Bispecific antibody compositions and methods of use thereof

Abstract

To provide antibodies, pharmaceutical compositions and therapeutic methods for cancer treatment.SOLUTION: The present invention provides an antibody having a N-terminal and a C-terminal, comprising a heavy chain and a light chain, wherein the heavy chain comprises in tandem from the N-terminal to the C-terminal, a variable component comprising a heavy chain scFv domain, a heavy chain linker, CH1, a hinge, CH2 and CH3 domains, wherein the light chain comprises in tandem from the N-terminal to the C-terminal, a variable component comprising a light chain scFv domain, a light chain linker and a CL domain, wherein the heavy chain scFv has specificity against a first target, wherein the light chain scFv has specificity against a second target, and wherein the first target and the second target are selected independently from a group consisting of CDH17, CD3, TROP2, GPC3, and HER.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application No. 62 / 672,325, filed May 16, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Technical Field The disclosure herein relates generally to the technical field of cancer immunotherapy, and more particularly to cadherin-17 (CDH17)-specific antibodies and cytotoxic cells for cancer treatment. [Background technology]

[0003] Unless otherwise stated herein, the materials described in this section are not prior art to the claims of this application and are not admitted to be prior art by inclusion in this section.

[0004] Despite recent advances in drug discovery and clinical imaging, cancer remains one of the deadliest diseases in humans. Our understanding of how tumors develop, survive under stress, colonize / metastasize to distant organs and sites, and become resistant to drugs remains limited. The American Cancer Society estimated that there were 1.6 million new cases of cancer in the United States in 2014, and no curative treatments have been approved for the majority of major cancers.

[0005] Gastrointestinal (GI) cancers (colorectal, gastric, pancreatic, esophageal, bile duct, and liver cancers) are a major cause of morbidity and mortality worldwide. Colorectal cancer (CRC) alone accounts for approximately 10% of all cancer diagnoses and is the second leading cause of cancer deaths worldwide. In China, liver and gastric cancers are among the most lethal of malignant tumors, accounting for more than half of diagnosed cases and causing over 1.42 million deaths annually globally, likely due to viral / bacterial epidemics (hepatitis B virus [HBV] and Helicobacter pylori infections), chemical poisoning, environmental pollution, and food contamination. No effective treatments are available. Therefore, novel biomarkers and therapeutic targets are needed for potential drug development against these aggressive cancers. Proven molecular-targeted drugs that can eliminate or inhibit the growth of these cancers would have significant clinical value and would have a significant impact on the market. If diagnosed early, these tumors can be effectively removed by surgery. Unfortunately, very often, most GI cancers are asymptomatic and are found at a very advanced stage when diagnosed in the clinic. Without effective treatment, these patients will either die shortly after diagnosis or relapse after salvage therapy.

[0006] CDH17 is a well-known cancer biomarker characterized by overexpression in both liver and gastric cancers, but is not expressed in normal tissues of healthy adults. Anti-CDH17 monoclonal antibodies exhibit growth-inhibitory effects on liver and gastric tumor cells. CDH17 is highly expressed in metastatic cancers, and blocking CDH17 expression and function significantly suppresses lung metastasis of hepatocellular carcinoma (HCC). These observations suggest that humanized anti-CDH17 antibodies could be developed as antibody therapeutics for treating cancer patients who exhibit the CDH17 biomarker in tumor tissues and / or serum samples.

[0007] In contrast to antibody therapeutics, which are characterized by the binding of monoclonal antibodies to cancer cells, multispecific antibody therapeutics can bind to T cells and mediate cytotoxicity against cancer cells. While bispecific antibodies are effective in treating hematological malignancies, their success in targeting solid tumors has been limited. Potential barriers include the lack of appropriate biomarkers for activated cytotoxic immune cells and limited access to solid tumor cells. Summary of the Invention

[0008] The disclosure herein provides compositions of multispecific antibodies and cytotoxic cells that target CDH17, as well as methods for treating cancer using the compositions and antibodies (or fragments thereof) disclosed herein.

[0009] In one aspect, the disclosure herein relates to a composition of a multispecific antibody that targets both a gastrointestinal-specific biomarker and CD3. In some embodiments, the antibody is a CDH17xCD3 bispecific antibody. This antibody can activate T cells and safely target CDH17-positive cells. In one embodiment, the CDH17xCD3 bispecific antibody can be used clinically to treat patients with CDH17-positive cancer.

[0010] In one embodiment, the disclosure herein provides an antibody having specificity for CDH17, comprising a heavy or light chain amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any other number therebetween) homology to an amino acid sequence selected from SEQ ID NOs: 15-33.

[0011] In some embodiments, the antibody is a monoclonal antibody. In one embodiment, the monoclonal antibody may be a murine antibody, a humanized antibody, or a human antibody. In some embodiments, the monoclonal antibody may be a human antibody isolated from a phage library screen.

[0012] In some embodiments, the antibody may comprise a light chain variable region (VL), a heavy chain variable region (VH), or a combination thereof. In one embodiment, the VL may comprise an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number in between) identical to an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, and 12. In some embodiments, the VH may comprise an amino acid sequence at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number in between) identical to an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, and 11.

[0013] In some embodiments, the antibody may comprise a conjugated cytotoxic moiety. In some embodiments, the conjugated cytotoxic moiety may comprise irinotecan, auristatins, PBDs, maytansines, amantines, spliceosome inhibitors, or combinations thereof. In some embodiments, the conjugated cytotoxic moiety may comprise a chemotherapeutic agent.

[0014] In some embodiments, the antibody is a bispecific antibody.

[0015] In some embodiments, the antibody may comprise specificity for a cellular receptor from a cytotoxic T cell or a NK cell. In some embodiments, the antibody is a bispecific antibody with specificity for both CDH17 and CD3. In some embodiments, the cellular receptor may comprise KIR2D52, KIR2D53, KIR2D54, KIR2D55, KIR3D51, CD16a, CD27, CD94, CD96, CD100, CD160, CD244, NKp30, NKp44, NKp46, NKp80, NKG2D, DNAM1, CRTAM, PSGL1, CEACAM1, NTB-A, SLAMF7, OX40, CD137, ICOS, CD28, TIM1, and TIM3, or a derivative or combination thereof.

