CEACAM5-dependent 4-1BB agonistic bispecific antibody

Abstract

Bispecific or multispecific antibodies are provided that include an anti-CEACAM5 moiety and one or more anti-4-1BB nanobodies whose agonistic activity depends on the presence of the CEACAM5 antigen. Such bispecific or multispecific antibodies are safe, effective in treating cancer, and have long-term protective immune memory against tumors. Methods of using the antibodies to treat diseases such as cancer are also provided. Bispecific antibodies that target CEACAM5 and 4-1BB are provided.

Description

[Background technology]

[0001] background 4-1BB (CD137, tumor necrosis factor receptor superfamily 9) 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. Activation of 4-1BB enhances immune responses and eliminates tumors in mice.

[0002] Commonly known anti-4-1BB antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513), are agonistic antibodies. These agonistic antibodies are associated with dose-limiting on-target toxicity. Bispecific antibodies containing a conditional agonist anti-4-1BB moiety and a second moiety that targets a tumor-associated antigen (TAA) have advantages over antibodies that use an agonistic anti-4-1BB moiety. The former are considered much safer because their anti-4-1BB moiety is only active in the presence of a TAA.

[0003] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as Cluster of Differentiation 66e (CD66e), is a TAA suitable for cancer immunotherapy. CEACAM5 is a member of the carcinoembryonic antigen (CEA) gene family. CEACAM5 is a biomarker for many types of malignant tumors, including colorectal cancer. The primary function of CEACAM5 in embryonic intestinal and colon tumors is epithelial cell-cell adhesion. CEACAM5 plays an important role in differentiation and inhibition of apoptosis in colonocytes. High CEACAM5 expression is positively associated with CD133-positive colorectal cancer stem cells. Summary of the Invention

[0004] overview A bispecific antibody targeting CEACAM5 and 4-1BB is provided. The anti-4-1BB portion comprises one or more anti-4-1BB nanobodies that do not have agonist activity by themselves. In the presence of a CEACAM5 antigen, such as a tumor cell expressing CEACAM5, the anti-4-1BB antibody is activated and exerts its immunomodulatory activity.

[0005] According to one embodiment of the present disclosure, there is provided a bispecific antibody comprising one or more anti-4-1BB single domain antibodies (VHHs) and an anti-CEACAM5 antibody or antigen-binding fragment thereof, wherein the one or more anti-4-1BB single domain antibodies each comprise complementarity determining region 1 (VHH CDR1), VHH CDR2, and VHH CDR3, wherein (a) VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, and VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 22 or 24; and VHH CDR3 comprises the amino acid sequence of SEQ ID NO: 23; or (b) VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 21 or 27; and VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 25 or 28, and VHH CDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0006] In some embodiments, the VHH CDR1 comprises the amino acid sequence of SEQ ID NO:21, the VHH CDR2 comprises the amino acid sequence of SEQ ID NO:22, and the VHH CDR3 comprises the amino acid sequence of SEQ ID NO:23.

[0007] In some embodiments, the one or more anti-4-1BB single domain antibodies each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-10 and 37-38. In some embodiments, the one or more anti-4-1BB single domain antibodies each comprise the amino acid sequence of SEQ ID NO: 37.

[0008] In some embodiments, the bispecific antibody further comprises an IgG Fc fragment. In some embodiments, the Fc fragment is N-terminal to the two anti-4-1BB single domain antibodies, optionally via a peptide linker. In some embodiments, the Fc fragment is C-terminal to the two anti-CEACAM5 fragments.

[0009] In some embodiments, the bispecific antibody comprises a full-size IgG anti-CEACAM5 antibody and two anti-4-1BB single domain antibodies fused to the C-terminus of the anti-CEACAM5 antibody. In some embodiments, the anti-CEACAM5 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 36.

[0010] In some embodiments, the anti-CEACAM5 antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO:29 and a VL comprising the amino acid sequence of SEQ ID NO:30.

[0011] Also provided is a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a bispecific antibody of the present disclosure, in some embodiments, the cancer is characterized by expression of CEACAM5. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a schematic diagram of the anti-CEACAM5-4-1BB bispecific antibody.

[0013] [Figure 2] Figures 2A-2B show that anti-41BB humanized nanobodies derived from parental clones VV02-1LP-263 and VV02-1SP(1)-73 bound to cell surface human 4-1BB in a concentration-dependent manner.

[0014] [Figure 3] FIG. 3A shows that all bispecific antibodies bound to human 4-1BB-overexpressing CHO-K1 cells in a concentration-dependent manner.

[0015] FIG. 3B shows that all bispecific antibodies bound to MKN-45 in a concentration-dependent manner.

