Multispecific antibody-drug conjugates (ADC) targeting CDH17-expressing tumors and method of making and using thereof

Abstract

The present disclosure provides a novel panel of fully humanized bispecific ADCs targeting CDH17 and another clinically validated target such as EGFR and HER2 for gastrointestinal cancers treatment. The bispecific ADCs offer improved target coverage, internalization rate and cytotoxicity over parental antibodies, as well as safety profile for a wider therapeutic window.

Description

[0001] MULTISPECIFIC ANTIBODY-DRUG CONJUGATES (ADC) TARGETING CDH17-EXPRESSING TUMORS AND METHOD OF MAKING AND USING THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 63 / 693,191 filed September 11, 2024, under 35 U.S.C. 119(e), the entire disclosures of which are incorporated by reference herein.

[0004] TECHNICAL FIELD

[0005] The present disclosure generally relates to the technical field of cancer immunotherapy, and more particularly to composition of antibody-drug conjugates. BACKGROUND

[0006] Antibody-drug conjugates (ADC) are a novel treatment for solid and hematological tumors, offering improved efficacy and reduced systemic toxicities comparing to conventional chemotherapy. Fifteen ADCs have been approved for marketing as of 2024 May. Despite the success of ADCs, clinical efficacy and safety are still limited by several hurdles: 1) on-target off- tumor toxicities due to target expression in normal tissues; 2) incomplete tumor eradication due to heterogeneity in target expression; 3) resistance by down-regulation of target. A potential approach to circumvent these challenges is to target two tumor targets by bispecific antibodies (BsAbs) and bispecific ADCs (BsADCs). By modulating the affinity and avidity, antibody can be engineered to bind preferentially to tumor cells over normal cells; targeting two antigens improves binding to heterogeneous tumors and resists target down-regulation. The present disclosure demonstrates the construction of BsADCs for gastrointestinal (Gl) cancer treatment.

[0007] Cadherin-17 (CDH17) is a novel tumor-associated antigen (TAA) expressed almost exclusively in healthy adult colon. It is commonly overexpressed in Gl cancer tumors and offers excellent safety profile. The present disclosure describes a BsADC targeting CDH17 and another well-known targets such as EGFR and HER2 to improve 1) target coverage; 2) affinity; 3) internalization rate and 4) cytotoxicity. The BsADC is fully-humanized, symmetric scFv-Fab- hlgGlFc format, bivalent to each target. The disclosure also describes biparatopic antibodies (BpAbs) and thereby biparatopic ADCs (BpADCs), which are bispecific to two epitopes of the sample target for improved efficacy. The affinity for each target can be modulated to optimize therapeutic window, i.e. maximize binding to tumor cells and minimize binding to normal tissues. A cytotoxic payload such as MMAE is conjugated to the antibody using a non-specific method such as maleimide-cysteine conjugation. Apart from efficacy and safety, the stability and developability of the BsAbs were also assessed as parameters of successful drug candidates.

[0008] SUMMARY

[0009] The following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description. The application provides, among others, antibody-drug conjugates (ADCs), bispecific tetravalent antibodies, methods of making the ADCs and bispecific antibodies, pharmaceutical compositions or preparations comprising ADCs or bispecific antibodies, use of ADCs or bispecific antibodies for treating disease including methods of treating cancer using the ADCs or the tetraspecific antibodies.

[0010] In one aspect, the application provides antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the ADC or a pharmaceutically acceptable salt or solvate thereof comprises a bispecific tetravalent antibody comprising two heavy chains (HCs) and two light chains (LCs) each having an N-terminal and a C- terminal, and a cytotoxin conjugated to the bispecific tetravalent antibody through a linker. Each HC may have a VH domain and a single-chain variable fragment (scFv) domain. Each LC may have a VL domain and the VH domain forming a Fab region. In one embodiment, the bispecific tetravalent antibody has a first binding specificity to human Cadherin-17 (CDH17) and a second binding specificity to a tumor-associated antigen (TAA). The TAA may be independently selected from EGFR, HER2, TROP2, a fragment or a derivative thereof.

[0011] In one embodiment, the scFv is connected to the HC at its N-terminal. In one embodiment, the scFv is connected to the HC at its C-terminal.

[0012] In one embodiment, the Fab domain may have the binding specificity to CDH17. In one embodiment, the scFv domain may have the binding specificity to CDH17. In one embodiment, the Fab domain has the binding specificity to EGFR, HER2, orTROP2.