[0016] In some embodiments, an antibody may comprise a first single-chain variable fragment (scFv) with specificity for CDH17 and a second single-chain variable fragment (scFv) with specificity for CD3 or TROP2. In one embodiment, the first scFv may comprise a first VH (variable heavy chain) and a first VL (variable light chain). In one embodiment, the second scFv may comprise a second VH and a second VL. In some embodiments, the first VH may comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number therebetween) homology to an amino acid sequence selected from SEQ ID NOs: 1, 3, 5, and 7. In some embodiments, the first VL may comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number therebetween) homology to an amino acid sequence selected from SEQ ID NOs: 2, 4, 6, and 8.

[0017] In some embodiments, the second VH may comprise a corresponding portion of an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number therebetween) homology to an amino acid sequence selected from amino acid sequences 9, 11, and 13.

[0018] In some embodiments, the second VL may comprise a corresponding portion of an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number therebetween) homology to amino acid sequence numbers 10, 12, and 14.

[0019] In some embodiments, the antibody may have specificity for an immune checkpoint inhibitor, which may include PD-1, PD-L1, CTL-A4, TIM3, LAG3, BTLA, CD96, TIGIT, CD226, or VISTA, or a combination thereof.

[0020] In some embodiments, the antibody may have specificity for an angiogenic factor, hi some embodiments, the angiogenic factor may include VEGF.

[0021] In some embodiments, the antibody may be configured to antagonize binding of the RGD site of CDH17 domain 6 to an integrin. In some embodiments, the integrin may comprise alpha2beta1.

[0022] In some embodiments, the antibody may be configured to bind to CDH17 ectodomain 5, domain 6, or domain 7 and antagonize CDH17 shedding.

[0023] In some embodiments, the antibody is a monoclonal antibody.

[0024] In some embodiments, the antibody relates to an IgG heavy chain for the antibody. In one embodiment, the antibody may comprise an IgG comprising a chain having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homology (or any other number in between) to SEQ ID NOs: 15, 16, 17, 20, 21, 22, 24, 25, 26, 28, 29, 30, 31, 32, 33.

[0025] Some embodiments relate to the light chain of the antibody. In one embodiment, the antibody may have a light chain having an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 18, 19, 23, or 27 (or any other number in between).

[0026] Some embodiments relate to antibody variable domains. In one embodiment, the variable domains may have amino acids with at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homology (or any other number in between) to SEQ ID NOs: 1-14.

[0027] In some embodiments, the antibody relates to an scFv or Fab with specificity for CDH17. In some embodiments, the antibody comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number in between) homology to an amino acid sequence selected from SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40.

[0028] In some embodiments, the scFv or Fab may comprise specificity for a cell receptor from a cytotoxic T cell or a NK cell. In some embodiments, the scFv or Fab may comprise specificity for an immune checkpoint inhibitor. In some embodiments, the scFv or Fab may comprise specificity for an angiogenic factor.

[0029] In some embodiments, the present invention relates to T or NK cells having specificity for CDH17. In one embodiment, the T or NK cells may comprise a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor may comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any other number in between) homology to an amino acid sequence selected from SEQ ID NOs: 41, 42, 43, 44, and 45.

[0030] Some embodiments relate to isolated nucleic acids encoding an antibody, IgG heavy chain, light chain, variable chain, or scFv or Fab described herein.

[0031] In some embodiments, the present disclosure relates to an expression vector comprising the isolated nucleic acid. In some embodiments, the vector is expressible in a cell.

[0032] Some embodiments relate to host cells comprising the nucleic acids described herein. Some embodiments relate to host cells comprising the expression vectors described herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0033] In one aspect, the present application provides a pharmaceutical composition for treating cancer. In one embodiment, the pharmaceutical composition comprises an antibody and a cytotoxic agent.

[0034] In some embodiments, the cytotoxic agent comprises cisplatin, gemcitabine, irinotecan, or an anti-tumor antibody.

[0035] In some embodiments, a pharmaceutical composition may comprise an antibody described herein and a pharmaceutically acceptable carrier. In a further aspect, the present application provides a method for treating a subject with cancer. In one embodiment, the method comprises administering to the subject an effective amount of an antibody, T cells, or NK cells. In some embodiments, an effective amount may be an amount that can treat cancer, alleviate symptoms, alter biomarkers to aid in the treatment of cancer, or a combination thereof. The subject may be a human or an animal.

[0036] In some embodiments, the cancer may be liver cancer, stomach cancer, colon cancer, pancreatic cancer, lung cancer, or a combination thereof.