[0016] [Figure 4] FIG. 4 shows that all bispecific antibodies activated CEACAM5-dependent 4-1BB signaling in a concentration-dependent manner in the presence of MKN45, which has endogenous CEACAM5 expression.

[0017] [Figure 5] FIG. 5 shows that the optimized bispecific antibody exhibited concentration-dependent binding activity to human 4-1BB-overexpressing CHO-K1 cells.

[0018] [Figure 6] FIG. 6 shows that the optimized bispecific antibody exhibited binding affinity to CEACAM5-bearing MM.1S cells similar to that of the parent bispecific antibody.

[0019] [Figure 7] FIG. 7 shows that the optimized bispecific antibody efficiently induced 4-1BB activation in the presence of target cells MKN-45.

[0020] [Figure 8] FIG. 8 shows that IMMU130-263-1-3 — 1 specifically bound to human CEACAM5 protein in a concentration-dependent manner, while there was no cross-reactivity to cynomolgus monkey CEACAM5.

[0021] [Figure 9]FIG. 9 shows that all tested antibodies exhibited typical sigmoidal binding behavior to CEACAM5_HEK293, MKN45, and LS174T cells, while showing no nonspecific binding activity to blank HEK-293 cells.

[0022] [Figure 10] FIG. 10 shows that bispecific antibody IMMU130-263-1-3_1 bound efficiently to human and cynomolgus 4-1BB-overexpressing CHO-K1 cells in a concentration-dependent manner.

[0023] [Figure 11] FIG. 11 shows that bispecific antibody IMMU130-263-1-3_1 induced significant 4-1BB activation in the presence of target cells with CEACAM expression.

[0024] [Figure 12] FIG. 12 shows that bispecific antibody IMMU130-263-1-3_1 induced significant IL-2 and IFN-γ production by primary PBMCs in the presence of target cells overexpressing CEACAM5.

[0025] [Figure 13] FIG. 13 shows that bispecific antibody IMMU130-263-1-3_1 demonstrated strong anti-tumor efficacy in a human 4-1BB transgenic mouse tumor model. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description definition It should be noted that the term "a" or "an" occurrence refers to one or more of that occurrence. 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.

[0027] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having 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 percent homology or percent sequence identity 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; description=50 sequences; sort criteria=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those polynucleotides that have the above-specified percent homology and encode polypeptides having the same or similar biological activity.

[0028] 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 to the nucleotide sequence of a nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence with a certain degree of homology to or its complement. In one embodiment, a homolog of a nucleic acid can hybridize to a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a certain degree of homology or sequence identity to 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.

[0029] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody or any antigen-binding fragment or single chain thereof. Thus, the term "antibody" encompasses any protein- or peptide-containing molecule that contains 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, a heavy or light chain complementarity-determining region (CDR) 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.

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

[0031] "Single-chain variable fragment" or "scFv" refers to a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L In some embodiments, the regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be glycine-rich for flexibility, serine- or threonine-rich for solubility, and V H N-terminus of V L The ScFv molecule can be 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.

[0032] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. 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 within each (e.g., γ1-γ4). It is the nature of the chain that determines the "class" of an antibody 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 confer 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 are therefore within the scope of this disclosure. While all immunoglobulin classes are clearly within the scope of this disclosure, 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 with a molecular weight of approximately 23,000 daltons and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. The four chains are typically joined 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.

[0033] 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 comprising either the VK or VH domains, 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). 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, IgAI, and IgA2), or subclass of immunoglobulin molecule.

[0034] By "specifically binds" or "having specificity for," it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, if an antibody binds to an epitope via its antigen-binding domain, the antibody is said to "specifically bind" to that epitope more readily than it would 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 higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with greater specificity than it has for the related epitope "D."

[0035] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (alleviate) an undesired physiological change or disorder, 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, stabilized (i.e., not worsening) disease, delayed or slowed disease progression, improved or alleviated disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving 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.

[0036] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, livestock animals, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0037] As used herein, phrases such as "patient in need of treatment" or "subject in need of treatment" include subjects, e.g., mammalian subjects, who would benefit from the administration of an antibody or composition of the present disclosure, e.g., used for detection, diagnostic procedures, and / or treatment.

[0038] CEACAM5-dependent 4-1BB agonistic bispecific antibody As mentioned above, major anti-4-1BB antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513), are agonistic antibodies. These antibodies can activate 4-1BB signaling by themselves. Due to their agnostic nature, these antibodies are thought to be associated with dose-limiting on-target toxicity. The inventors have developed anti-4-1BB single-domain antibodies (sdAbs, or nanobodies) that are not independently agonistic. These nanobodies can bind to 4-1BB protein but cannot activate 4-1BB signaling by themselves. When incorporated into a bispecific format with another antibody or antigen-binding fragment targeting a tumor-associated antigen (TAA), the bispecific antibody can activate 4-1BB signaling in the presence of the TAA. Such antibodies are referred to herein as "TAA-dependent 4-1BB agonistic bispecific antibodies."

