Also provided are humanized antibodies, such as those provided in SEQ ID NOs: 26-30 for antibody B125. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, 74A, 84P, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, and 74A, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, and 74A, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 43K, 44E, 45R, 47G, 74A, 84P and 94T, according to Kabat numbering.

[0048] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:26. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:27. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:28. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:29. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:30. In some embodiments, the antibody comprises the set forth CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs:3 and 26-30.

[0049] In another embodiment, a single domain antibody is provided that comprises a CDR1, CDR2 and CDR3 having the CDR1, CDR2 and CDR3 sequences, respectively, of antibody B164 (SEQ ID NO: 4), or a polypeptide comprising the single domain antibody. In some embodiments, the CDR1, CDR2 and CDR3 comprise the amino acid sequences of SEQ ID NOs: 15-17, respectively. In some embodiments, the antibody comprises the set forth CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 4.

[0050] In another embodiment, a single domain antibody is provided that comprises a CDR1, CDR2 and CDR3 having the CDR1, CDR2 and CDR3 sequences, respectively, of antibody B210 (SEQ ID NO: 5), or a polypeptide comprising the single domain antibody. In some embodiments, the CDR1, CDR2 and CDR3 each comprise the amino acid sequences of SEQ ID NOs: 18-20.

[0051] Also provided are humanized antibodies, such as those provided in SEQ ID NOs: 26-30 for antibody B210. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 27L, 29L, 37F, 44E, 45R, 47G, 89D, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 27L, 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering. In some embodiments, the humanized antibody comprises a back mutation selected from the group consisting of 27L, 37F, 44E, 45R, 47G, and 94T, according to Kabat numbering.

[0052] In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:35. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:36. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:37. In some embodiments, the antibody comprises the humanized sequence of SEQ ID NO:38. In some embodiments, the antibody comprises the set forth CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs:5 and 35-38.

[0053] In some embodiments, anti-4-1BB antibodies and antigen-binding fragments are also provided that compete with any of the antibodies disclosed herein in binding to human 4-1BB. In some embodiments, anti-4-1BB antibodies and antigen-binding fragments are also provided that bind to the same epitope as any of the antibodies disclosed herein. In some embodiments, anti-4-1BB antibodies and antigen-binding fragments are also provided that include CDR1, CDR2 and CDR3 of the antibodies disclosed herein.

[0054] Compositions comprising an antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided.

[0055] It will also be understood by those skilled in the art that the antibodies disclosed herein can be modified to differ in amino acid sequence from the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a designated protein can be similar to the starting sequence, e.g., have a certain percent identity to the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence. In some embodiments, the modified antibody or fragment retains the designated CDR sequence.

[0056] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties that are not normally associated with the antibody.Exemplary modifications are described in more detail below.For example, the antibody of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0057] Bispecific and multispecific antibodies comprising one, two, three or four units of the single domain anti-4-1BB antibodies disclosed herein and one or more other specificities (other than 4-1BB) are also provided. Bispecific and multispecific antibodies and chimeric antigen receptors (CARs)

[0058] As provided, the anti-4-1BB antibodies disclosed herein are particularly useful for preparing bispecific and multispecific antibodies, as well as chimeric antigen receptors (CARs), due at least to the improved therapeutic index of these antibodies and their small size.

[0059] Thus, in one embodiment, a bispecific antibody is provided that comprises an anti-4-1BB nanobody, or antigen-binding fragment thereof, of the present disclosure and a second antibody or antigen-binding fragment that has binding specificity for a target antigen that is not 4-1BB. In some embodiments, a third and fourth specificity are further included.

[0060] In some embodiments, the target antigen that is not 4-1BB is a tumor antigen.Many tumor antigens are known in the art, and new tumor antigens can be easily identified by 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, FOLR1, 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 are Her2, EpCAM, CD33, CD47, CD133, CEA, gpA33, mucin, TAG-72, CIX, PSMA, GD2, GD3, GM2, integrins, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin.