[0037] Objects and advantages of the present disclosure will become apparent from the following detailed description of embodiments thereof, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0038] Embodiments according to the disclosure herein may be described with reference to the figures, where like reference numerals indicate like elements. [Figure 1] FIG. 1 shows structural variants of an exemplary bispecific antibody against CDH17 and CD3, designated as scFv4-Ig or tB (tetraB), IgG-scFv or fL (full length), and taFv-Fc or Fc (bait-Fc). [Figure 2] FIG. 2 shows a sequence alignment of exemplary variable domains of humanized CDH17 antibodies of SEQ ID NOs: 1-8, TROP2 antibodies of SEQ ID NOs: 9 and 10, and CD3 antibodies of SEQ ID NOs: 11 and 12. [Figure 3] FIG. 3 shows CDH17 expression in DLD-1 (colon cancer) and AGS (gastric cancer) tumor cell lines using the CDH17xCD3 bispecific antibody ARB201 (h3G1Fc) and flow cytometry analysis in an example. [Figure 4] Figure 4 shows live cell images of ARB201 antibody-induced retargeted T cell cytotoxicity against DLD-1 spheroids. DLD-1 cells were stained with CellBrite™ Green and grown as spheroids. Cells were incubated for 48 hours in the presence or absence of PBMCs and / or ARB201 (Ab). Retargeted T cell cytotoxicity was monitored by red fluorescent staining of dead target cells; brightfield, green: GFP filter set; red: PI filter set; and live cell images were acquired and analyzed with an automated fluorescent imager. [Figure 5] Figure 5 shows the concentration response of ARB201 in 2D and 3D DLD-1 models. DLD-1 cells were incubated with fresh PBMCs in the presence of different concentrations of ARB201. DLD-1 cell death was assessed at 48 hours. Cytotoxicity of retargeted T cells was monitored with Dead Red dye. IC50 values ​​were calculated using nonlinear regression fitting data to a sigmoidal four-point, four-parameter log-logistic dose-response model. [Figure 6] Figure 6 shows the concentration response of ARB201 in 2D and 3D AGS models. AGS cells were incubated with fresh PBMCs in the presence of different concentrations of ARB201. AGS cell death was assessed at 16 hours. The cytotoxicity of retargeted T cells was monitored with Dead Red dye. IC50 values ​​were calculated using nonlinear regression fitting data to a sigmoidal four-point, four-parameter log-logistic dose-response model. [Figure 7]Figure 7 shows an illustration of ARB201 retargeting T cell cytotoxicity. A) ARB201 binds to both T cells (red) and tumor cells (green), supporting contact between T cells and tumor target cells. B) Brightfield. C) Binding of ARB201 to CD3 / TCR stimulates a cytotoxic T cell response with the release of perforin and granzymes, which induce pore formation and apoptosis, respectively. [Figure 8] FIG. 8 shows binding of exemplary CDH17xCD3 bispecific antibodies h5G1fL and h5G4fL to CDH17 as determined by ELISA. [Figure 9] FIG. 9 shows binding of exemplary CDH17xCD3 bispecific antibodies h5G1fL and h5G4fL to CD3 on Jurkat T cells. [Figure 10] FIG. 10 shows the binding of exemplary CDH17xCD3 bispecific antibodies h10G1fL and h10G4fL to CDH17 as determined by ELISA. [Figure 11] FIG. 11 shows the binding of exemplary CDh17xCD3 bispecific antibodies, h10G1fL and h10G4fL, to CD3 on Jurkat T cells. [Figure 12] FIG. 12 shows the binding of exemplary CDH17xCD3 bispecific antibodies h10G1tB and h10G4tB to CD3. [Figure 13] FIG. 13 shows the binding of exemplary CDH17xCD3 bispecific antibodies h10G1tB and h10G4tB to CD3 on Jurkat T cells. [Figure 14] FIG. 14 shows the safety profile of exemplary CDH17xCD3 bispecific antibodies h10G1fL, h10G4fL, h10G4tB, and h3G4tB, which do not activate T cells in the absence of tumor cells. [Figure 15] FIG. 15 shows tumor cell-dependent T cell activation by the exemplary CDH17xCD3 bispecific antibody h10G4fL using PBMCs and AsPC1 tumor cells. [Figure 16]FIG. 16 shows an exemplary CDH17xCD3 bispecific antibody h10G4fL that redirects T cell cytotoxicity toward CDH17-positive pancreatic and colon cancer cell lines in a concentration-dependent manner. [Figure 17] Figure 17 shows pharmacokinetic analysis of serum concentrations following intravenous injection of exemplary CDH17xCD3 bispecific antibody h10G4fL: A) 3 mg / kg in mice (A), and B) 3 mg / kg and 10 mg / kg in non-human primate (NHP) models. [Figure 18] FIG. 18 shows histopathological analysis of the exemplary CDH17xCD3 bispecific antibody h10G4fL in (A) necropsy samples and (B) NHP colon and pancreas from an in vivo model. [Figure 19] Figure 19 shows that the exemplary CDH17xCD3 bispecific antibody ARB202 can inhibit tumor growth in a mouse model of AsPC-1 cell-derived pancreatic cancer. A: Determination of tumor volume over a 4-week period in mice treated with RPMI (vehicle), activated T cells (T cells) derived from PBMCs, T cells plus 0.05 mg / kg ARB202, or T cells plus 0.5 mg / kg ARB202 at the indicated time points by intratumoral administration. And B: Only treatment with ARB202 resulted in increased levels of human IL-2 in plasma. DETAILED DESCRIPTION OF THE INVENTION

[0039] This application provides antibodies specific for both cadherin-17 (CDH17) and CD3, antibodies that target tumor cells, and antitumor immunotherapies using such antibodies, including antibodies with different modes of cytotoxicity or chimeric antigen receptors that stimulate T cell or NK cell cytotoxicity.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure herein, the methods and materials are described. For purposes of this disclosure, the following terms are defined:

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0042] "About" means an amount, level, value, number, frequency, percentage, size, dimensions, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, value, number, frequency, percentage, size, dimensions, weight, or length.

[0043] "Coding sequence" refers to any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not contribute to the coding of the polypeptide product of a gene.

[0044] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" may be understood to mean the inclusion of stated steps or elements or groups of steps or elements but not to the exclusion of other steps or elements or groups of steps or elements.

[0045] "Consisting of" means inclusive of, and limited to, what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present.

[0046] "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or action identified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that these other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0047] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "AGT" is complementary to the sequence "TCA." Complementarity can be "partial," where only some of the nucleic acids' bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0048] "Corresponds to" or "corresponding to" means (a) a polynucleotide having a nucleotide sequence that is substantially identical to or complementary to all or a portion of a reference polynucleotide sequence, or a polynucleotide that encodes an amino acid sequence identical to that of a peptide or protein, or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to that of a reference peptide or protein.

[0049] As used herein, terms such as "function" and "functional" refer to biological, binding, or therapeutic functions.

[0050] "Gene" means a unit of heredity that occupies a specific locus on a chromosome and consists of transcriptional and / or translational regulatory sequences and / or coding regions and / or untranslated sequences (introns, 5' and 3' untranslated sequences).

[0051] "Homology" refers to the percentage of amino acids that are identical or that make conservative substitutions. Homology can be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395), which is incorporated herein by reference. In this manner, sequences of similar or substantially different lengths to those cited herein can be compared by inserting gaps into the alignment, with such gaps determined, for example, by the comparison algorithm used by GAP.

[0052] The term "host cell" includes an individual cell or cell culture that can be or has been a recipient of any of the recombinant vectors or isolated polynucleotides disclosed herein. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and / or change. A host cell includes cells that have been transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide disclosed herein. A host cell containing a recombinant vector disclosed herein is a recombinant host cell.

[0053] An "isolated" antibody is one that has been identified and separated and / or recovered from components of its natural environment, which are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0054] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.

[0055] However, isolated nucleic acid molecules typically include nucleic acid molecules contained in cells that express the antibody, where, for example, the nucleic acid molecule may be in a chromosomal location different from that of natural cells.