[0039] CEACAM5-dependent 4-1BB agonistic bispecific antibodies have been developed in the present disclosure. CEACAM5 is a member of the carcinoembryonic antigen (CEA) gene family and is overexpressed in many cancer types, including colorectal cancer. CEACAM5 plays an important role in differentiation and inhibition of apoptosis in cancer cells. The bispecific antibodies tested herein have high binding affinity to both GPRC4D and 4-1BB. In addition, these bispecific antibodies can activate CEACAM5-dependent 4-1BB signaling in a concentration-dependent manner.

[0040] In vivo studies showed that treatment with the bispecific antibodies of the present disclosure resulted in complete tumor regression. Importantly, and unexpectedly, further tumor rechallenge of treated animals without further medication did not result in tumor growth ( FIG. 13 ). This indicates that these bispecific antibody treatments induced long-term protective immune memory.

[0041] The bispecific antibody format tested is shown in Figure 1, although other commonly used formats may also work. In Figure 1, two anti-4-1BB nanobodies are fused to the C-terminus of the Fc fragment of a full-sized conventional Fab antibody targeting CEACAM5.

[0042] Thus, in one embodiment, there is provided a bispecific (or multispecific) antibody comprising 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 CEACAM5, and in some embodiments further comprising a third or fourth specificity.

[0043] In one embodiment, the bispecific antibody comprises a full-size conventional IgG antibody against CEACAM5 and two anti-4-1BB nanobodies, as shown in Figure 1. In an alternative embodiment, the bispecific antibody comprises an anti-4-1BB nanobody that replaces one of the VH-CH1 / VL-CL pairs of the conventional anti-CEACAM5 antibody. In another embodiment, the bispecific antibody comprises two tandemly linked (concatenated) anti-4-1BB nanobodies in a single chain that replaces one of the VH-CH1 / VL-CL pairs of the conventional anti-CEACAM5 antibody.

[0044] In another embodiment, the bispecific antibody comprises one, two, or four anti-4-1BB nanobodies fused to the N-terminus of one or more variable regions of an anti-CEACAM5 antibody. For example, two anti-4-1BB nanobodies can be fused to the N-terminus of two VH chains. In another example, two anti-4-1BB nanobodies can be fused to the N-terminus of two VL chains. In another example, four anti-4-1BB nanobodies can be fused to the N-terminus of all VH / VL chains.

[0045] In some embodiments, the bispecific antibody comprises a constant region of a human IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant region is mutated to prevent post-translational modification (e.g., N297A). In some embodiments, the constant region is modified to increase or eliminate ADCC activity. In some embodiments, the Fc is an IgG1 Fc. In some embodiments, the IgG1 Fc has N297A (Eu numbering) to reduce or eliminate effector function. In some embodiments, the Fc is an IgG4 Fc. In some embodiments, the Fc has reduced or eliminated binding to Fc receptors (e.g., FcγRI and FcγRII). In some embodiments, the Fc is an IgG4 Fc with FALA substitutions (F234A, L235A, EU numbering).

[0046] Anti-4-1BB nanobody The present disclosure provides nanobodies (including humanized nanobodies) against the human 4-1BB protein that can be used in bispecific antibodies. These antibodies have high binding affinity for 4-1BB and can effectively block the interaction between 4-1BB and its ligand. These antibodies demonstrated 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 nanobodies of the present invention are particularly suitable for development into bispecific or multispecific antibodies.

[0047] Thus, in one embodiment of the disclosure, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises CDR1, CDR2, and CDR3. In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 21, CDR2 comprises the amino acid sequence of SEQ ID NO: 22 or 24, and CDR3 comprises the amino acid sequence of SEQ ID NO: 23.

[0048] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:21, CDR2 comprises the amino acid sequence of SEQ ID NO:22, and CDR3 comprises the amino acid sequence of SEQ ID NO:23.

[0049] In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 4. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 37.In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:38.

[0050] In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 21, 24, and 23, respectively. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0051] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 21 or 27, CDR2 comprises the amino acid sequence of SEQ ID NO: 25 or 28, and CDR3 comprises the amino acid sequence of SEQ ID NO: 26.

[0052] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO:21, CDR2 comprises the amino acid sequence of SEQ ID NO:25, and CDR3 comprises the amino acid sequence of SEQ ID NO:26.