[0013] In one embodiment, the scFv domain has the binding specificity to EGFR, HER2, or TROP2.

[0014] In one embodiment, the Fab domain has the binding specificity to CDH17, and the scFv domain has the binding specificity to EGFR, HER2, or TROP2. In one embodiment, the scFv domain has the binding specificity to HER2. In one embodiment, the scFv domain has the binding specificity to TROP2. In one embodiment, the scFv domain has the binding specificity to EGFR.

[0015] In one embodiment, the Fab domain has the binding specificity to EGFR, HER2, orTROP2, and the scFv domain has the binding specificity to CFH17. In one embodiment, the Fab domain has the binding specificity to HER2. In one embodiment, the Fab domain has the binding specificity to TROP2. In one embodiment, the Fab domain has the binding specificity to EGFR. In one embodiment, the VH domain comprises the amino acid sequence having at least

[0016] 60%, 70% 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2, 5, 6, 7, or 8.

[0017] In one embodiment, the VL domain comprises an amino acid sequence having at least 60%, 70% 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 1, 3, or 4.

[0018] In one embodiment, the VH domain comprises 3 complementary determining regions (CDRs) from SEQ ID NO. 2, 5, 6, 7, or 8. In one embodiment, the VH domain comprises 3 CDRs selected from: (1) SEQ ID NO. 18, 19 and 20, (2) SEQ ID NO. 24, 25 and 26, (3) SEQ ID NO. 30, 31, and 32, and (4) SEQ ID NO. 36, 37, and 38. In one embodiment, the VL domain comprises 3 CDRs from SEQ ID NO. 1, 3, or 4. In one embodiment, the VL domain comprises 3 CDRs selected from: (1) SEQ ID NO. 15, 16, and 17, (2) SEQ ID NO. 21, 22, and 23, (3) SEQ ID NO. 27, 28, and 29, and (4) SEQ ID NO. 33, 34, and 35.

[0019] In one embodiment, the scFv domain comprises an amino acid sequence having at least 60%, 70% 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 9, 10, 11, 12, 13, or 14. In one embodiment, the scFv domain comprises CDRs from SEQ ID NO. 9, 10, 11, 12, 13, or 14.

[0020] The cytotoxin may be an anti-microtubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizing agent, a DNA alkylating agent, or a combination thereof. In one embodiment, the cytotoxin is an anti-microtubule agent selected from the group consisting of a maytansinoid, an auristatin, and a tubulysin. In one embodiment, the cytotoxin is an auristatin selected from a group consisting of monomethyl auristatin E (MMAE). In one embodiment, the linker comprises a maleimidocaproyl (MC) group, a valine-citruline p- aminobenzylcarbamate (VC-PAB) unit, or a combination thereof.

[0021] In one aspect, the application provides method for making the antibody-drug conjugate (ADCs). In one embodiment, the method includes the step of conjugating the bispecific tetravalent antibody with the cytotoxin through the linker.

[0022] In one aspect, the application provides the pharmaceutical composition , comprising the antibody-drug conjugate as disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may further include a therapeutic agent. The representative therapeutic agent useful for the pharmaceutical composition may be an antibody, a chemotherapy agent, an enzyme, a radionuclide, or a combination thereof.

[0023] In one aspect, the application provides methods for treating or preventing cancer in a subject. In one embodiment, the method may include the step of administering to the subject a pharmaceutical composition comprising the antibody-drug conjugate disclosed herein. In one embodiment, the method includes the step of co-administering an effective amount of a therapeutic agent. Representative therapeutic agent may include an antibody, a chemotherapy agent, an enzyme, a radionuclide, ora combination thereof. The ADC may be used to treat cancer such as a cancer of gastric, esophageal, colorectal, or breast. In one embodiment, the subject may be a mammal. In one embodiment, the subject may be a human.

[0024] In one aspect, the application provides a solution comprising the antibody-drug conjugate disclosed herein. In one embodiment, the solution comprises a diagnostic agent. In one embodiment, the solution comprises blood.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:

[0027] Figure 1 depicts configurations of bispecific antibodies (BsAbs): (A) the BsAb is fully-humanized, symmetric and bivalent to each target, typically comprising of an scFv, an Fab and a hlgGlFc; (B) the scFv-IgG format showed preserved binding to both antigens whilst IgG-scFv showed reduced binding for the scFv; and (C) protein A-MMAE cytotoxicity assay was consistent with ELISA binding, showing that scFv-IgG format was more potent;

