Anti-4-1BB nanobody

Abstract

Anti-4-1BB nanobody and bispecific or multispecific antibodies incorporating the nanobody are provided. Methods of using the antibodies to treat and diagnose diseases such as cancer are also provided. Compositions comprising the antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided. Also provided are isolated cells comprising one or more polynucleotides encoding the antibody or polypeptide, one or more polynucleotides encoding the antibody or fragment thereof.

Description

[Background technology]

[0001] (background) 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) is a member of the TNF receptor superfamily (TNFRSF) and 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. Therefore, it has been suggested that 4-1BB is a promising target molecule in cancer immunology.

[0002] Single domain antibodies (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric antibody variable domain. Nanobodies made from camelids and certain other animals are also called VHH fragments. Like whole antibodies, nanobodies can selectively bind to a specific antigen. With a molecular weight of only 12 kDa to 15 kDa, single domain antibodies are considerably smaller than common antibodies (150 kDa to 160 kDa). Given their small size and single-chain nature, single domain antibodies may be particularly suitable for inclusion as fragments in other proteins (e.g., chimeric antigen receptors (CARs) and bispecific antibodies). Summary of the Invention [Means for solving the problem]

[0003] (overview) Anti-human 4-1BB Nanobodies suitable for inclusion in bispecific or trispecific antibodies are provided. Thus, in one embodiment of the disclosure, a single domain antibody or a polypeptide comprising said single domain antibody is provided, said single domain antibody having binding specificity to human 4-1BB protein, said single domain antibody comprising complementarity determining region 1 (CDR1), CDR2 and CDR3, said CDR1, CDR2 and CDR3 being selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 58 and the amino acid sequence of SEQ ID NO: 32; (b) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 59 and the amino acid sequence of SEQ ID NO: 38; (c) the amino acid sequence of SEQ ID NO: 17, the amino acid sequence of SEQ ID NO: 24 and the amino acid sequence of SEQ ID NO: 31; (d) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 32; (e) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 26 and the amino acid sequence of SEQ ID NO: 33; (f) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 34; (g) the amino acid sequence of SEQ ID NO: 18 (h) the amino acid sequence of SEQ ID NO:19, the amino acid sequence of SEQ ID NO:28 and the amino acid sequence of SEQ ID NO:35; (i) the amino acid sequence of SEQ ID NO:20, the amino acid sequence of SEQ ID NO:28 and the amino acid sequence of SEQ ID NO:35; (j) the amino acid sequence of SEQ ID NO:19, the amino acid sequence of SEQ ID NO:28 and the amino acid sequence of SEQ ID NO:35; (k) the amino acid sequence of SEQ ID NO:21, the amino acid sequence of SEQ ID NO:29 and the amino acid sequence of SEQ ID NO:36; (l) the amino acid sequence of SEQ ID NO:22, the amino acid sequence of SEQ ID NO:29 and the amino acid sequence of SEQ ID NO:36; (m) the amino acid sequence of SEQ ID NO:21, the amino acid sequence of SEQ ID NO:29 and the amino acid sequence of SEQ ID NO:36; (n) the amino acid sequence of SEQ ID NO:21, the amino acid sequence of SEQ ID NO:29 and the amino acid sequence of SEQ ID NO:36; (o) the amino acid sequence of SEQ ID NO:19, the amino acid sequence of SEQ ID NO:26 and the amino acid sequence of SEQ ID NO:33; (p) the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:28 and the amino acid sequence of SEQ ID NO:37;(q) the amino acid sequence of SEQ ID NO:23, the amino acid sequence of SEQ ID NO:30, and the amino acid sequence of SEQ ID NO:38; or (r) the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:28, and the amino acid sequence of SEQ ID NO:39;

[0004] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 18, the CDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the single domain antibody or polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 40-48 and 60-62.

[0005] In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO: 18, the CDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the single domain antibody or polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 49-57.

[0006] In some embodiments, the single domain antibody or polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:1-16.

[0007] Compositions comprising the antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided. Also provided are isolated cells comprising one or more polynucleotides encoding the antibody or polypeptide, one or more polynucleotides encoding the antibody or fragment thereof.

[0008] 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 the 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]

[0009] [Figure 1-1] 1A-1B show that the anti-41BB nanobodies bound to soluble human 4-1BB and soluble cynomolgus 4-1BB in a dose-dependent manner. [Figure 1-2] Same as above.