[0053] In some embodiments, the CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 21, 25, and 26, respectively. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 16. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the antibody comprises the recited CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 19. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 20.

[0054] In some embodiments, CDR1, CDR2, and CDR3 comprise the amino acid sequences of SEQ ID NOs: 27, 28, and 26, respectively. In some embodiments, the antibody comprises the listed CDR1, CDR2, and CDR3 and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 11.

[0055] In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 5.

[0056] In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody) is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 10.

[0057] In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody), is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, a single domain antibody, or a polypeptide comprising a single domain antibody (e.g., a bispecific or multispecific antibody), is provided, wherein the single domain antibody comprises the amino acid sequence of SEQ ID NO: 38.

[0058] Anti-CEACAM5 antibodies and fragments Also provided are anti-CEACAM5 antibodies and antigen-binding fragments suitable for inclusion in the bispecific / multispecific antibodies of the present disclosure. In some embodiments, the anti-CEACAM5 antibody or antigen-binding fragment comprises a VH and a CL, each comprising CDR1, CDR2, and CDR3.

[0059] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 31, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 32, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 33, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 34, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 35, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 36.

[0060] In some embodiments, the VH comprises the recited VH CDR sequences and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 29. In some embodiments, the VL comprises the recited VL CDR sequences and has at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 30.

[0061] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:29 and the VL comprises the amino acid sequence of SEQ ID NO:30.

[0062] In some embodiments, the bispecific antibody comprises (1) one or more anti-4-1BB nanobodies, each having a CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs: 21, 22, and 23, respectively, and (2) one or more anti-CEACAM5 antigen-binding fragments, each having a VH CDR1 of SEQ ID NO: 31, a VH CDR2 of SEQ ID NO: 32, a VH CDR3 of SEQ ID NO: 33, a VL CDR1 of SEQ ID NO: 34, a VL CDR2 of SEQ ID NO: 35, and a VL CDR3 of SEQ ID NO: 36.

[0063] In some embodiments, a bispecific antibody comprises (1) one or more anti-4-1BB Nanobodies, each having the amino acid sequence of SEQ ID NO: 37, and (2) one or more anti-CEACAM5 antigen-binding fragments, each having a VH of SEQ ID NO: 29 and a VL of SEQ ID NO: 30. In some embodiments, a bispecific antibody comprises (1) one or more anti-4-1BB Nanobodies, each having the amino acid sequence of SEQ ID NO: 38, and (2) one or more anti-CEACAM5 antigen-binding fragments, each having a VH of SEQ ID NO: 29 and a VL of SEQ ID NO: 30.

[0064] In some embodiments, the bispecific antibody comprises a full-size IgG anti-CEACAM5 antibody and two anti-4-1BB nanobodies fused to the C-terminus of the Fc fragment of the anti-CEACAM5 antibody.

[0065] Also provided are compositions comprising the antibody or polypeptide and a pharmaceutically acceptable carrier.

[0066] It will also be understood by those skilled in the art that the antibodies disclosed herein may be modified to have different amino acid sequences from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a specified protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., 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.

[0067] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure may comprise a flexible linker sequence or may be modified to attach a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0068] Polynucleotides encoding antibodies and methods for preparing antibodies The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0069] Methods for producing antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be produced as described herein using techniques described in the art. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene locus has been disabled. Exemplary techniques that can be used to produce such antibodies are described in U.S. Patent No. 6,150,584, U.S. Patent No. 6,458,592, and U.S. Patent No. 6,420,140 (these are incorporated by reference in their entirety).

[0070] Cancer treatment As described herein, the antibodies, bispecific antibodies, polypeptides, variants or derivatives of the present disclosure may be used in certain treatment and diagnostic methods.

[0071] The present disclosure is further directed to antibody-based therapies comprising administering an antibody of the present disclosure to a patient, e.g., an animal, mammal, or human, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, an antibody of the present disclosure (including its variants and derivatives as described herein) and a nucleic acid or polynucleotide encoding an antibody of the present disclosure (including its variants and derivatives as described herein).

[0072] In some embodiments, methods of treating cancer in a patient in need thereof are provided. The methods, in one embodiment, involve 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.