[0028] Figure 2 shows cell binding affinity / avidity of BsAbs: (A) the binding of hlOGl (CDH17) and BsAbs CXshlOGl (CDH17 / EGFR) and TZshlOGl (CDH17 / HER2) to cell lines, indicating that hlOGl bound CDH17+ but not CDH17- cells, while BsAbs bound both cell lines; and (B) the binding of BsAbs CXsh5Gl (CDH17 / EGFR) and TZsh5Gl (CDH17 / HER2) to cell lines, indicating that BsAbs bound stronger to double positive cells than CDH17- EGFR+ HER2+ cells;

[0029] Figure 3 shows internalization rate of BsAbs: (A) BpAb hlO / h9E9 internalized faster than both parental antibodies; (B) BsAb hl0C12 / cetuximab internalized faster in CDH17+ AGS cell line than cetuximab-scFv alone; and (C) BpAb h3sh5Gl demonstrated higher internalization rate than both parental antibodies by pHRodo Red method;

[0030] Figure 4 shows the results of cytotoxicity screening of BsAbs by protein A-MMAE: (A) BpAbs induced cytotoxicity on AGS cell line much more potent than (B) parental antibodies h3Gl, h5Gl, h9E9 and hlOGl; (C) anti-CDH17 / HER2 BsAbs killed AGS cell line more potent than parental antibodies; and (D) anti-CDH17 / EGFR BsAbs were also more potent than parent antibodies;

[0031] Figure 5 shows the characterization of anti-CDH17 / HER2 BsAb-drug conjugate as an example for generating BsADC: (A) TZshlOGl-MMAE was generated by maleimide-based conjugation of MMAE to TZshlOGl; (B) MMAE conjugation was verified by bridging ELISA with CDH17 protein coated on plate, and anti-MMAE detection antibody; and (C) TZshlOGl-MMAE demonstrated cytotoxicity on both CDH17+ HER2+ and CDH17- HER2+ cell lines, whilst ARB102A (hlOGl-MMAE) only killed CDH17+ cell lines;

[0032] Figure 6 shows the results of screening of anti-CDH17 / TROP2 BsADC in the configuration of IgG- scFv (hlOG4h8v4) versus scFv-IgG (h8v4hlOGl): (A) anti-TROP2 clone h8 had much higher potency than anti-CDH17 clone hlO; (B) and (C) IgG-scFv format reduced potency of h8 comparing to scFv-IgG in AsPCl (CDH17+ / TROP2-) and AGC A4 (CDH17- / TROP2+) cells; (D) IgG- scFv was still more potent than hlO; and (E) h8 with S122D or D124E were generated and h8 with S122D was selected as it demonstrated improved killing over parentals in double positive cells;

[0033] Figure 7 shows the characterization of anti-CDH17 / CDH17 BpAb-drug conjugate as an example for generating BpADC: (A) h3sh5Gl-MMAE showed improved total CDH17 binding in ELISA compared to parental antibodies; (B) h3sh5Gl-MMAE showed >10-fold improved cytotoxicity on CDH17+ cell line AsPCl; and (C) h3sh5Gl-MMAE demonstrated potent killing on CDH17+ pancreatic cancer cell lines; Figure 8 shows the potency of BpADC against standard-of-care (SOC): h3sh5Gl-MMAE showed 100-1000-fold greater potency over pancreatic cancer SOC gemcitabine;

[0034] Figure 9 shows the selectivity and specificity of the h3sh5Gl-MMAE conjugate: (A) h3sh5Gl- MMAE showed strong binding to CDH17+ AsPCl cells comparable to naked antibody; and (B) h3sh5Gl-MMAE showed binding to human and cynomolgus CDH17 but not human CDH16 nor E- cadherin;

[0035] Figure 10 shows the secondary MoAs of h3sh5Gl: h3sh5Gl-MMAE induced C3b deposition comparable to naked antibody, and the payload MMAE induced immunogenic cell death (ICD) as assayed by surface CRT MFI;

[0036] Figure 11 shows developability of h3sh5Gl: (A) and (B) h3sh5Gl was stable for short-term ambient storage, as well as up to 5 freeze-thaw cycles; and (C) when spiked in human plasma ~90% activity was retained after 7 days incubation at 37°C; and

[0037] Figure 12 shows the yield and purity of BsAb production: CHO-S cells were transfected to generate stable cell pools, and the (A) yield and (B) monomer % were compared against anti- CDH17 monospecific antibody, hlOGl.

[0038] DETAILED DESCRIPTION

[0039] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0040] The terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate.