[0061] In some embodiments, the bispecific antibody has specificity for 4-1BB and claudin 18.2.

[0062] Also provided is a chimeric antigen receptor (CAR) comprising the nanobody of the present disclosure. In the CAR, the nanobody can serve as an antigen recognition domain. In addition, in some embodiments, the CAR also comprises an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0063] The hinge, also called the spacer, is a small structural domain between the antigen recognition region and the outer membrane of the cell. A suitable hinge improves the flexibility of the scFv receptor head, thus reducing the spatial constraint between the CAR and its target antigen. Examples of hinge sequences are based on the juxtamembrane regions from immune molecules, such as IgG, CD8 and CD28.

[0064] The transmembrane domain is a structural component consisting of a hydrophobic alpha helix that spans the cell membrane. It anchors the CAR to the plasma membrane, thus bridging the extracellular hinge and antigen recognition region with the intracellular signaling region. Typically, a transmembrane domain from the juxtamembrane component of the endodomain, such as the CD28 transmembrane domain, can be used.

[0065] The intracellular T cell signaling domain resides in the endodomain of the receptor inside the cell. When an antigen is bound to the outer antigen recognition domain, the CAR receptors cluster with each other and transmit an activation signal. As a result, the inner cytoplasmic tail of the receptor perpetuates the signal transduction into the T cell. To mimic this process, the cytoplasmic domain of CD3-zeta is commonly used as the main CAR endodomain component.

[0066] In addition to CD3 signaling, T cells also require costimulatory molecules to persist after activation. In some embodiments, the endodomain of the CAR receptor also contains one or more chimeric domains from costimulatory proteins, such as CD28, CD27, CD134 (OX40), and CD137 (4-1BB). Polynucleotides encoding antibodies and methods for preparing antibodies

[0067] The present disclosure also provides an isolated polynucleotide or nucleic acid molecule encoding the antibody of the present disclosure, its variant or derivative. The polynucleotide of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotide of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.

[0068] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as 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 administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety. Cancer Treatment

[0069] As described herein, the antibodies, bispecific antibodies, polypeptides, variants or derivatives of the disclosure can be used in certain treatment and diagnostic methods.

[0070] The present disclosure further relates to antibody-based therapies, including administering the antibodies of the present disclosure to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including variants and derivatives thereof as described herein), and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0071] In some embodiments, a method for treating cancer in a patient in need thereof is provided. In one embodiment, the method involves administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient overexpresses a tumor antigen, such as claudin 18.2.

[0072] Cell therapy, for example, chimeric antigen receptor (CAR) T cell or NK cell therapy, is also provided in the present disclosure. Suitable cells can be used that are contacted with the antibody or CAR of the present disclosure (or alternatively, are engineered to express the antibody or CAR of the present disclosure). Thus, by such contact or operation, the cells can be introduced into a cancer patient that needs treatment. The cancer patient can have any type of cancer as disclosed herein. The cells (for example, T cells or NK cells) can be, for example, but are not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0073] In some embodiments, the cells are isolated from the cancer patient himself or herself. In some embodiments, the cells are provided by a donor or from a cell bank. If the cells are isolated from the cancer patient, unwanted immune responses can be minimized.

[0074] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including sarcomas and carcinomas, such as fibrosarcomas. , myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, 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 These include, but are not limited to, myeloid leukoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Diagnostic methods

[0075] Overexpression of 4-1BB is found in certain tumor samples, and patients with 4-1BB overexpressing cells are more likely to respond to treatment with the anti-4-1BB antibody of the present disclosure. Therefore, the antibody of the present disclosure can also be used for diagnostic and prognostic purposes.

[0076] A sample preferably containing cells can be obtained from a patient, which can be a cancer patient or a patient who wants to be diagnosed. The cells can be from tumor tissue or tumor block, blood sample, urine sample or any sample from a patient. Once the sample is optimally pretreated, the sample can be incubated with the antibody of the present disclosure under conditions that allow the antibody to interact with 4-1BB protein that may be present in the sample. Methods such as ELISA can be used to detect the presence of 4-1BB protein in a sample by taking advantage of anti-4-1BB antibody.