[0056] As used herein, the term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, optional operability sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0057] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Typically, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0058] As used herein, the designation "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA. The term refers to a polymeric form of nucleotides, typically at least 10 bases in length, either ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide. The term includes single- and double-stranded forms of DNA and RNA.

[0059] Terms such as "polynucleotide variant" and "variant" refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or that hybridize to a reference sequence under stringent conditions, as defined below. These terms also encompass polynucleotides that differ from a reference polynucleotide by the addition, deletion, or substitution of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted, or substituted with different nucleotides. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide such that the modified polynucleotide retains the biological function or activity of the reference polynucleotide or has increased (i.e., optimized) activity relative to the reference polynucleotide. Polynucleotide variants include, for example, polynucleotides having at least 50% sequence identity (and at least 51% to at least 99% and all integer percentages therebetween, e.g., 90%, 95%, or 98%) to a reference polynucleotide sequence described herein. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants and orthologs encoding these enzymes.

[0060] "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, and to variants and synthetic analogs thereof. Thus, these terms apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids. In certain aspects, polypeptides may include enzymatic polypeptides, or "enzymes," which typically catalyze (i.e., increase the rate of) various chemical reactions.

[0061] A polypeptide "variant" refers to a polypeptide that differs from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain embodiments, a polypeptide variant differs from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, a polypeptide variant includes conservative substitutions; in this regard, it is well understood in the art that some amino acids may be changed to other amino acids with broadly similar properties without altering the properties of the polypeptide's activity. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or substituted with a different amino acid residue.

[0062] The term "reference sequence" generally refers to a nucleic acid coding sequence or an amino acid sequence that is compared to another sequence. All polypeptide and polynucleotide sequences described herein are included as reference sequences.

[0063] As used herein, "sequence identity," or designations including, for example, "50% sequence identity," refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., the Sequence Listing), and typically, polypeptide variants retain at least one biological activity of the reference polypeptide.

[0064] "Statistically significant" means that the results are unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability that the observed event would occur if the null hypothesis were true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0065] "Substantially" or "essentially" means nearly completely or entirely, for example, 95%, 96%, 97%, 98%, 99% or more of a specified amount.

[0066] "Treating" or "treatment" or "palliative" refers to both therapeutic and prophylactic or preventative treatment. Here, the objective is to prevent or slow (reduce) the targeted pathological condition or disorder. For example, in the case of cancer, this includes a reduction in the number of cancer cells or their absence, a reduction in tumor size, inhibition (i.e., slowing to some extent, preferably halting) of tumor metastasis, inhibition to some extent of tumor growth, an increase in the length of some remission and / or remission of one or more symptoms associated with a particular cancer, a reduction in morbidity and mortality, and an improvement in quality of life issues. A reduction in signs or symptoms of the disease may also be felt by the patient. Treatment can achieve a complete response, defined as the disappearance of all signs of cancer, or a partial response, where tumor size is preferably reduced by more than 50%, more preferably by more than 75%. A patient is also considered treated if they experience stable disease. In one embodiment, the patient is cancer progression-free after one year, preferably after 15 months. These parameters for assessing successful treatment and disease improvement are readily measurable by routine procedures well known to a physician of appropriate skill in the art.

[0067] The terms "modulate" and "alter" include "increase" and "enhance," as well as "decrease" or "reduce," typically by a statistically significant amount or physiologically significant amount or degree compared to a control. In certain embodiments, immunological rejection associated with transplantation of a blood substitute is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% compared to unmodified or modified stem cells.

[0068] An "increased" or "enhanced" amount is typically a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50-fold or more (e.g., 100-fold, 500-fold, 1000-fold) (including all integers and decimal points between greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amounts or levels described herein.

[0069] A "decreased" or "reduced" or "less" amount is typically a "statistically significant" amount and may include a decrease of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50-fold or more (e.g., 100-fold, 500-fold, 1000-fold) (including all integers and decimal points between greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amounts or levels described herein.

[0070] "Derived from" means, for example, that a sample, such as a polynucleotide or polypeptide, is isolated or derived from a particular source, such as a desired organism or a particular tissue within a desired organism. "Derived from" can also refer to situations in which a polynucleotide or polypeptide sequence is isolated or derived from a particular source, such as a desired organism or a particular tissue within a desired organism. For example, polynucleotide sequences encoding the reference polypeptides described herein may be isolated from various prokaryotic or eukaryotic organisms, or from particular tissues or cells within a particular eukaryotic organism. A "therapeutically effective amount" refers to an amount of an antibody or drug effective to "treat" a disease or disorder of interest. In the case of cancer, a therapeutically effective amount of a drug may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, suppress tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. See the definition of "treatment" above.

[0071] "Chronic" administration refers to administration of the agent in a continuous, as opposed to acute, mode to maintain the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is not given continuously without interruption, but rather in cycles.

[0072] "Vector" includes shuttle and expression vectors. Typically, a plasmid construct contains an origin of replication (e.g., ColE1 replication origin) and a selectable marker (e.g., ampicillin or tetracycline resistance) for replication and selection of the plasmid in bacteria, respectively. "Expression vector" refers to a vector containing the control sequences or regulatory elements necessary for expressing antibodies, including the antibody fragments disclosed herein, in bacteria or eukaryotic cells. Suitable vectors are described below.

[0073] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or function.

[0074] An "antibody fragment" comprises a portion of a full-length antibody, generally the antigen-binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0075] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable domain and one light-chain variable domain tightly bound by noncovalent bonds. The folding of these two domains generates six hypervariable loops (three loops each from the H chain and L chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific complementarity-determining regions (CDRs)) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0076] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for minor amounts of naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the disclosure herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0077] The term "variable" refers to the fact that certain segments of the variable domain (V domain) vary extensively in sequence among antibodies. V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, they are not evenly distributed across the 10-amino acid span of the variable domain. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs), separated by shorter regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable domains each contain four framework regions (FRs), largely adopting a β-sheet structure, connected by three hypervariable regions, which form connecting loops and, in some cases, part of the β-sheet structure. The hypervariable regions of each chain are held in close proximity by FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved in directly binding the antibody to an antigen but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0078] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable regions generally comprise amino acid residues in the CDRs (e.g., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL, and about residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from the "hypervariable loops" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the VL, and residues 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in the VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0079] A "chimeric" antibody (immunoglobulin) is an antibody in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited, as well as fragments of such antibodies (U.S. Pat. No. 4,816,567, and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). As used herein, humanized antibodies are a subset of chimeric antibodies.