[0041] The term "antigen" refers to an entity or fragment thereof which can induce an immune response in an organism, particularly an animal, more particularly a mammal including a human. The term includes immunogens and regions thereof responsible for antigenicity or antigenic determinants.

[0042] The term "epitope", also known as "antigenic determinant", is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells, and is the specific piece of the antigen to which an antibody binds.

[0043] The term "immunogenic" refers to substances which elicit or enhance the production of antibodies, T-cells, or other reactive immune cells directed against an immunogenic agent and contribute to an immune response in humans or animals. An immune response occurs when an individual produces sufficient antibodies, T-cells, and other reactive immune cells against administered immunogenic compositions of the present application to moderate or alleviate the disorder to be treated. The term "tumor antigen" as used herein means an antigenic molecule produced in tumor cells. A tumor antigen may trigger an immune response in the host. In one embodiment, the tumor cells express tumor antigens, including without limitation, tumor-specific antigens (TSA), neoantigens, and tumor-associated antigens (TAA).

[0044] The term “ antibody ” is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, as well as antibody fragments, such as Fab, F(ab')2, and Fv, so long as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, chimeric, single chain, multi-specific, multi-effective, human and humanized antibodies. Examples of active antibody fragments that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, as well as the products of a Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above. In some embodiments, antibody may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically bind to an antigen. The immunoglobulin can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG 1, lgG2, lgG3, lgG4, IgAl and lgA2) or subclasses of immunoglobulin molecule. In one embodiment, the antibody may be a whole antibody and any antigen-binding fragment derived from the whole antibody. Atypical antibody refers to heterotetrameric protein comprising typically of two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain moiety is comprised of a light chain moiety variable domain (abbreviated as VL) and a light chain moiety constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementarity determining regions (CDR), and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino-terminus to carboxy-terminus. Within the variable regions of the heavy and light chain there are binding regions that interacts with the antigen.

[0045] The term "bi-specific antibody (BsAb)" refers to an engineered antibody that has two binding sites each having a binding affinity to an epitope of an antigen.

[0046] The term "biparatopic antibody (BpAb)" refers to an engineered antibody that simultaneously bind to two distinct, non-overlapping epitopes on the same antigen, offering significant advantages over conventional monospecific antibodies. These enhanced antibodies provide increased binding affinity, superior cell surface receptor blockade, potent internalization and downregulation of the target, and can overcome resistance mechanisms, making them promising agents in cancer therapy and other disease treatments.

[0047] The term "humanized antibody" antibody refers to an engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity. Methods to obtain "humanized antibodies" are well known to those skilled in the art (see Queen et al., Proc. Natl Acad Sci USA, 1989; Hodgson et al., Bio / Technology, 1991). In one embodiment, the "humanized antibody" may be obtained by genetic engineering approach that enables production of affinity- matured humanlike polyclonal antibodies in large animals such as, for example, rabbits (see U.S. Pat. No. 7,129,084). The term "specific binding to" or "specifically binds to" or "specific for" a particular antigen or an epitope as used herein means the binding that is measurably different from a nonspecific interaction. Specific binding can be measured by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. Specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or an epitope can be exhibited by an antibody having a KD for an antigen or epitope of at least about 10-4 M, at least about 10-5 M, at least about 10-6 M, at least about 10-7 M, at least about 10-8 M, at least about 10-9, alternatively at least about 10-10 M, at least about 10-11 M, at least about 10-12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. In some embodiments, a multi-specific antibody that specifically binds to an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope. Also, specific binding for a particular antigen or an epitope can be exhibited by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction.

[0048] The term "antibody drug conjugate" as used herein refers to the linkage of an antibody or an antigen binding fragment thereof with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, and the like. The linkage can be covalent bonds, or non-covalent interactions such as through electrostatic forces. Various linkers, known in the art, can be employed to form the immunoconjugate.

[0049] The term "cytotoxin", or "cytotoxic agent" as used herein, refers to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.

[0050] The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0051] The term "drug moiety" or "payload" as used herein refers to a chemical moiety that is conjugated to an antibody or antigen binding fragment of the invention, and can include any therapeutic or diagnostic agent, for example, an anti-cancer, anti-inflammatory, anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic, anti-viral), or an anesthetic agent. For example, the drug moiety can be an anti-cancer agent, such as a cytotoxin. In certain embodiments, a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. Methods for attaching each of these to a linker compatible with the antibodies and method of the invention are known in the art. In addition, a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide.