[0073] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed, and / or prognosed by the antibodies or variants, or derivatives thereof, of the present disclosure include malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, 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, osteogenic sarcoma, chordoma, angiosarcoma, hemangioendothelial sarcoma, and endothelial sarcoma). ehosarcoma), lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct In some embodiments, the cancer is multiple myeloma, including, but not limited to, progression and / or metastasis of tumors including, but not limited to, carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0074] composition The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0075] In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Furthermore, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0076] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a 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 pharmaceutical compositions are administered intravenously. 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, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetate, citrate, or phosphate. Antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tonicity such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained-release formulations. 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 E.W. Martin, incorporated herein by reference. Such compositions contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The parental preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0077] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately in unit dosage form or mixed together, for example, as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet, indicating the quantity of active ingredient. If the composition is to be administered by injection, the composition can be dispensed into an infusion bottle containing sterile pharmaceutical-grade water or saline. 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 before administration. [Example]

[0078] Example 1: Generation and humanization of nanobodies against human 4-1BB This example describes the generation of nanobodies against the human 4-1BB protein.

[0079] Llamas were immunized with recombinant ECD of human 4-1BB fused to a human immunoglobulin Fc domain. Llamas whose serum contained sufficient titers of anti-4-1BB antibodies were selected for phage library construction. Briefly, lymphocytes were isolated from peripheral blood collected from immunized llamas. Lymphocyte RNA was extracted, and cDNA encoding the VHH domain was amplified by PCR and used to construct an M13 phage display-based nanobody library. Several rounds of panning were applied to screen the phage library expressing anti-4-1BB nanobodies.

[0080] All positive clones were screened by ELISA and FACS assays before sequencing. Based on sequence diversity, 16 unique clones were selected. The anti-4-1BB nanobody fused to a human IgG1 Fc fragment carrying the N297A mutation at its C-terminus was characterized for its specificity and activity by a series of functional assays, including binding, ligand competition, and 4-1BB signal activation, resulting in the identification of a lead nanobody for further humanization.

[0081] In follow-up studies, the binding properties of the resulting anti-4-1BB nanobodies to human and cynomolgus monkey 4-1BB proteins were tested by ELISA assay for their ability to block 4-1BB binding to its ligand, and their functional properties were evaluated by 4-1BB reporter assay. The results from these studies helped to determine two of them, VV02-1LP-263 and VV02-1SP(1)-73, as lead antibodies. These lead antibodies were subjected to humanization.

[0082] Humanization was performed by a CDR-grafting and backmutation strategy. The sequences of the VV02-1LP-263 and VV02-1SP(1)-73 antibodies and their humanized versions are shown in Table 1. [Table 1-1] [Table 1-2]

[0083] For VV02-1LP-263, two mutations (D54G and D61E, Kabat numbering) were introduced into CDR2 to improve developability. For VV02-1SP(1)-73, a mutation (N31S, Kabat numbering) and two mutations (D54S and D61E, Kabat numbering) were introduced into CDR1 and CDR2, respectively, to improve developability. The original and optimized CDR sequences are shown in Table 2 below. [Table 2]

[0084] To assess antigen-binding properties in a cell-based setting, 4-1BB humanized nanobodies were analyzed for their binding to 4-1BB overexpressed on CHO-K1 cells by FACS.

[0085] Briefly, CHO-K1 cells overexpressing human 4-1BB or cynomolgus 4-1BB were incubated with serially diluted anti-4-1BB nanobodies for 30 minutes at 4°C. The cells were then incubated with an Alexa Fluor 633-conjugated anti-human Fc secondary antibody. Binding was measured using an Agilent flow cytometer. The results showed that all tested humanized nanobodies exhibited typical sigmoid binding behavior to cell surface human 4-1BB and cynomolgus 4-1BB (Figures 2A-B). Accordingly, the binding EC 50 are shown in Tables 3 and 4. [Table 3] [Table 4]

[0086] Example 2: Generation of anti-CEACAM5-4-1BB bispecific antibody The anti-CEACAM5-4-1BB bispecific antibody was designed in a tetravalent IgG(H)-VHH fusion format (Figure 1), an IgG1 backbone in which the Fc domain contains the N297A mutation, which abolishes Fcγ function. The anti-CEACAM5 moiety was located within the intact IgG moiety, while the anti-4-1BB nanobody was a VHH located at the C-terminal end of the Fc fragment via a (G4S)4 (SEQ ID NO: 39) linker. The sequence of the anti-CEACAM5 arm was adapted from labetuzumab (Immunomedics, or Immu130; Table 1). The sequences of previously identified anti-4-1BB nanobodies used to construct the bispecific antibody are listed in Table 5. [Table 5] [Table 5A]

[0087] The resulting bispecific antibodies were transiently produced in CHO-K1 cells and purified by Protein A affinity chromatography. Well-qualified bispecific antibodies were subjected to in vitro characterization, including cell-based 4-1BB binding, CEACAM5 binding, and CEACAM5-dependent 4-1BB activation reporter assays.