[0010] [Diagram 2] FIG. 2 shows that anti-41BB nanobodies were able to block the binding of the 4-1BB ligand to 4-1BB in a concentration-dependent manner.

[0011] [Figure 3-1] 3A-B show that in the presence of Fc cross-linking, anti-41BB nanobodies efficiently induced 4-1BB-mediated NFκB activity. [Figure 3-2] Same as above.

[0012] [Figure 4-1] Figures 4A-B show that anti-41BB humanized nanobodies derived from the parent clone VV02-1LP-263 bound to cell-surface human 4-1BB or cell-surface cynomolgus 4-1BB in a concentration-dependent manner. [Figure 4-2] Same as above.

[0013] [Diagram 5] FIG. 5 shows that the anti-41BB humanized nanobody derived from the parental clone VV02-1SP(1)-73 bound to cell surface human 4-1BB in a concentration-dependent manner.

[0014] [Figure 6] FIG. 6 shows that the optimized anti-4-1BB sequence maintained binding activity to 4-1BB-bearing CHO-K1 cells comparable to the parental sequence.

[0015] [Figure 7] FIG. 7 shows that the optimized anti-4-1BB-based bispecific antibodies had comparable potency to the parental antibody TAA-263-1-3 in inducing 4-1BB activation in the presence of target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] (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 refer to one or more antibodies. Thus, the terms "a" or "an," "one or more," and "at least one" can be used interchangeably herein.

[0017] 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" with another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and percent homology or percent sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Preferably, default parameters are used for alignment. One alignment program is BLAST, which uses default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above specified percentage of homology and encode polypeptides having the same or similar biological activity.

[0018] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity with the nucleotide sequence of the nucleic acid or its complement. A double-stranded nucleic acid homolog is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology with its complement or its complement. In one embodiment, a nucleic acid homolog is hybridizable with the nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a certain 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%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%. In some embodiments, the equivalent polypeptide or equivalent polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof when compared to the reference polypeptide or reference polynucleotide. In some embodiments, the equivalent sequence retains an activity (eg, epitope binding) or structure (eg, salt bridges) of the reference sequence.

[0019] 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 may be a whole antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule, including at least a portion of an immunoglobulin molecule, that has the biological activity of binding to its antigen. Examples include, but are not limited to, the complementarity determining region (CDR) of the heavy or light chain or a ligand-binding portion thereof, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0020] The term "antibody fragment" or "antigen-binding fragment" as used herein is 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 to 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 engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0021] "Single-chain variable fragment" or "scFv" refers to a fragment of the heavy chain variable region of an immunoglobulin (V H ) and the light chain variable region (V L In some embodiments, the regions are linked by a short linker peptide of 10 amino acids to about 25 amino acids. The linker can be rich in glycine for flexibility, rich in serine or threonine for solubility, and rich in V. H N-terminus of V L The ScFv molecule can be either linked to the C-terminus of the ScFv polypeptide or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0022] The term antibody includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses therein (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Subclasses (isotypes) of immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc.) are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art given the scope of this disclosure, and thus are within the scope of this disclosure. All classes of immunoglobulins are clearly within the scope of this disclosure, and the following discussion will generally relate to immunoglobulin molecules of the IgG class. 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.

[0023] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present 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 containing either the VK domain or the VH, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies, including, for example, anti-Id antibodies against the LIGHT antibody disclosed herein. The immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecule.

[0024] By "specifically bind" or "have specificity for", it is generally meant that an antibody binds to an epitope through its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. In accordance with this definition, an antibody is said to "specifically bind" to an epitope when the antibody binds to the epitope through its antigen-binding domain more readily than the antibody would bind to a random and unrelated epitope. The term "specificity" is used herein to quantify 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 antibody "A" has for related epitope "D".

[0025] 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 (e.g., the progression of cancer). Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or alleviation of disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0026] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals, sport animals, or pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc.).

[0027] As used herein, phrases such as "to a patient in need of treatment" or "subject in need of treatment" include subjects (e.g., mammalian subjects) who would benefit from administration of an antibody or composition of the disclosure for use, e.g., for detection, diagnostic procedures, and / or treatment.