[0052] Several short peptidic compounds have been isolated from the marine mollusc Dolabella auricularia and found to have biological activity. Analogs of these compounds have also been prepared, and some were found to have biological activity (see Pettit et al. Anti-Cancer Drug Design 13:243-277, 1998). For example, auristatin E is a synthetic analogue of the marine natural product Dolastatin 10, an agent that inhibits tubulin polymerization by binding to the same domain on tubulin as the anticancer drug vincristine (US 5,635,483). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds, and a C-terminal amide. The auristatin peptides, auristain E (AE) and monomethylauristatin (MMAE) are synthetic analogs of dolastatin. MMAE is an antimitotic agent which inhibits cell division by blocking the polymerisation of tubulin. The linker to the monoclonal antibody is stable in extracellularfluid but is cleaved by cathepsin once the conjugate has entered a tumor cell, thus activating the antimitotic mechanism. MMAE is one of the most commonly used payloads to make ADCs, including anti-CD20 ADC, RITUXAN® (WO 2004 / 032828) for the treatment of CD20-expressing cancers and immune disorders, ADCETRIS® (brentuximab vedotin), PADCEV® (enfortumab vedotin-ejfv), and POLIVY® (polatuzumab vedotin-piiq).

[0053] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms "patient" or "subject" are used herein interchangeably.

[0054] EXAMPLES

[0055] Example 1. Construction of BsAbs against CDH17 and EGFR / HER2

[0056] A BsAb was constructed by fusing an scFv to the C-terminal of a human lgG4 antibody (hlgG4)(lgG-scFv) or the N-terminal of a human IgGl antibody (hlgGl)(scFv-lgG) (FIG. 1A). The scFv-IgG format was found to bind both antigens equally whilst in IgG-scFv format scFv binding was greatly hindered (FIG. IB). The difference in binding was confirmed with protein A-MMAE assay as scFv-IgG showed stronger killing (FIG. 1C), therefore the former format was chosen for further development.

[0057] An anti-CDH17 clone 10C12 was humanized and cloned into hlgGl backbone (hlOGl) and was shown to bind only CDH17+ cell line OE19 but not the CDH17- cell line OE33. BsAbs were constructed by fusing cetuximab scFv and trastuzumab scFv to hlOGl to target CDH17 / EGFR (CXshlOGl) and CDH17 / HER2 (TZshlOGl), respectively. The BsAbs bound OE19 with similar affinity to parental hlOGl but with high maximum binding signal as it bound to more antigens on cell surface; the BsAbs bound OE33 as it was EGFR+ HER2+ (FIG. 2A). The BsAbs therefore offered the advantage of wider coverage of TAAs. BsAbs were constructed similarly with another a nti- CDH17 clone h5 and they bound to OE19 and OE33 with similar affinity. Binding signal to OE19 was much higher as it expressed CDH17 as well as EGFR and HER2 (FIG. 2B). The BsAb therefore bound double positive cells stronger than single positive cells, offering more specificity to target tumor cells.

[0058] Example 2. BsAbs to improve internalization and cytotoxicity

[0059] By fusing two anti-CDH17 clones, hlOGl targeting domain 1 of CDH17 and h9E92.2 targeting domain 3 of CDH17, the resultant biparatopic antibodies (BpAb) engaging both domain 1 and 3 showed improved internalization rate versus their parental antibodies (FIG. 3A). By fusing an anti-CDH17 clone h5Gl and cetuximab scFv, the BsAb demonstrated improved internalization in CDH17+ EGFR+ AGS cell line (FIG. 3B). Another BpAb h3sh5Gl engaged domain 1 and 2 showed higher internalization versus the parental antibodies, as shown by pHRodo Red flow cytometry assay (FIG. 3C).

[0060] Example 3. Protein A-MMAE as a screening platform of BsADC potency

[0061] To increase the throughput of ADC potency screening, protein A-MMAE was used instead of conjugating each BsAb / BpAb candidate. A panel of BpAbs were generated with humanized anti-CDH17 clones h3, h5, h9E9 and hlO. Three of the BpAbs tested displayed improved EC50s and maximum killing comparing to parental antibodies (FIG. 4A, FIG. 4B). For BsAbs against CDH17 / EGFR and CDH17 / HER2, anti-CDH17 clones h5 and hlO were fused with scFvs of cetuximab (CX) and trastuzumab (TZ) to form two pairs of BsAbs. CXsh5Gl and TZsh5Gl demonstrated potent killing comparable to free MMAE and were more potent than parental antibodies (FIG. 4C, FIG. 4D).