[0080] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of the recipient have been replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some embodiments, humanized antibodies are antibodies derived from human cells or transgenic animals (typically mice) expressing human antibody genes.

[0081] In one embodiment, the antibody or antigen-binding fragment thereof provided herein is an antibody or antigen-binding fragment thereof having specificity for CDH17. Tumor-associated antigens can serve as targets for anti-tumor immunotherapy by inhibiting their tumor growth-promoting activity and inducing cytotoxic activity against tumor cells. CDH17 is a type 1 integral transmembrane glycoprotein belonging to the cadherin superfamily of cell adhesion molecules. CDH17 is a non-classical cadherin with seven cadherin or cadherin-like repeats in its ectodomain. CDH17 is a tumor-associated antigen and is involved in tumor growth. CDH17 expression is typically restricted to intestinal epithelial cells of the colon, small intestine, and pancreatic duct, but is overexpressed in several tumors, including colon adenocarcinoma, gastric adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, esophageal adenocarcinoma, and pancreatic adenocarcinoma. Its tumor growth-promoting activity may be related to the binding of the RGD motif in CDH17 domain 6 to integrins such as α2β1. Abnormally elevated levels of CDH17 in blood and exosomes may serve as a prognostic marker for cancer.

[0082] Through extensive research using proteomics and oncogenomics approaches, a therapeutic target, hepato-intestinal cadherin (CDH17), is disclosed herein. This target is overexpressed in a large proportion of gastric cancer (GC) and hepatocellular carcinoma (HCC), as well as pancreatic cancer (panCA), colon cancer (CRC), ovarian cancer, and lung cancer. RNAi silencing of the CDH17 gene could suppress tumor growth and metastatic spread in established HCC mouse models (both xenografts and allografts). The antitumor mechanism is based on inactivation of Wnt signaling, accompanied by reactivation of tumor suppressor pathways.

[0083] The anti-CDH17 antibodies described herein have demonstrated anti-tumor effects in multiple in vitro and in vivo systems for liver cancer and gastric cancer. Such antibodies have in vitro and in vivo uses for purification, detection, diagnostics, and therapeutics. Such antibodies support anti-tumor activity by selectively binding to tumor cells and may be developed to stimulate complement fixation, antibody-dependent cellular cytotoxicity, binding agent-mediated cytotoxicity, lymphocyte-mediated cytotoxicity, and NK-mediated cytotoxicity. Provided herein are antibodies and humanized antibodies, antigen-binding fragments, or chimeric antibody proteins comprising a heavy chain variable region having the amino acid sequence of the corresponding SEQ ID NO: shown below.

[0084] CDH17 antibody sequences may include various types of antibodies, such as murine antibodies (5F6, 9B5, 9C6, 10C12, 8B5) and their humanized variants (Figures 1 and 2), as well as bispecific antibodies including various artificial antibody fragments (Fab, scFv, diabodies, etc.). Exemplary forms include "tB", "fL", and "Fc" (Figure 1).

[0085] In some embodiments, the humanized CDH17xCD3 bispecific antibodies, h5G1fL, h5G4fL, h10G1fL, h10G4fL, h10G1tB, and h5G4tB, exhibit the ability to bind to CDH17 in an ELISA assay, as shown in Figures 8, 10, and 12. The ability to bind to CD3 is demonstrated by flow cytofluorometry in Figures 9, 11, and 13.

[0086] In another aspect, certain CDH17xCD3 bispecific antibodies, h10G1fL, h10G4fL, h10G4tB, and h3G4tB, exhibit a safety profile in that they do not induce cytotoxic T cell responses when incubated with PBMCs in the absence of tumor cells (Figure 14).

[0087] In one embodiment, antibodies may be identified that bind to any of the C-terminal ectodomains of CDH17, such as D5, D6, or D7. This binding may prevent CDH17 from being cleaved and shredded, enabling a novel mechanism for unique therapeutic activity. Such anti-CDH17 antibodies may be used to construct bispecific or trispecific antibodies that prevent CDH17 release while supporting T cell or NK killing of tumor cells. The second or third specificity of such antibodies may be CD3 or an NK cell receptor. [Example]

[0088] The present disclosure will be further described with reference to the following examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present disclosure should not be interpreted as being limited to the following examples, but rather as encompassing any variations that become apparent as a result of the teachings provided herein.

[0089] Example 1. Construction of CDH17xCD3 bispecific antibodies CDH17xCD3 bispecific antibodies were generated and grouped based on their structural topology (scFv4-Ig or tB (tetraB), IgG-scFv or fL (full-length), and taFv-Fc or Fc (bait-Fc) as shown in Figure 1 and Table 1). All three designs contain U1, a humanized UCHT-1 scFv with binding specificity for CD3. fL (full-length) represents a group of humanized anti-CDH17 antibodies, while tB (tetraB) and Fc (bait-Fc) contain the anti-CDH17 scFv and humanized UCHT-1 scFv, respectively. As shown in Figure 2, the variable domains of the CDH17 murine antibodies m5F6, m9B5, m9C6, and m10C12 and the TROP2 murine antibody m8B5 were aligned with the homologous human germline and humanized VH and VL sequences, h5F6, h9B5, h9C6, h10C12, and h8B5. The humanized sequences contain mutations that may contain mouse or human germline residues at any "X" position. The mutations may contain substitutions at one or more positions. The variable domains of the anti-CD3 antibody UCHT-1, i.e., SEQ ID NOS: 11 and 12, were first humanized in 1992 (Beverley 1981 and Shalaby 1992). The amino acids at the positions designated "X" form hydrogen bonds with CD3 epsilon (Arnett 2004). Specific substitutions of these residues may result in reduced affinity for CD3.

[0090] Example 2. Characterization of the h3 / Fc group of the CDH17xCD3 bispecific antibody Of all CDH17xCD3 bispecific antibodies listed in Table 1, h10G4fL was named ARB202, and the monospecific version of ARB202 was named ARB102. ARB201 is identical to h3G1Fc, and the sequence of the humanized variable domain h3 or Lic3 is disclosed in WO2017 / 120557A1, so it is not listed in Table 1. However, ARB201 was used to demonstrate CDH17 expression in DLD-1 (colon cancer) and AGS (gastric cancer) tumor cell lines in flow cytometry analysis (Figure 3).