[0028] (Anti-4-1BB nanobody) The present disclosure provides nanobodies (including humanized nanobodies) against human 4-1BB protein. These antibodies have high binding affinity to 4-1BB and can effectively block the interaction between 4-1BB and its ligand. As shown in Example 4, these antibodies showed a strong ability to induce 4-1BB-mediated NF-κB activity only in the presence of Fc cross-linking. Thus, these antibodies are non-agonistic antibodies that do not activate 4-1BB signaling by themselves. However, when combined with a second antibody, the resulting bispecific antibody can activate 4-1BB signaling in the presence of the target antigen of the second antibody. In other words, the present nanobodies are particularly suitable for development into bispecific or multispecific antibodies.

[0029] Thus, in one embodiment of the disclosure, a single domain antibody or a polypeptide comprising this single domain antibody is provided, which comprises a CDR1, a CDR2 and a CDR3, each of which has the CDR1, CDR2 and CDR3 sequence of any one of the antibodies in Table 1.

[0030] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-317 (SEQ ID NO:1). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:17, the amino acid sequence of SEQ ID NO:24, and the amino acid sequence of SEQ ID NO:31, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0031] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-263 (SEQ ID NO:2). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:25, and the amino acid sequence of SEQ ID NO:32, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:2.

[0032] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-355 (SEQ ID NO: 3). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 26, and the amino acid sequence of SEQ ID NO: 33, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3.

[0033] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-3LP-66 (SEQ ID NO:4). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:27, and the amino acid sequence of SEQ ID NO:34, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:4.

[0034] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-265 (SEQ ID NO:5). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:28, and the amino acid sequence of SEQ ID NO:35, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:5.

[0035] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-2LP-13 (SEQ ID NO:6). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:19, the amino acid sequence of SEQ ID NO:28, and the amino acid sequence of SEQ ID NO:35, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:6.

[0036] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-3LP-142 (SEQ ID NO:7). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:20, the amino acid sequence of SEQ ID NO:28, and the amino acid sequence of SEQ ID NO:35, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:7.

[0037] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-3LP-83 (SEQ ID NO: 8). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 35, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:8.

[0038] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-417 (SEQ ID NO:9). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:21, the amino acid sequence of SEQ ID NO:29, and the amino acid sequence of SEQ ID NO:36, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:9.

[0039] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-422 (SEQ ID NO: 10). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 10.

[0040] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-238 (SEQ ID NO: 11). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 11.

[0041] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1LP-288 (SEQ ID NO: 12). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0042] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1SP(1)-168 (SEQ ID NO: 13). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 26, and the amino acid sequence of SEQ ID NO: 33, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 13.

[0043] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1SP(1)-55 (SEQ ID NO: 14). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 37, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 14.

[0044] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1SP(1)-73 (SEQ ID NO: 15). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 23, the amino acid sequence of SEQ ID NO: 30, and the amino acid sequence of SEQ ID NO: 38, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 15.

[0045] In some embodiments, the CDR1, CDR2, and CDR3 are the CDR1, CDR2, and CDR3 of antibody VV02-1SP(1)-358 (SEQ ID NO: 16). In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 39, respectively. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 16.

[0046] Humanized antibodies (e.g., the humanized antibodies provided in SEQ ID NOs: 40-48 and 60-62 for antibody VV02-1LP-263, and the humanized antibodies provided in SEQ ID NOs: 49-57 for antibody VV02-1SP(1)-73) are also provided. For VV02-1LP-263 (CDR1 in SEQ ID NO: 18, CDR2 in SEQ ID NO: 25, and CDR3 in SEQ ID NO: 32), the CDR2 may incorporate either or both of the mutations (D54G and D61E in Kabat numbering). Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 58, and the amino acid sequence of SEQ ID NO: 32, respectively. In some embodiments, the antibody comprises any one of SEQ ID NOs: 40-48 and 60-62. In some embodiments, the antibody comprises the described CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 40-48 and 60-62.

[0047] Of these humanized sequences, 263 huNb-1-3_1 (SEQ ID NO: 60), 263 huNb-1-3_2 (SEQ ID NO: 61), and 263 hnNb-1-3_3 (SEQ ID NO: 62) are further optimized humanized sequences, which have been shown to be suitable for further clinical development (Example 7 and Figures 6-7). In some embodiments, the antibody comprises SEQ ID NO: 60. In some embodiments, the antibody comprises the CDR1, CDR2, and CDR3 as set forth and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 60. In some embodiments, the antibody comprises SEQ ID NO: 61. In some embodiments, the antibody comprises the CDR1, CDR2, and CDR3 as set forth and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 61. In some embodiments, the antibody comprises SEQ ID NO: 62. In some embodiments, the antibody comprises the described CDR1, CDR2 and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:62.