[0077] The presence of 4-1BB protein in a sample (along with the amount or concentration, if appropriate) can be used to diagnose cancer, either as an indication that the patient is suitable for treatment with the antibody, or as an indication that the patient is (not) responding to cancer treatment. For prognostic methods, once cancer treatment has been initiated, detection can be performed once, twice or more times at a particular stage to indicate the progress of the treatment. composition

[0078] The present disclosure also provides pharmaceutical compositions. Such compositions include an effective amount of the antibody and an acceptable carrier. In some embodiments, the composition further includes a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0079] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0080] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. 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 purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0081] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. EXAMPLES

[0082] Example 1 Generation of VHH antibodies against human 4-1BB This example describes the generation of single domain (VHH) antibodies against the human 4-1BB protein.

[0083] Immunization: To generate VHH antibodies against human 4-1BB, two alpacas were immunized with human 4-1BB protein. After two rounds of immunization, the serum of the immunized alpacas was subjected to antibody titer evaluation by ELISA.

[0084] Immunized library construction: Phage libraries were constructed using phagemid vectors consisting of VHH gene fragments amplified from PBMCs of 4-1BB immunized alpacas. The antibody format is VHH fragments in the phage display library. Three immunized libraries were generated from PBMCs of different alpacas in different immunization rounds. All library sizes were 1×10 9More extensively, sequence diversity was analyzed by picking 48 clones from each library and further sequencing. The sequences showed sufficient diversity of the CDRs for these three libraries.

[0085] Phage panning and clone selection: 4-1BB protein was used as the antigen for phage library panning.

[0086] Phage library solution panning or solid-phase panning against human 4-1BB: Bound phages were eluted by Gly-Hcl. The resulting phages are output 1. Bound phages were incubated with SS320 cells and plated on 2YT plates for the next round of panning screening. There were a total of three rounds of panning screening. Phage ELISA of output 1, output 2 and output 3 showed enriched 4-1BB binders after three rounds of screening.

[0087] Single clones were picked up from output 2 and output 3 phages. Phages of these clones were subjected to antigen-binding ELISA. Clones that showed good binding activity were selected for subsequent sequencing.

[0088] The five candidate sequences were cloned into the pcDNA 3.4 vector and expressed in 293F cells. Monoclonal antibodies were purified from the culture supernatant by protein G. The purified antibodies were subjected to ELISA binding evaluation against 4-1BB-His protein.

[0089] The amino acid sequences of the single variable domains of B31, B16, B125, B164, B210 are listed in Table 1 below. The CDR sequences are summarized in Tables 1A-1E. Some of the CDR sequences contain dipeptides such as NG and DG that may be susceptible to post-translational modifications. Certain "risk-averse" versions of these sequences that are believed to reduce the risk of such post-translational modifications but retain antibody activity are also provided in the table. [Table 1] [Table 1A] [Table 1B] [Table 1C] [Table 1D] [Table 1E]

[0090] Example 2 Binding activity to 4-1BB antigen This example examined the binding activity of antibodies against the 4-1BB protein. 2.1 ELISA binding to 4-1BB

[0091] To evaluate the binding activity of clones B31, B16, B125, B164 and B210, chimeric mAbs from these clones were subjected to ELISA tests together with the reference anti-4-1BB agonist antibody, urelumab.

[0092] Briefly, microtiter plates were coated with 100 μl / well of human 4-1BB-His protein at 0.5 μg / ml in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of B31, B16, B125, B164 and B210 antibodies starting at 3 μg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween® and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugated for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Figure 1 and Table 2, all of these clones bound human 4-1BB with high affinity.

[0093] Microtiter plates were coated with 100 μl / well of 0.5 μg / ml cynomolgus 4-1BB-His protein in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of B31, B16, B125, B164 and B210 antibodies starting at 15 μg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugated for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Figure 1 and Table 2, all of these clones bound with high affinity to cynomolgus 4-1BB.