[0088] Cell-based 4-1BB binding To evaluate the 4-1BB-binding activity of the bispecific antibodies, CHO-K1 cells overexpressing human 4-1BB were incubated with various concentrations of anti-CEACAM5-4-1BB bispecific antibodies for 30 minutes at 4°C. The cells were then washed twice with FACS buffer and stained with PE-conjugated secondary antibodies for 30 minutes at 4°C. After washing twice with FACS buffer, the MFI of PE was analyzed using a NovoCyte flow cytometer. As shown in Figure 3A, all bispecific antibodies bound to human 4-1BB-overexpressing CHO-K1 cells in a concentration-dependent manner.

[0089] Cell-based CEACAM5 binding To evaluate the CEACAM5-binding potency of the bispecific antibodies, MKN-45 cells, which endogenously express CEACAM5, were incubated with various concentrations of anti-CEACAM5-4-1BB bispecific antibodies at 4°C for 30 minutes. The cells were then washed twice with FACS buffer and stained with PE-conjugated secondary antibodies at 4°C for 30 minutes. After washing twice with FACS buffer, the MFI of PE was analyzed using a NovoCyte flow cytometer. As shown in Figure 3B, all bispecific antibodies bound to MKN-45 in a concentration-dependent manner.

[0090] CEACAM5-dependent 4-1BB activation To evaluate the ability of anti-CEACAM5-4-1BB bispecific antibodies to activate 4-1BB signaling, we used a reporter gene assay. In this assay, Jurkat cells engineered to express 4-1BB on their surface and carry an NF-κB luciferase reporter construct were used as effector cells. MKN-45 cells were used as target cells. Briefly, effector cells at a density of 1E5 cells per well were co-incubated with 1E4 target cells in the presence of 4-fold serial dilutions of anti-CEACAM5-4-1BB bispecific antibodies at 37°C in a 5% CO2 incubator. After overnight incubation, luminescence was obtained by adding luciferase substrate and measured using a microplate reader. As shown in Figure 4, all bispecific antibodies activated CEACAM5-dependent 4-1BB signaling in the presence of MKN-45 target cells.

[0091] Example 3: Optimization of anti-CEACAM5-4-1BB bispecific antibodies To further increase the developability of the candidate, bispecific antibody IMMU130-263-1-3 was selected to be optimized by site-directed mutations in the anti-4-1BB unit. Similar to the previous format, the optimized anti-4-1BB was placed at the C-terminal end of an IgG1 Fc fragment carrying the N297A mutation via a G4S linker (SEQ ID NO: 39). The optimized sequence of 263 huNb 1-3 is shown in Table 6. [Table 6]

[0092] The resulting optimized bispecific antibodies were subjected to in vitro characterization including cell-based 4-1BB binding, CEACAM5 binding, and CEACAM5-dependent 4-1BB activation reporter assays according to the assays described above.

[0093] Figure 5 shows that the optimized bispecific antibody exhibited binding activity comparable to that of the parental antibody to human 4-1BB-overexpressing CHO-K1 cells. Figure 6 shows that the optimized bispecific antibody exhibited similar binding affinity to CEACAM5-bearing MKN-45 cells as the parental bispecific antibody. Figure 7 shows that the optimized bispecific antibody efficiently induced 4-1BB activation in the presence of target cells MKN-45.

[0094] Example 4: Antigen binding of anti-CEACAM5-4-1BB bispecific antibodies In this example, the binding activity of the bispecific antibody IMMU130-263-1-3_1 in Example 3 against CEACAM5 and 4-1BB was evaluated in comparison with a reference antibody using protein-based and cell-based assays.

[0095] ELISA binding to CEACAM5 The anti-CEACAM5-4-1BB bispecific antibody IMMU130-263-1-3_1 was tested for in vitro binding to soluble recombinant CEACAM5 from various species, along with the parent CEACAM5 antibody labetuzumab, whose sequences were used to construct our bispecific antibody candidate, and the reference antibody civisatamab as a comparison. For standard ELISA, 100 μL of 2 μg / mL human CEACAM5 protein (Sino-11077-H08H-100UG) diluted in PBS was coated onto a microplate overnight at 4°C, followed by blocking with 200 μL / well of blocking buffer (1x DPBS containing 2% BSA). Five-fold dilutions of the antibody, starting from 200 nM, were added and incubated at 25°C for 60 minutes. The plates were then washed twice with ELISA wash buffer (1×DPBS containing 0.5% Tween®-20) and incubated with peroxidase-conjugated secondary antibody for 60 minutes at 25° C. After washing four times with ELISA wash buffer, the plates were detected with TMB-ELISA Substrate Solution, stopped with ELISA Stopping Solution, and analyzed at OD450nm and OD405nm using a microplate reader.