[0091] To determine whether ARB201 is sufficient to mediate retargeted T cell cytotoxicity against tumor cells, we used standard two-dimensional (2D) tumor cell and three-dimensional (3D) tumor cell spheroid models. CDH17-expressing colon cancer cells (DLD-1) were cultured in CellBrite. TM The tumor cells were labeled with Ficoll-Paque™ Green (Biotium, Cat. No. 30021) and plated in microtiter wells containing RPMI supplemented with 100% FCS. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and separated by density gradient centrifugation using Ficoll-Paque™ Plus (GE Healthcare) for use as effector cells. In the 3D model, tumor cells were plated in SQ 384-well Elplasia plates pre-coated with pHEMA hydrogel. TM Spheroids were formed after seeding in RPMI, 5% FBS, 2 mM L-alanyl-L-glutamine, 1 mM sodium pyruvate, and 1% penicillin / streptomycin medium in plates. For this assay, cells were incubated with or without ARB201 for 16–48 hours until spheroids formed. Dead cells, which have compromised plasma membranes, were stained with a red fluorescent dye kit (EthD-III, Biotium, Cat. No. 30002). After 1 hour, brightfield, GFP, and Texas Red were visualized using a fluorescent imager. (R) Cell analysis was performed under a filter set. IC of ARB201 50 As shown in Figure 5, the IL-10 concentration was 0.002 μg / ml in the 2D model and 0.008 μg / ml in the 3D model. This data demonstrates high efficacy, with only a four-fold decrease in the 3D model (47 pM) compared to the 2D model. 3D models may be more predictive of solid tumor cytotoxicity than 2D models. This result indicates that ARB201 is capable of mediating retargeted T cell cytotoxicity to tumor cells.

[0092] In addition to the DLD-1 cell model, gastric cancer cells (AGS) were used in CellBrite TMThe assay was performed in 2D and 3D tumor models as described for DLD-1 cells, except that the cells were labeled with GFP Green and assays were measured after 16 hours. As shown in Figure 6, the IC value of ARB201 in this assay was 0.01. 50 The concentration of ARB201 was 0.001 μg / ml in both the 2D and 3D models. This data demonstrates high efficacy in the 3D model without a decrease in efficacy compared to the 2D model. Furthermore, in live imaging studies, as shown in Figure 7, the addition of ARB201 appeared to efficiently attract individual T cells and tumor cells together. This finding supports the idea that ARB201 stimulates a cytotoxic T cell response accompanied by the release of perforin and granzymes, which induce pore formation and apoptosis, respectively.

[0093] Thus, ARB201, the Fc group of the CDH17xCD3 bispecific antibody, has high efficacy (low pM IC50) in both 2D and 3D tumor models using CRC and GC tumor cells. No reduction in efficacy was observed in the 3D GC model, and only a 4-fold reduction was observed in the 3D CRC model. Efficient killing in 3D models can be translated into clinical efficacy against solid tumors.

[0094] Example 3. Characterization of the h5 / fL group of CDH17xCD3 bispecific antibodies The CDH17xCD3 bispecific antibodies h5G1fL and h5G4fL were used to characterize the h5 / fL group of antibodies. To determine binding specificity, h5G1fL and h5G4fL were expressed and produced in CHO cells, respectively. Different clones were incubated with conditioned medium in microtiter wells coated with recombinant CDH17 or anti-human IgG. ELISA was used. Binding of h5G1fL and h5G4fL to either CDH17 or anti-human IgG (to determine production) was detected using an anti-human Fc-HRP conjugate in ELISA. Relative binding activity was measured and compared, as shown in Figure 8. The results indicated that the h5 / fL group of antibodies had binding specificity for CDH17 comparable to that of the control anti-CDH17 antibody.

[0095] Next, h5G1fL and h5G4fL were each incubated with Jurkat T cells, after which binding was detected by subsequent binding of an anti-human IgG Alexa647 conjugate in flow cytofluorimetry analysis, as shown in Figure 9, demonstrating that the anti-CD3 scFv was fully functional.

[0096] Example 4. Characterization of the h10 / fL group of CDH17xCD3 bispecific antibodies The CDH17xCD3 bispecific antibodies h10G1fL and h10G4fL were used to characterize the h10 / fL group of antibodies. To determine binding specificity, h10G1fL and h10G4fL were expressed and produced, respectively, in CHO cells. Different clones were incubated with conditioned medium in microtiter wells coated with recombinant CDH17 or anti-human IgG. ELISA was used. Binding of h10G1fL and h10G4fL to either CDH17 or anti-human IgG (to determine production) was detected using an anti-human Fc-HRP conjugate in ELISA. Relative binding activity was measured and compared, as shown in Figure 10. The results indicated that the h10 / fL group of antibodies had binding specificity for CDH17 comparable to that of the control anti-CDH17 antibody.

[0097] Next, h10G1fL and h10G4fL were each incubated with Jurkat T cells. As shown in Figure 11, binding was detected by flow cytofluorimetry analysis with subsequent binding of an anti-human IgG Alexa647 conjugate, indicating that the anti-CD3 scFv was fully functional.

[0098] Example 5. Characterization of the h10 / tB group of CDH17xCD3 bispecific antibodies The CDH17xCD3 bispecific antibodies h10G1tB and h10G4tB were used to characterize the h10 / tB group of antibodies. To determine binding specificity, h10G1tB and h10G4tB were expressed and produced in CHO cells, respectively. Different clones were incubated with conditioned medium in microtiter wells coated with recombinant CDH17 or anti-human IgG. ELISA was used. Binding of h10G1tB and h10G4tB to either CDH17 or anti-human IgG (to determine production) was detected using an anti-human Fc-HRP conjugate in ELISA. Relative binding activity was measured and compared, as shown in Figure 12. The results indicated that the h10 / tB group of antibodies had binding specificity for CDH17 comparable to that of the control anti-CDH17 antibody.

[0099] Next, h10G1fL and h5G4fL were each incubated with Jurkat T cells. As shown in Figure 13, binding was detected by flow cytofluorimetry analysis with subsequent binding of an anti-human IgG Alexa647 conjugate, indicating that the anti-CD3 scFv was fully functional.