[0094] Microtiter plates were coated with 100 μl / well of mouse 4-1BB-His protein at 0.5 μg / ml in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of B31, B16, B125, B164 and B210 antibodies starting at 15 μg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugated for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Figure 1 and Table 2, only B16, B125 and B210 bound to mouse 4-1BB. [Table 2] 2.2 Cell-based binding to 4-1BB

[0095] To evaluate 4-1BB binding properties, anti-CLDN18.2-4-1BB bispecific antibodies were analyzed by FACS for their binding to HEK293 expressing 4-1BB. A total of 1×10 5 HEK293-4-1BB cells were incubated with 4-fold serial dilutions of antibodies starting from 100 nM in FACS buffer for 30 min at 4°C. After washing with FACS buffer, PE-conjugated anti-human IgG antibodies were added to each well and incubated for 30 min at 4°C. After washing, the MFI of PE was evaluated by a MACSQuant Analyzer 16. As shown in Figure 2, the tested anti-CLDN18.2-4-1BB bispecific antibodies showed a concentration-dependent binding ability to 4-1BB. 2.3 Protein complete kinetics for 4-1BB

[0096] Binding of B31, B16, B125, B164 and B210 antibodies to recombinant 4-1BB protein (human 4-1BB-his tag) was tested on Biacore using the capture method. B31, B16, B125, B164 and B210 mAbs were captured using a Protein A chip. Serial dilutions of human 4-1BB-his tag protein were injected over the captured antibody for 30 seconds at a flow rate of 30 μl / min. Antigen was left to dissociate for 360 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Figure 3 and Table 3 below. [Table 3] 2.4 Cross-binding activity to OX40 and CD40

[0097] To evaluate the cross-binding activity of clones B31, B16, B125, B164 and B210 with human CD40 and human OX40, chimeric mAbs from these clones were subjected to ELISA tests.

[0098] Briefly, microtiter plates were coated with 100 μl / well of human CD40 protein or human OX40 protein at 0.5 μg / ml in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Five-fold dilutions of B31, B16, B125, B164 and B210 antibodies starting at 3 μg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugated for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Figure 4, all these clones do not cross-link to human CD40 or human OX40.

[0099] Example 3 Functional activity of 4-1BB nanobody This example tested the functional activity of the antibody and shows that, unlike urelumab, the antibody did not activate 4-1BB signaling. Cell line-based functional characterization of 4-1BB monoclonal antibody

[0100] To evaluate the ability of 4-1BB monoclonal antibody to activate the 4-1BB signaling pathway, a commercially available 4-1BB NF-κB luciferase reporter system was used. In this assay, HEK-4-1BB NF-κB is used as the reporter cell line. The HEK-4-1BB NF-κB cell line has been genetically modified to stably express 4-1BB and luciferase downstream of a response element (Genomeditech, Cat. No. GM-C04832). Luciferase expression is induced by antibody binding to the 4-1BB receptor. Briefly, reporter cells were plated at 2.5×10 cells per well. 4 The antibodies were serially diluted and added to white 96-well assay plates at final concentrations ranging from 150 nM to 0.0000768 nM for the B31 molecule and 100 nM to 0.000381 nM for the B16, B125, B164 and B210 molecules. After 6 h of incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed by GraphPad software.

[0101] As shown in Figure 5, urelumab monoclonal antibody activated 4-1BB signaling in a dose-dependent manner, whereas B31, B16, B125, B164 and B210 antibodies did not boost 4-1BB induction in the same experimental setting.