[0048] For VV02-1SP(1)-73 (CDR1 in SEQ ID NO:23, CDR2 in SEQ ID NO:30, and CDR3 in SEQ ID NO:38), one mutation (N31S in Kabat numbering) may be incorporated in the CDR1, or any of two mutations (D54S and D61E in Kabat numbering) may be incorporated in the CDR2, respectively. Thus, in some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequence of SEQ ID NO:18, the amino acid sequence of SEQ ID NO:59, and the amino acid sequence of SEQ ID NO:38, respectively. In some embodiments, the antibody comprises any one of SEQ ID NOs:49-57. In some embodiments, the antibody comprises the described CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs:49-57.

[0049] In some embodiments, also provided is a Nanobody or a polypeptide comprising a Nanobody, which comprises a CDR1 having an amino acid sequence selected from SEQ ID NOs: 17-23, a CDR2 having an amino acid sequence selected from SEQ ID NOs: 24-30 or 58-59, and a CDR3 having an amino acid sequence selected from SEQ ID NOs: 31-39.

[0050] In some embodiments, a Nanobody or a polypeptide comprising the Nanobody is also provided, which comprises a CDR1 having an amino acid sequence selected from SEQ ID NOs: 21-22, a CDR2 having the amino acid sequence of SEQ ID NO: 29, and a CDR3 having the amino acid sequence of SEQ ID NO: 36.

[0051] 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.

[0052] Compositions comprising the antibody or polypeptide and a pharma- ceutically acceptable carrier are also provided.

[0053] It is also understood by those skilled in the art that the antibody disclosed herein may be modified to have a different amino acid sequence from the naturally occurring binding polypeptide from which it is derived.For example, the polypeptide sequence or amino acid sequence derived from a specified protein may be similar, for example, have a certain percent identity to the starting sequence, for example, it may 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 specified CDR sequence.

[0054] 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).

[0055] Bispecific and multispecific antibodies are also provided, which contain 1 unit, 2 units, 3 units or 4 units of a single domain anti-4-1BB antibody disclosed herein and one or more other specificities (not 4-1BB).

[0056] Bispecific and Multispecific Antibodies, and Chimeric Antigen Receptors (CARs) 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 enhanced therapeutic index of these antibodies and their small size.

[0057] Thus, in one embodiment, a bispecific antibody is provided comprising an anti-4-1BB Nanobody or antigen-binding fragment thereof of the present disclosure and a second antibody or antigen-binding fragment thereof having binding specificity for a target antigen that is not 4-1BB. In some embodiments, a third or fourth specificity is further included.

[0058] The non-4-1BB target antigen, in some embodiments, is a tumor antigen. Many tumor antigens are known in the art, and novel tumor antigens can be readily 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, Nectin4, 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, Integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

[0059] Also provided are chimeric antigen receptors (CARs) comprising the nanobodies of the present disclosure. In the CAR, the nanobody can serve as an antigen recognition domain. Furthermore, in some embodiments, the CAR also comprises an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

[0060] The hinge (also called the spacer) is a small structural domain that exists between the antigen recognition region and the outer membrane of the cell. A suitable hinge increases the flexibility of the head of the scFv receptor, reducing the spatial constraint between the CAR and its target antigen. Exemplary hinge sequences are based on the membrane proximal regions from immune molecules (e.g., IgG, CD8, and CD28).

[0061] The transmembrane domain is a structural component that consists of a hydrophobic α-helix that spans the cell membrane. It anchors the CAR to the plasma membrane and bridges its extracellular hinge domain and antigen recognition domain with the intracellular signaling region. Typically, a transmembrane domain derived from the membrane-proximal component of the internal domain (e.g., CD28 transmembrane domain) can be used.

[0062] The intracellular T cell signaling domain resides in the endodomain of the receptor inside the cell. After antigen is bound to the external antigen recognition domain, the CAR receptors cluster together and transmit an activation signal. The internal cytoplasmic tail of the receptor then perpetuates the signaling inside the T cell. To mimic this process, the cytoplasmic domain of CD3-zeta is commonly used as the main CAR endodomain component.