[0096] As shown in Figure 8, IMMU130-263-1-3_1 specifically bound to human CEACAM5 protein in a concentration-dependent manner. The EC50 was estimated to be 0.41 nM, comparable to the EC50 of the reference antibody. However, IMMU130-263-1-3_1 did not show cross-reactivity with cynomolgus monkey CEACAM5.

[0097] Cell-based binding to CEACAM5 To evaluate its antigen-binding properties in a cell-based setting, IMMU130-263-1-3_1 was analyzed for its binding to CEACAM5 overexpressed on HEK293 cells and tumor cell lines with endogenous CEACAM5 expression by flow cytometry as described above. The parent CEACAM5 antibody labetuzumab and the reference antibody civisatamab were included in parallel for comparison. As shown in Figure 9, all tested antibodies exhibited typical sigmoidal binding behavior to CEACAM5_HEK293, MKN45, and LS174T cells, while showing no nonspecific binding activity to blank HEK-293 cells. IMMU130-263-1-3_1 bound efficiently to human CEACAM5-expressing HEK-293, MKN45, and LS174T cells in a concentration-dependent manner, with EC50 values ​​of 3.78 nM, 4.71 nM, and 5.99 nM, respectively.

[0098] Cell-based binding to 4-1BB The anti-CEACAM5-4-1BB bispecific antibody IMMU130-263-1-3_1 was tested for in vitro binding to 4-1BB expressed on the surface of a CHO-K1 cell line, along with the parent anti-4-1BB antibody 263-1-3-1 as a reference. Additionally, urelumab, developed by Bristol-Myers Squibb Company, and 1A10, developed by ABL Bio, Inc., were also compared in parallel with IMMU130-263-1-3_1. As shown in Figure 10, the bispecific antibody efficiently bound to CHO-K1 cells overexpressing human 4-1BB and cynomolgus monkey 4-1BB in a concentration-dependent manner. The average EC50 of the bispecific antibody was estimated to be 1.07 nM for human 4-1BB and 0.39 nM for cynomolgus monkey 4-1BB. Notably, all the antibodies tested did not exhibit non-specific binding activity to blank CHO-K1 cells, as shown in FIG. 10C.

[0099] Example 5: CEACAM5-dependent 4-1BB signal activation in 4-1BB reporter assay In this example, we used a classical reporter assay to investigate the ability of the anti-CEACAM5-4-1BB bispecific antibody IMMU130-263-1-3_1 to activate 4-1BB signaling. In this assay, engineered Jurkat cells stably expressing 4-1BB and carrying an NK-κB luciferase reporter construct integrated into their genome were used as effector cells. HEK-293 cells engineered to overexpress CEACAM5 and the tumor cell line MKN45, which endogenously expresses CEACAM5, were used as target cells.

[0100] After 4-1BB activation, endogenous NF-κB transcription factors bind to the DNA response element to induce transcription of the luciferase gene, and the protein product is then quantified by measuring the luminescence signal after adding the substrate. In this study, effector and target cells were co-incubated with various concentrations of IMMU130-263-1-3_1 or a reference antibody overnight at 37°C in a 5% CO2 incubator. Luciferase substrate was then added, and luminescence intensity was determined using a microplate reader. The parent anti-4-1BB antibody 263-1-3-1 and urelumab, developed by Bristol-Myers Squibb Company, were used as reference antibodies.

[0101] As shown in Figure 11, the bispecific antibody IMMU130-263-1-3_1 induced significant 4-1BB activation in the presence of target cells expressing CEACAM. 50 The estimated potency of IMMU130-263-1-3_1 was 0.12 nM with human CEACAM5_HEK-293 as target cells and 0.11 nM with MKN45 as target cells. Notably, IMMU130-263-1-3_1 was unable to activate 4-1BB signaling in the presence of target cells that do not express CEACAM5, indicating that IMMU130-263-1-3_1-mediated 4-1BB activation is dependent on CEACAM5 engagement.

[0102] Example 6: CEACAM5-dependent primary T cell activation To test the ability of the anti-CEACAM5-4-1BB bispecific antibody IMMU130-263-1-3_1 to activate human T cells, human PBMCs (4E5 cells / well) were cocultured with HEK-293 cells overexpressing CEACAM5 (2E4 cells / well) in the presence of human anti-CD3 antibody (0.25 μg / mL OKT3) and serially diluted bispecific antibody. IL-2 levels in the culture supernatant were determined after 24 hours of incubation using a Human IL-2 ELISA Kit (CAT# Mabtech-3445-1H-20). IFN-γ levels were determined after 48 hours of incubation using a Human IFN-γ ELISA Kit (CAT# Mabtech-3420-1H-20). As shown in Figure 12, compared to the parental 4-1BB antibody, the CEACAM5-4-1BB bispecific antibody induced significant cytokine production by primary PBMCs. 50 was estimated to be 0.073 nM for IL-2 and 0.20 nM for IFN-γ.