[0102] Example 4 Functional activity of anti-claudin 18.2 / 4-1BB bispecific antibody In this example, a bispecific antibody incorporating an anti-4-1BB nanobody and also having specificity for claudin 18.2 was generated and tested. 4.1 Cell-line-based functional characterization of claudin 18.2-4-1BB bispecific antibodies

[0103] To evaluate the ability of the generated anti-claudin 18.2 / 4-1BB bispecific antibodies to activate the 4-1BB signaling pathway, a commercially available 4-1BB NF-κB luciferase reporter system was used. In this assay, H_TNFRSF9(4-1BB)NFκB-reporter Jurkat (Genomeditech, Cat. No. GM-C09468) was used as the effector cells and CHO-K1 with or without expressing claudin 18.2 was used as the target cells. The H_TNFRSF9(4-1BB)NFKB-reporter Jurkat cell line has been genetically modified to stably express 4-1BB and luciferase downstream of a response element. Luciferase expression is induced by antibody binding to the 4-1BB reporter. Briefly, 2.5 × 10 cells per well were cultured. 4 Effector cells at a density of 2.5 x 10 4 Target cells were co-cultured (E / T ratio = 1:1) in white 96-well plates. Antibodies were serially diluted 4-fold and added to white 96-well assay plates at final concentrations ranging from 100 nM to 0.000381 nM. After 6 h of incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0104] As shown in Figure 6, urelumab monoclonal antibody could boost 4-1BB signaling in both CHO-K1 and CHO-claudin18.2-overexpressing cells in a dose-dependent manner, while the activity of anti-claudin18.2 / 4-1BB bispecific antibodies B31-BiAb, B16-BiAb, B125-BiAb, B164-BiAb and B210-BiAb was dependent on the expression of claudin18.2 on the cells. 4.2 Activity of bispecific antibodies to enhance human peripheral blood mononuclear cell (PBMC) immune responses

[0105] To investigate the ability of claudin 18.2-4-1BB bispecific antibody to enhance the response of stimulated human PBMCs, IL-2 cytokine release was examined by the LANCE Ultra TR-FRET detection kit. Human PBMCs stimulated with 0.5 μg / ml human anti-CD3 antibody were used as effector cells. CHO-K1 expressing claudin 18.2 were used as target cells. Human PBMCs (1 × 10 5 ) to CHO-K1-claudin18.2 or parental CHO-K1 cells (2.5 × 10 4 ) and (E / T ratio = 4:1) in the presence of human anti-CD3 antibody. Five-fold serial dilutions of the bispecific antibody were added to the culture medium at final concentrations starting from 100 nM. After 48 h, the secreted levels of IL2 in the culture medium were measured using an IL-2 (human) LANCE Ultra TR-FRET detection kit (PerkinElmer). As shown in Figure 7, the bispecific antibody was exclusively able to activate PBMC responses in the presence of claudin 18.2-overexpressing cells.

[0106] Example 5 Humanization of B31 / B125 / B16 / B210 VHH antibodies The B31 / B125 / B16 / B210 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the amino acid sequence of the B31 / B125 / B16 / B210 VHH was compared to available databases of human Ig gene sequences to find the best overall match of the human germline Ig gene sequence. Then, the humanized variable domain sequence of B31 / B125 / B16 / B210 was designed; where CDRH1, H2 and H3 are on the framework sequences of the VH gene.

[0107] The amino acid and nucleotide sequences for portions of the humanized antibodies are listed in Table 4 below. [Table 4A] [Table 4B] [Table 4C-1] [Table 4C-2] [Table 4D]

[0108] Reversion mutations in B31 include 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 78M, 82(82A)D, and 94A. More specifically, VHH-v2 contained the reverse mutations 37Y, 44Q, 45R and 94A; VHH-v3 contained the reverse mutations 23A, 37Y, 44Q, 45R, 49A, 74N and 94A; VHH-v4 contained the reverse mutations 23A, 37Y, 44Q, 45R, 49A, 78M, 82(82A)D and 94A; VHH-v5 contained the reverse mutations 23A, 37Y, 40P, 41Q, 44Q, 45R, 49A, 74N, 82(82A)D and 94A; and VHH-v6 contained the reverse mutations 37Y, 44Q, 45R and 49A.