[0063] T cells also require costimulatory molecules in addition to CD3 signaling to persist after activation. In some embodiments, the internal domain of the CAR receptor also contains one or more chimeric domains derived from costimulatory proteins (e.g., CD28, CD27, CD134 (OX40), and CD137 (4-1BB)).

[0064] Polynucleotides encoding the antibodies and methods for preparing the antibodies 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 chain variable region and the entire light chain variable region of the present antigen-binding polypeptide, its variant or derivative, on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotide of the present disclosure may encode a portion of the heavy chain variable region and a portion of the light chain variable region of the present antigen-binding polypeptide, its variant or derivative, on the same polynucleotide molecule or on separate polynucleotide molecules.

[0065] Methods for making antibodies are well known in the art and 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 particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous locus has been disabled.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 by reference in their entirety.

[0066] (Cancer Treatment) As described herein, the antibodies, bispecific antibodies, polypeptides, variants or derivatives of the disclosure may be used in certain treatment and diagnostic methods.

[0067] The present disclosure further relates to antibody-based therapeutic methods for treating one or more of the disorders or conditions described herein, which comprise administering an antibody of the present disclosure to a patient (e.g., an animal, a mammal, and a human).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).

[0068] In some embodiments, a method for treating cancer in a patient in need thereof is provided. The method, in one embodiment, comprises 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.

[0069] Cell therapy (e.g., chimeric antigen receptor (CAR) T cell therapy or chimeric antigen receptor (CAR) NK cell therapy) is also provided in the present disclosure. Suitable cells can be used that are placed in contact with the antibody or CAR of the present disclosure (or alternatively, engineered to express the antibody or CAR of the present disclosure). Once such contacted or engineered, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient can have any of the types of cancer disclosed herein. The cells (e.g., 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 a combination thereof.

[0070] In some embodiments, the cells are isolated from the cancer patient themselves. 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 may be minimized.

[0071] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed using the antibodies or variants or derivatives thereof of the present disclosure include leukemias (acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, (lymphangioendotheliosarcoma), 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, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal These include, but are not limited to, progression and metastasis of malignant diseases and related disorders, such as, but not limited to, cancer, Wilms' tumor, cervical cancer, testicular cancer, 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.

[0072] (Diagnostic Method) Overexpression of 4-1BB has been observed in certain tumor samples, and patients with cells that overexpress 4-1BB may respond to treatment with the anti-4-1BB antibody of the present disclosure. Thus, the antibody of the present disclosure may also be used for diagnostic or prognostic purposes.

[0073] A sample preferably containing cells can be obtained from a patient, and the patient can be a cancer patient or a patient who wants to be diagnosed. The cells can be tumor tissue or tumor block, blood sample, urine sample, or any sample cell from the patient. The sample can be pre-treated as necessary, and 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 the sample, utilizing anti-4-1BB antibody.

[0074] The presence of 4-1BB protein in the sample (along with the amount or concentration as appropriate) can be used for cancer diagnosis, as an indication that the patient is suitable for treatment with the antibody, or as an indication that the patient has responded (or has not) to cancer treatment. For prognostic methods, the detection can be performed once, twice, or more times at a particular stage to initiate cancer treatment and indicate the progress of the treatment.

[0075] (composition) The present disclosure also provides a pharmaceutical composition. Such a composition comprises an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer drug (e.g., an immune checkpoint inhibitor).

[0076] In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, more particularly humans. Moreover, a "pharmaceutically acceptable carrier" is generally a non-toxic, solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0077] 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 oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used 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, dried skim milk, 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. Antimicrobial agents (e.g., benzyl alcohol or methyl parabens); antioxidants (e.g., ascorbic acid or sodium bisulfite); chelating agents (e.g., ethylenediaminetetraacetic acid); and tonicity adjusters (e.g., 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 may be formulated as suppositories, with traditional binders and carriers (e.g., triglycerides). Oral formulations may contain standard carriers (e.g., pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like). Examples of suitable pharmaceutical carriers are described in EW Martin, Remington's Pharmaceutical Sciences, incorporated herein by reference. Such compositions 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 should suit the mode of administration.The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0078] In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container (e.g., an ampoule or sachette) indicating the quantity of active substance. When the composition is to be administered by injection, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is 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

[0079] (Example) Example 1: Preparation of nanobodies against human 4-1BB This example describes the generation of nanobodies against the human 4-1BB protein.