[0100] Example 6. CDH17xCD3 bispecific antibodies with an IgG4 isotype do not activate T cells in the absence of tumor cells The CDH17xCD3 bispecific antibodies h10G1fL, h10G4fL, h10G4tB, and h3G4tB were used in this analysis. Fresh PBMCs were incubated with each of the four CDH17xCD3 bispecific antibodies at concentrations ranging from 0 to 4 μg / ml for 24 hours at 37°C in microtiter wells. T cell activation and cytotoxic responses were determined by staining cells with anti-CD107a antibody and anti-mIgG fluorescent conjugates by flow cytofluorometry. The percentage of CD107a-positive cells, indicating cytotoxic T cell activation, was plotted against antibody concentration.

[0101] As shown in Figure 14, neither fL nor IgG4 tB antibodies induced CD107a expression, whereas only tB with an IgG1 isotype induced CD107a expression. This result demonstrates the safety profile of the CDH17xCD3 bispecific antibody, which has a unique substructure morphology, and does not induce cytotoxic T cell responses as determined by CD107a expression, even when incubated with PBMCs in the absence of tumor cells.

[0102] Example 7. CDH17xCD3 bispecific antibody h10G4fL mediates tumor cell-dependent T cell activation To characterize tumor cell-dependent T cell activation by the CDH17xCD3 bispecific antibody, PBMCs and h10G4fL were incubated with or without the tumor cell line AsPC1 at a 5:1 ratio for 16 hours. T cell activation was determined by measuring IL2 production using a quantitative ELISA kit. As shown in Figure 15, in the presence of AsPC1, IL2 was activated by EC 50 At EC50 = 30 pM (A), IL2 was induced in a concentration-dependent manner by h10G4fL. In the absence of AsPC1, IL2 > (B) CDH17xCD3 bispecific antibody h10G4fL thus exhibited a potential therapeutic index of over 600-fold.

[0103] Example 8. h10G4fL redirects T cell cytotoxicity to CDH17-positive tumor cells To further characterize the function of h10G4fL, its ability to redirect T cell cytotoxicity was assessed using both CDH17-positive and -negative tumor cells. Human PBMC-derived activated T cells and h10G4fL (or no antibody) were cocultured with labeled tumor cells at a 5:1 ratio for 16 hours. At the end of the incubation, each mixture was washed, substrate was added, and the remaining viable cells were quantified and the killing rate was calculated. T cell activation was determined by measuring IL2 production using a quantitative ELISA kit. As shown in Figure 16, the results indicate that h10G4fL exhibited concentration-dependent cytotoxicity against CDH17-positive luciferase-labeled pancreatic and colonic cell lines (Figures 16A and 16B), but not against the CDH17-negative colon tumor cell line SW40 (Figure 16D). However, ectopically expressed CDH17 in SW40 cells conferred susceptibility to h10G4fL-dependent killing (FIG. 16C), demonstrating specificity for target tumor cells.

[0104] Example 9. Pharmacokinetic and toxicological analysis of h10G4fL / ARB202 To determine the pharmacokinetics of h10G4fL, mice and non-human primates were used as small and large animal models. Figure 17 shows the time course of serum concentrations of h10G4fL / ARB202 after intravenous injection at 3 mg / kg in mice (Figure 17A) and non-human primate (NHP) models (Figure 17B). For comparison, h10G1 / ARB102, an isotype variant of h10G4fL / ARB202, was used at 1 mg / kg in mice (Figure 17A) and 10 mg / kg in NHPs (Figure 17B).

[0105] Next, both h10G4fL / ARB202 and h10G1 / ARB102 were used in a preclinical cynomolgus monkey toxicity study at Charles River Laboratories. This study was designed as a 14-day single-dose study. It was previously determined that the CDH17xCD3 bispecific antibody can recognize and bind to cynomolgus monkey CDH17 using cell transfectants. This antibody also binds to monkey CDH17 in necropsy colon tissue, as shown by immunohistochemistry (IHC) analysis. However, there was no evidence that the CDH17xCD3 bispecific antibody can access and bind to CDH17 in the colon at the life stage. This issue was addressed by using post-necropsy colon tissue and an anti-human IgG antibody for IHC analysis, as shown in Figure 18. Furthermore, no diarrhea or dose-dependent fecal occult blood, which could be indicative of inflammatory tissue damage associated with antibody treatment, was observed. Overall, the pathology report concluded that animals randomized to this study were free of pathology and no gross or microscopic findings were attributable to either ARB102 or ARB202. This data supports the safety concept that CDH17, or at least its epitopes, is inaccessible in the normal colon and that therapeutic treatment may spare normal tissue.

[0106] Example 10. Efficacy analysis of ARB202 To determine the efficacy of ARB202 for the in vivo treatment of CDH17-positive tumors, a mouse xenograft model was used. Pancreatic tumor models were established in NSB mice via subcutaneous injection of AsPC-1 pancreatic tumor cells. Mice were then treated via intratumoral administration of vehicle (RPMI), T cells, T cells plus 0.05 mg / kg ARB202, or T cells plus 0.5 mg / kg ARB202 at various time points, as shown in Figure 19. Tumor volumes were determined over a 4-week period. Results show that only low and high doses of ARB202 significantly reduced tumor growth compared to vehicle (P<0.05 compared to RPMI injection). The non-statistically significant reduction in tumor growth observed with T cells alone is likely due to the high NK activity in the expanded T cell population and the NK sensitivity of AsPC-1. To further validate T cell activation during this process, serum IL-2 was analyzed. Results showed that only ARB202 treatment resulted in increased levels of human IL-2 in plasma. Thus, ARB202 provides proof of concept that CDH17xCD3 bispecific antibodies can be used to treat CDh17-positive tumors.

[0107] Pharmaceutical Composition The term "effective amount" refers to an amount of drug effective to achieve a desired effect, e.g., an amount effective to ameliorate a disease in a subject. When the disease is cancer, an effective amount of drug can inhibit (e.g., slow to some extent, inhibit, or stop) cancer cell proliferation, cancer cell division, cancer cell motility, cancer cell invasion into peripheral organs, tumor metastasis, and one or more abnormal characteristics of tumor growth, including, but not limited to, cancer cell proliferation, cancer cell division, cancer cell motility, cancer cell invasion into peripheral organs, tumor metastasis, and one or more abnormal characteristics of tumor growth. When the disease is cancer, an effective amount of drug, when administered to a subject, can alternatively do one or more of the following: slow or stop tumor growth, reduce tumor size (e.g., volume or mass), alleviate to some extent one or more symptoms associated with cancer, extend progression-free survival, induce an objective response (including, e.g., partial response or complete response), and extend overall survival. To the extent a drug can prevent growth and / or kill existing cancer cells, it is cytostatic and / or cytotoxic.