[0109] The back mutations of B16 include 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P and 94T. More specifically, VHH-v2 included back mutations 37F, 44E, 45R, 47G and 94T; VHH-v3 included back mutations 29L, 30D, 37F, 44E, 45R, 47G and 94T; VHH-v4 included back mutations 29L, 30D, 37F, 44E, 45R, 47G, 74A, 84P and 94T.

[0110] The back mutations of B125 include 37F, 43K, 44E, 45R, 47G, 74A, 84P and 94T. More specifically, VHH-v2 includes back mutations 37F, 44E, 45R, 47G and 94T; VHH-v3 includes back mutations 37F, 43K, 44E, 45R, 47G and 74A; VHH-v4 includes back mutations 37F, 43K, 44E, 45R, 47G, 74A and 94T; VHH-v5 includes back mutations 37F, 43K, 44E, 45R, 47G, 74A, 84P and 94T.

[0111] The back mutations of B210 include 27L, 29L, 37F, 44E, 45R, 47G, 89D and 94T. More specifically, VHH-v2 includes back mutations 37F, 44E, 45R, 47G and 94T; VHH-v3 includes back mutations 27L, 37F, 44E, 45R, 47G and 94T; VHH-v4 includes back mutations 27L, 29L, 37F, 44E, 45R, 47G and 94T; VHH-v5 includes back mutations 27L, 29L, 37F, 44E, 45R, 47G, 89D and 94T.

[0112] The humanized VHH genes were cloned into pcDNA3.4 vector and transfected into 293F cells for further analysis.

[0113] Example 6 Antigen-binding properties of humanized antibodies Full kinetic affinity of humanized antibodies by Biacore Binding of humanized antibodies to recombinant human 4-1BB protein (human 4-1BB-his tag) was tested on Biacore using the capture method. B31-V2, B31-V3, B31-V4, B31-V5 and B31-V6 were captured using a Protein A chip. Serial dilutions of human 4-1BB-his tag protein were injected over the captured antibody for 30 seconds at a flow rate of 10 μl / min. Antigen was left to dissociate for 360 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. The results are shown in Table 5 below. All humanized antibodies showed strong binding. [Table 5-1] [Table 5-2]

[0114] Example 7 Functional activity of humanized bispecific antibodies In this example, an anti-claudin18.2 / 4-1BB bispecific antibody was prepared and tested using the humanized 4-1BB nanobody. 7.1 Cell Line-Based Functional Characterization of Claudin 18.2-4-1BB Humanized Bispecific Antibody

[0115] To evaluate the ability of the claudin 18.2-4-1BB humanized bispecific antibody to activate the 4-1BB signaling pathway, a commercially available 4-1BB NF-κB luciferase reporter system was used. In this assay, H_TNFRSF9(4-1BB)NFκB-reporter Jurkat (Genomeditech, Cat. No. GM-C09468) was used as the effector cells and CHO-K1 with or without expressing claudin 18.2 was used as the target cells. The H_TNFRSF9(4-1BB)NFKB-reporter Jurkat cell line has been genetically modified to stably express 4-1BB and luciferase downstream of a response element. Luciferase expression is induced by antibody binding to the 4-1BB receptor. Briefly, 2.5 × 10 cells per well were cultured. 4 Effector cells at a density of 2.5 x 10 4 Target cells were co-cultured (E / T ratio = 1:1) in white 96-well plates. Antibodies were serially diluted 3-fold and added to white 96-well assay plates at final concentrations ranging from 100 nM to 0.005 nM. After 6 h of incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed using GraphPad software.