[0080] Llamas were immunized with recombinant ECD of human 4-1BB fused to human immunoglobulin Fc domain. Llamas whose sera contained sufficient titers of anti-4-1BB antibodies were selected for the generation of a phage library. Briefly, lymphocytes were isolated from peripheral blood collected from immunized llamas. Their RNA was extracted and cDNAs encoding the VHH domains were amplified by PCR and used for the construction of an M13 phage display-based nanobody library. Several rounds of panning were applied to screen the phage library expressing anti-4-1BB nanobodies.

[0081] All the positive clones were screened through ELISA and FACS assays prior to sequencing. Based on their sequence diversity, 16 unique clones were selected. Their sequences are shown in Table 1. The anti-4-1BB nanobodies fused with human IgG1 Fc fragment containing the N297A mutation at the C-terminus were characterized for their specificity and activity through a series of functional assays (including binding, ligand competition and 4-1BB signal activation), resulting in the identification of lead nanobodies for further humanization. (Table 1. Sequences of 16 selected unique clones) [Table 1-1] [Table 1-2]

[0082] Their CDR sequences are summarized below in Table 1A. (Table 1A. Sequences of 16 selected unique clones) [Table 1A]

[0083] Antibodies VV02-1LP-417, VV02-1LP-422, VV02-1LP-238, and VV02-1LP-288 appear to share very similar CDRs, while the remaining antibodies also have homologous CDRs.

[0084] Example 2. Soluble 4-1BB binding properties of anti-4-1BB nanobodies This example tested the binding properties of the resulting anti-4-1BB nanobodies to human 4-1BB protein and cynomolgus monkey 4-1BB protein by ELISA assay.

[0085] His-tagged 4-1BB was coated overnight at 2 μg / ml and then blocked with 2% BSA in PBS. Serially diluted anti-4-1BB nanobodies were incubated with the coated antigen for 1 h at room temperature with a reference antibody "Reference Monoclonal Antibody (Ref mAb)". The resulting plates were washed with PBS / T and incubated with goat anti-human IgG-HRP for 1 h at room temperature. The plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. The results of these ELISA assays are shown in Figure 1 and Table 2, which shows the EC20 binding to human 4-1BB protein and cynomolgus 4-1BB protein. 50 Shows. Table 2. Binding EC for human 4-1BB protein and cynomolgus monkey (Cyno) 4-1BB protein 50( nM)) [Table 2]

[0086] Example 3. Blockade of 4-1BB / 4-1BB Ligand Interaction by Anti-41BB Nanobodies In this example, anti-41BB nanobodies were examined for their ability to block the binding of 4-1BB to its ligand.

[0087] CHO-K1 cells overexpressing human 4-1BB were incubated with biotinylated human 4-1BB ligand (0.3 μg / ml) in the presence of serially diluted anti-41BB nanobodies for 1 h at 4° C., with the reference monoclonal antibody (Ref mAb) used as a reference antibody. The cells were then incubated with Alexa Fluor 633-conjugated streptavidin. Binding was measured using an Agilent flow cytometer. As shown in FIG. 2 and Table 3, all of the tested nanobodies were able to block the binding of the 4-1BB ligand to 4-1BB in a concentration-dependent manner, while the reference monoclonal antibody (Ref mAb) had no such effect. (Table 3. Competitive IC for 4-1BB / 4-1BB ligand interaction 50( nM)) [Table 3]

[0088] Example 4. Functional characterization of anti-41BB nanobodies in the Jurkat-41BB NFκB reporter assay This example evaluated the functional properties of anti-41BB nanobodies in a 4-1BB reporter assay.

[0089] In this assay, the effector cells were 4-1BB NFκB reporter Jurkat cell line, which stably expresses human 4-1BB and has an NFκB luciferase reporter construct integrated into its genome. After 4-1BB activation, endogenous NFκB transcription factors bind to DNA response elements and induce transcription of the luciferase gene. The protein product of the luciferase gene is then quantified by measuring its luminescence signal. The 4-1BB NFκB reporter Jurkat cell line was co-cultured with the target cell line CHO-K1 or the FcγRIIB-CHO-K1 cell line overexpressing human Fcγ receptor II B. Antibodies were serially diluted and added to white 96-well assay plates. After 16 hours of incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured by a microplate reader.