[0103] Example 7: In vivo tumor growth inhibition by bispecific antibodies To evaluate the anti-tumor efficacy of the anti-CEACAM5-4-1BB bispecific antibody IMMU130-263-1-3_1, a syngeneic mouse tumor model was used, using human 4-1BB transgenic mice inoculated with MC38 cells ectopically expressing human CEACAM5. Humanized 4-1BB mice were inoculated with 1 x 10 6 hCEACAM5-MC38 cells were subcutaneously implanted. The average tumor volume was 65 mm 3 When tumors reached a size of 1000, tumor-bearing mice were randomized into two groups (N=6 / group) and intraperitoneally administered vehicle (PBS) or IMMU130-263-1-3_1, respectively. Antibody was administered twice weekly. Tumor volume was monitored by caliper measurement three times weekly throughout the experimental period.

[0104] As shown in Figure 13, injection of IMMU130-263-1-3_1 resulted in significant tumor inhibition, with five of the six mice in the treatment group becoming tumor-free by approximately one month after the start of treatment. When rechallenged with a second dose of the same tumor cells, these five tumor-free mice resisted the tumor rechallenge and were considered tumor-free, whereas tumor cells continued to grow in naive mice. This suggests that the anti-CEACAM5-4-1BB bispecific antibody IMMU130-263-1-3_1 exhibited strong antitumor efficacy and induced long-term protective immune memory. * * *

[0105] The present disclosure should not be limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the disclosure; any functionally equivalent compositions or methods are within the scope of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the disclosed methods and compositions without departing from the spirit or scope of the disclosure. Therefore, the present disclosure is intended to cover the modifications and variations of the present disclosure provided they come within the scope of the appended claims and their equivalents.

[0106] 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 bispecific antibody comprising one or more anti-4-1BB single domain antibodies (VHH) and an anti-CEACAM5 antibody or antigen-binding fragment thereof, wherein the one or more anti-4-1BB single domain antibodies each comprise a complementarity determining region 1 (VHH CDR1), a VHH CDR2, and a VHH CDR3; (a) the VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, the VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 22 or 24; and the VHH CDR3 comprises the amino acid sequence of SEQ ID NO: 23; or (b) a bispecific antibody, wherein said VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 21 or 27; said VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 25 or 28, and said VHH CDR3 comprises the amino acid sequence of SEQ ID NO:

26.

2. 2. The bispecific antibody of claim 1 , wherein the VHH CDR1 comprises the amino acid sequence of SEQ ID NO: 21, the VHH CDR2 comprises the amino acid sequence of SEQ ID NO: 22, and the VHH CDR3 comprises the amino acid sequence of SEQ ID NO:

23.

3. 3. The bispecific antibody of claim 2, wherein the one or more anti-4-1BB single domain antibodies each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-10 and 37-38.

4. 3. The bispecific antibody of claim 2, wherein the one or more anti-4-1BB single domain antibodies each comprise the amino acid sequence of SEQ ID NO:

37.

5. 10. The bispecific antibody of any preceding claim, further comprising an IgG Fc fragment.

6. The bispecific antibody of claim 5, wherein the Fc fragment is N-terminal to two anti-4-1BB single domain antibodies, optionally via a peptide linker.

7. The bispecific antibody of claim 5 or 6, wherein the Fc fragment is C-terminal to the two anti-CEACAM5 fragments.

8. 10. The bispecific antibody of any preceding claim, comprising a full-size IgG anti-CEACAM5 antibody and two anti-4-1BB single domain antibodies fused to the C-terminus of said anti-CEACAM5 antibody.

9. 3. The bispecific antibody of any preceding claim, wherein the anti-CEACAM5 antibody or antigen-binding fragment comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:

36.

10. The bispecific antibody of claim 9 , wherein the anti-CEACAM5 antibody or antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 29 and a VL comprising the amino acid sequence of SEQ ID NO:

30.

11. 11. One or more polynucleotides encoding the bispecific antibody of any one of claims 1 to 10.

12. A cell comprising the polynucleotide of claim 11.

13. A composition comprising the bispecific antibody of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

14. 11. A method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a bispecific antibody according to any one of claims 1 to 10.

15. 15. The method of claim 14, wherein the cancer is characterized by expression of CEACAM5.

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