[0108] For formulating compositions suitable for administration to a subject, such as a human patient in need of treatment, the antibodies disclosed herein may be mixed or combined with pharmaceutically acceptable carriers known in the art, depending on the route of administration selected. There is no particular limitation on the method of administration of the antibodies disclosed herein, and the selection of an appropriate route of administration and an appropriate composition is known in the art and can be made without undue experimentation.

[0109] While many dosage forms are possible, an exemplary dosage form may be a solution for injection, particularly a solution for intravenous or intraarterial injection. Typically, a suitable pharmaceutical composition for injection may include a pharmaceutically suitable carrier or excipient, such as, but not limited to, a buffer, a surfactant, or a stabilizer. Exemplary buffers may include, but are not limited to, acetate, phosphate, or citrate buffers. Exemplary surfactants may include, but are not limited to, polysorbates. Exemplary stabilizers may include, but are not limited to, human albumin.

[0110] Similarly, one of ordinary skill in the art is capable of determining the effective amount or concentration of the antibodies disclosed therein to effectively treat a condition such as cancer. Other parameters, such as the proportions of various components of the pharmaceutical composition, dosage, and frequency of administration, can be determined by one of ordinary skill in the art without undue experimentation. For example, a solution suitable for injection may contain, but is not limited to, about 1 to about 20, or about 1 to about 10 mg of antibody per ml. Exemplary dosages may be, but are not limited to, about 0.1 to about 20, or about 1 to about 5 mg / kg body weight. Exemplary administration frequencies may be, but are not limited to, once daily or three times a week.

[0111] Although the disclosure herein has been described with reference to particular embodiments or examples, it can be understood that the embodiments are illustrative and that the scope of the disclosure is not so limited. Alternative embodiments of the disclosure herein may become apparent to those skilled in the art to which the disclosure herein applies. Such alternative embodiments are considered to be encompassed within the scope of the disclosure herein. Accordingly, the scope of the disclosure herein is defined by the appended claims and supported by the foregoing description.

[0112] In summary, described herein is a 2D / 3D platform for studying the retargeting T cell cytotoxicity of ARB201, a bispecific antibody targeting CDH17 and CD3. ARB201 exhibited an IC of 0.002 μg / ml in the 2D model and 0.008 μg / ml in the 3D model. 50 ARB201 induced retargeting T cell cytotoxicity in DLD-1 colon adenocarcinoma cells. ARB201 also induced retargeting T cell cytotoxicity in AGS gastric adenocarcinoma cells with an IC of 0.001 μg / ml in both 2D and 3D models. 50 This study demonstrated that ARB201 efficiently and innovatively killed tumor cells in 3D models with nearly the same efficiency as in 2D models. Efficient killing in 3D models can be translated into clinical efficacy against solid tumors.

[0113] Although the disclosure herein has been described with reference to specific embodiments, it can be understood that the embodiments are illustrative and that the scope of the disclosure is not so limited. Alternative embodiments of the disclosure herein will become apparent to those skilled in the art to which the disclosure herein applies. Such alternative embodiments are considered to be encompassed within the scope of the disclosure herein. Accordingly, the scope of the disclosure herein is defined by the appended claims and supported by the foregoing description.

[0114] The embodiments are merely for illustrating the disclosure of the present specification and are not intended to limit the scope of the disclosure. It should be understood by those skilled in the art that certain modifications and improvements may be made and should be considered under the protection of the disclosure of the present specification without departing from the principles of the disclosure of the present specification. [Table 1] [Table 2] [Table 3] JPEG0007813469000004.jpg227163JPEG0007813469000005.jpg232163JPEG0007813469000006.jpg232163JPEG0007813469000007.jpg23316 3JPEG0007813469000008.jpg227163JPEG0007813469000009.jpg227163JPEG0007813469000010.jpg233163JPEG0007813469000011.jpg93163

Claims

1. an antibody having an N-terminus and a C-terminus, comprising, in tandem from the N-terminus to the C-terminus, a first scFv domain, a second scFv domain, a hinge, a CH2 domain, and a CH3 domain; the first scFv domain has specificity for a first target; the second scFv domain has specificity for a second target, and the first target is CDH17 and the second target is CD3; An antibody comprising the amino acid sequence shown in SEQ ID NO: 34 or 35.

2. The antibody of claim 1, wherein the CDH17 comprises CDH17 ectodomains D1, D2, D3, D4, D5, D6 and D7.

3. The antibody of any one of claims 1 to 2, further comprising a conjugated cytotoxic moiety.

4. The antibody of claim 3, wherein the conjugated cytotoxic moiety comprises irinotecan, auristatins, PBDs, maytansines, amantines, spliceosome inhibitors, or combinations thereof.

5. The antibody of claim 3 , wherein the conjugated cytotoxic moiety comprises a chemotherapeutic agent.

6. An expression vector comprising an isolated nucleic acid encoding the antibody of any one of claims 1 to 5.

7. The expression vector of claim 6 , wherein the vector is capable of being expressed in a cell.

8. A host cell comprising a nucleic acid encoding the antibody of any one of claims 1 to 5.

9. A host cell comprising the expression vector of claim 7.

10. The host cell of claim 9 , wherein the host cell is a prokaryotic or eukaryotic cell.

11. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 5 and a cytotoxic agent.

12. 12. The pharmaceutical composition of claim 11, wherein the cytotoxic agent comprises cisplatin, gemcitabine, irinotecan, or an anti-tumor antibody.

13. A pharmaceutical composition comprising the antibody of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for treating a subject with cancer, comprising an effective amount of the antibody of any one of claims 1 to 5.

15. 15. The pharmaceutical composition of claim 14, wherein the cancer is liver cancer, stomach cancer, colon cancer, pancreatic cancer, lung cancer, esophageal cancer, or a combination thereof.

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