[0090] As shown in Figure 3 and Table 4, the tested 4-1BB nanobodies induced 4-1BB-mediated NF-κB activity in the presence of Fc cross-linking and EC 50 The range was 0.002 nM to 0.081 nM. However, in the absence of Fc cross-linking, the antibodies tested were unable to activate NF-κB signaling. (Table 4. EC in Jurkat-41BB NFκB reporter assay) 50( nM)) [Table 4]

[0091] Example 5. Anti-41BB Nanobody Humanization Humanization of the lead nanobodies (VV02-1LP-263 and VV02-1SP(1)-73) was performed by CDR grafting and backmutation strategy. Their humanized sequences are shown in Table 5. Table 5. Sequences of humanized lead nanobodies. [Table 5-1] [Table 5-2]

[0092] For VV02-1LP-263, two mutations (D54G and D61E in Kabat numbering) were introduced in CDR2 to improve developability. For VV02-1SP(1)-73, one mutation (N31S in Kabat numbering) was introduced in CDR1 and two mutations (D54S and D61E in Kabat numbering) were introduced in CDR2 to improve developability. A comparison of their sequences is shown in Table 5A below. (Table 5A. Updated CDRs) [Table 5A]

[0093] Example 6. Cell surface 4-1BB binding properties of anti-4-1BB humanized nanobodies To evaluate the antigen-binding properties in a cell-based setting, the 4-1BB humanized nanobodies were analyzed by FACS for their binding to 4-1BB overexpressed on CHO-K1 cells.

[0094] Briefly, CHO-K1 cells overexpressing human 4-1BB or cynomolgus 4-1BB were incubated with serially diluted anti-4-1BB nanobodies for 30 min at 4°C. The cells were then incubated with Alexa Fluor 633-conjugated anti-human Fc secondary antibody. Binding was measured on an Agilent flow cytometer. The results showed that all tested humanized nanobodies exhibited typical sigmoidal binding behavior to cell surface human 4-1BB and cell surface cynomolgus (cyno) 4-1BB (Figures 4 and 5). Their binding EC 50 are shown in Tables 6 and 7 as appropriate. Table 6. Binding EC of humanized nanobodies derived from parent clone VV02-1LP-263 on cell surface 4-1BB 50 (nM) [Table 6] Table 7. Binding EC of humanized nanobodies derived from parent clone VV02-1SP(1)-73 on cell surface 4-1BB 50 (nM) [Table 7]

[0095] Example 7. Sequence optimization of anti-4-1BB nanobody by site-specific mutagenesis To further increase the developability of these candidates, anti-4-1BB nanobody VV02-1LP-263 huNb_1_3 was selected and optimized by site-directed mutagenesis, the optimized sequence of which is shown in Table 8. (Table 8. Optimized sequence of VV02-1LP-263 huNb_1_3) [Table 8]

[0096] To evaluate the feasibility of the optimized anti-4-1BB sequence for constructing bispecific antibodies, the optimized anti-4-1BB was fused to the heavy chain C-terminus of an IgG1 antibody targeting a tumor-associated antigen (TAA) with an N297A mutation in the Fc fragment via a G4S linker. Then, the light chain expression vector and the heavy chain expression vector were co-transfected into CHO-K1 cells. After transient transfection, the bispecific antibodies were purified from the culture medium by protein A affinity chromatography. Fully qualified antibodies were applied for in vitro characterization, including cell-based 4-1BB binding, 4-1BB activation and developability evaluation.

[0097] (Cell-based 4-1BB binding) The optimized 4-1BB nanobody-based bispecific antibodies were analyzed for their binding to 4-1BB overexpressed on CHO-K1 cells according to the protocol described above. As shown in Figure 6, the optimized anti-4-1BB sequences maintained binding activity comparable to the parental sequence to 4-1BB-bearing CHO-K1 cells.

[0098] (TAA-dependent 4-1BB activation) A classical reporter gene assay was used to evaluate the ability of anti-TAA-4-1BB bispecific antibodies to activate 4-1BB signaling. In this assay, engineered Jurkat cells stably expressing 4-1BB and carrying an NF-κB luciferase reporter construct integrated into their genome were used as effector cells. CHO-K1 cells engineered to overexpress TAA or blank CHO-K1 cells were used as target cells. These two types of cells were co-incubated with various concentrations of antibodies overnight at 37°C in a 5% CO2 incubator. Then, luciferase substrate was added and the luminescence intensity was determined by a microplate reader.