[0116] As shown in FIG. 8, the activity of the anti-claudin 18.2 / 4-1BB humanized bispecific antibody was comparable to that of the B31 chimeric antibody. 7.2 Activity of humanized bispecific antibodies to stimulate human peripheral blood mononuclear cell (PBMC) immune responses

[0117] To investigate the ability of claudin18.2-4-1BB humanized bispecific antibody to enhance the response of stimulated human PBMCs, IL-2 cytokine release was examined by the LANCE Ultra TR-FRET detection kit. Human PBMCs stimulated with 0.5 μg / ml human anti-CD3 antibody were used as effector cells. CHO-K1 expressing claudin18.2 were used as target cells. Human PBMCs (1 × 10 5) to CHO-K1-claudin18.2 or parental CHO-K1 cells (2.5 × 10 4 ) (E / T ratio = 4:1) in the presence of human anti-CD3 antibody. Five-fold serial dilutions of humanized bispecific antibodies were added to the culture medium at final concentrations starting from 100 nM. After 48 h, the secreted levels of IL2 in the culture medium were measured using an IL-2 (human) LANCE Ultra TR-FRET detection kit (PerkinElmer). As shown in Figure 9, the IL-2 secretion induced by the humanized bispecific antibodies was comparable to that of the B31 chimeric antibody.

[0118] Example 8 Tumor growth inhibition by anti-claudin 18.2 / 4-1BB bispecific antibody In this example, humanized mice expressing the extracellular domain of human 4-1BB were used to test the activity of bispecific antibodies in inhibiting tumor growth.

[0119] Mouse colon adenocarcinoma cells (MC38) were engineered to express human CLDN18.2. Humanized mice (h4-1BB) were implanted subcutaneously with MC38-hCLDN18.2 cells. Mice were administered the following antibodies intraperitoneally once a week for 3 weeks: 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), B210 anti-CLDN18.2 / 4-1BB bispecific antibody (1 mg / kg). Tumor volumes were monitored by caliper measurements twice weekly for the duration of the experiment. As shown in Figure 10, urelumab, B31, B16, B125, B164 and B210 are all capable of inhibiting tumor growth by 62.4%-96.9% TG1. [Table 6] * * *

[0120] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present 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 the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0121] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A single-domain antibody, or a polypeptide comprising the single-domain antibody, wherein the single-domain antibody has binding specificity to human 4-1BB protein and comprises complementarity-determining regions 1 (CDR1), 2 and 3. (a) CDR1 contains the amino acid sequence of SEQ ID NO: 15, CDR2 contains the amino acid sequence of SEQ ID NO: 16, and CDR3 contains the amino acid sequence of SEQ ID NO: 17; or (b) An antibody or polypeptide wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 18, CDR2 comprises the amino acid sequence of SEQ ID NO: 19, and CDR3 comprises the amino acid sequence of SEQ ID NO:

20.

2. The antibody or polypeptide according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 15, CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and CDR3 comprises the amino acid sequence of SEQ ID NO:

17.

3. The antibody or polypeptide according to claim 2, comprising the amino acid sequence of SEQ ID NO:

4.

4. The antibody or polypeptide according to claim 1, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 18, CDR2 comprises the amino acid sequence of SEQ ID NO: 19, and CDR3 comprises the amino acid sequence of SEQ ID NO:

20.

5. The antibody or polypeptide according to claim 4, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 5 and 35-38.

6. The antibody or polypeptide according to any one of claims 1 to 5, wherein the polypeptide is a chimeric antigen receptor (CAR) or a bispecific antibody having binding specificity to an antigen different from 4-1BB.

7. A bispecific antibody comprising the antibody according to any one of claims 1 to 5 and a secondary antibody or antigen-binding fragment having binding specificity to a target antigen other than 4-1BB.

8. The bispecific antibody according to claim 7, wherein the target antigen is selected from the group consisting of claudin 18.2, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin.

9. A polynucleotide encoding the antibody or polypeptide described in claim 1.

10. A cell comprising the polynucleotide described in claim 9.

11. A composition comprising the antibody or polypeptide described in claim 1 and a pharmaceutically acceptable carrier.

12. A composition for treating cancer in patients who require it, comprising the antibody or polypeptide described in claim 1.

13. Use of the antibody or polypeptide according to claim 1 for the preparation of a pharmaceutical for treating cancer.

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