[0099] As shown in FIG. 7, these optimized anti-4-1BB-based bispecific antibodies had comparable potency to the parent antibody TAA-263-1-3 in inducing 4-1BB activation in the presence of target cells.

[0100] (Thermal stability evaluation) Forced degradation studies were performed to evaluate the thermal stability of the TAA-4-1BB bispecific antibodies. The antibodies were incubated for 2 weeks at 40° C. in a buffer containing 20 mM His, 6% sucrose + 0.02% PS80 pH 6.0. Representative quality attributes (particularly aggregation and fragmentation) were monitored at 2 weeks using SEC-HPLC. Bispecific antibodies derived from the parent anti-4-1BB sequence showed poor thermal stability as evidenced by aggregation and fragmentation during incubation. In contrast, the new TAA-4-1BB bispecific antibodies based on the optimized 4-1BB sequence showed acceptable thermal stability for 2 weeks at 40° C., indicating reasonable promising development potential moving forward (Table 9). Table 9. SEC-HPLC characterization of TAA-4-1BB bispecific antibody in forced degradation studies. [Table 9] * * *

[0101] The present disclosure should not be limited in scope by the described specific embodiments, which are intended as individual 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 is clear to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to include modifications and variations of the present disclosure, provided that the modifications and variations are within the scope of the appended claims and their equivalents.

[0102] 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 said single domain antibody, wherein said single domain antibody has binding specificity for human 4-1BB protein, said single domain antibody comprising complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein said CDR1, CDR2 and CDR3 are, respectively: (a) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 58, and the amino acid sequence of SEQ ID NO: 32; (b) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 59, and the amino acid sequence of SEQ ID NO: 38; (c) the amino acid sequence of SEQ ID NO: 17, the amino acid sequence of SEQ ID NO: 24, and the amino acid sequence of SEQ ID NO: 31; (d) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 25, and the amino acid sequence of SEQ ID NO: 32; (e) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 26, and the amino acid sequence of SEQ ID NO: 33; (f) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 27, and the amino acid sequence of SEQ ID NO: 34; (g) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 35; (h) the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 35; (i) the amino acid sequence of SEQ ID NO: 20, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 35; (j) the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 35; (k) the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36; (l) the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36; (m) the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36; (n) the amino acid sequence of SEQ ID NO: 21, the amino acid sequence of SEQ ID NO: 29, and the amino acid sequence of SEQ ID NO: 36; (o) the amino acid sequence of SEQ ID NO: 19, the amino acid sequence of SEQ ID NO: 26, and the amino acid sequence of SEQ ID NO: 33; (p) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 37; (q) the amino acid sequence of SEQ ID NO: 23, the amino acid sequence of SEQ ID NO: 30, and the amino acid sequence of SEQ ID NO: 38; or (r) the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of SEQ ID NO: 39 A single domain antibody or a polypeptide comprising said single domain antibody, comprising:

2. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 18, the CDR2 comprises the amino acid sequence of SEQ ID NO: 58, and the CDR3 comprises the amino acid sequence of SEQ ID NO:

32.

3. The antibody or polypeptide of claim 2, comprising the amino acid sequence of any one of SEQ ID NOs: 40-48 and 60-62.

4. The antibody or polypeptide of claim 1, wherein the CDR1 comprises the amino acid sequence of SEQ ID NO: 18, the CDR2 comprises the amino acid sequence of SEQ ID NO: 59, and the CDR3 comprises the amino acid sequence of SEQ ID NO:

38.

5. The antibody or polypeptide of claim 4, comprising the amino acid sequence of any one of SEQ ID NOs: 49 to 57.

6. The antibody or polypeptide of claim 1, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 16.

7. The antibody or polypeptide of claim 1, wherein the polypeptide is a chimeric antigen receptor (CAR) having binding specificity for an antigen different from that of 4-1BB or a bispecific antibody having binding specificity for an antigen different from that of 4-1BB.

8. A bispecific antibody comprising the antibody of claim 1 and a second antibody or antigen-binding fragment having binding specificity for a target antigen other than 4-1BB.

9. One or more polynucleotides encoding the antibody or polypeptide of any one of claims 1 to 8.

10. A cell comprising the polynucleotide of claim 9.

11. A composition comprising the antibody or polypeptide of any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

12. A composition for treating cancer in a patient in need thereof, comprising an antibody or polypeptide according to any one of claims 1 to 8.

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