[0062] Example 4. Construction of anti-CDH17 / HER2 BsADCs

[0063] By maleimide-based non-specific conjugation, BsADCs CXshlOGl and TZshlOGl-MMAE were generated with payload-linker mc-Val-Cit-PAB-MMAE, which conjugates via free cysteine residues on the antibody (FIG. 5A). The conjugation was verified by sandwich ELISA with CDH17 protein and anti-MMAE detection antibody (FIG. 5B). In an in vitro cytotoxicity assay, TZshlOGl- MMAE killed both CDH17+ / HER2+ and CDH17- / HER2+ cell lines, while the parental antibody ARB102A (hlOGl-MMAE) only killed CDH17+ cell lines. This demonstrated wider target coverage offered by BsADCs.

[0064] Example 5. Lead optimization of CDH17 / TROP2 BsAb by affinity and format modulation

[0065] To generate a CDH17 / TROP2 BsAb, the potency to each antigen must be carefully considered. CDH17 is considered a more specific and safer target than TROP2, therefore an antibody more potent against CDH17+ and CDH17+TROP2+ than TROP2+ cells is ideal. Anti- CDH17 clone hlO and anti-TROP2 clone h8 were selected to generate a BsAb, and IgG-scFv and scFv-IgG formats were tested (FIG. 6A). Using protein-MMAE as a screening platform, h8 was shown to be much more potent than hlO (FIG. 6B), effort was needed to reduce potency of h8 to improve the safety. Consistent with data in FIG. 1, placing h8 in IgG-scFv format reduced its potency without affect hlO (FIG. 6C), but it was still slightly more potent than hlO (FIG. 6D). Several CDR mutants were further generated with the aim to lower h8 affinity, and S122 and D124E were selected to incorporate into IgG-scFv format. S122D was shown to have greatly reduced potency, and the candidate hlOG4h8(S122D) showed stronger cytotoxicity against CDH17+ and CDH17+TROP2+ cells (FIG. 6E), accomplishing the aim of this study.

[0066] Example 6. Construction of CDH17 BpADC by maleimide-based non-specific conjugation

[0067] An anti-CDH17 BpADC h3sh5Gl-MMAE was generated in the same way with payloadlinker mc-Val-Cit-PAB-MMAE. The conjugate retained binding to CDH17 (FIG. 7A) and demonstrated 30-fold improved potency on CDH17+ cell line AsPCl. No killing was observed on CDH17- cell line Capan2, demonstrating target specificity and that cytotoxicity was not due to residual free linker-payload (FIG. 7B). The BpADC was further characterized on a panel of cell lines, showing EC50s of sub-nanomolar range on CDH17+ but no killing on CDH17- cell lines (FIG. 7C).

[0068] Example 7. h3sh5Gl-MMAE as drug candidate to treat pancreatic cancers To show that h3sh5Gl-MMAE could be a second-line or third-line pancreatic cancer therapeutic, its potency was compared to gemcitabine, a standard-of-care (SOC) drug for the indication and showed up to 100-1000-fold lower EC50 than the latter (FIG. 8). h3sh5Gl was notably also capable of killing gemcitabine-resistant AsPCl effectively, suggesting it could be prescribed if previous gemcitabine treatment was ineffective. The BpADC was shown to bind CDH17+ AsPCl but not CDH17- PaTu-8988S, showing selectivity across cell lines (FIG. 9A). Crossreactivity was shown against cynomolgus CDH17, making cynomolgus monkey a useful model for toxicology study. Cross-reactivity against human CDH16 and E-cadherin was absent suggesting the antibody was specific to its intended target CDH17 (FIG. 9B). Apart from payload toxicity, secondary mechanisms-of-action (MoAs) such as complement-dependent-cytotoxicity (CDC) was assayed by C3b deposition, where the conjugate and naked antibody were both shown to be able to recruit C3b (FIG. 10A). The MMAE payload was also capable of inducing immunogenic cell death (ICD) as assayed by surface CRT MFI (FIG. 10B).

[0069] Example 8. Developability of BsAbs

[0070] Developability of anti-CDH17 BpAb h3sh5Gl was assessed by SEC-HPLC. Despite having initial aggregation at ~20%, the %monomer was stable at room temperature storage in up to 5 days (FIG. 11A); % monomer was also stable up to five free-thaw cycles (FIG. 11B). h3sh5Gl was spiked in human plasma and incubated at 37oC for one week and ~80% of activity (as assayed by CDH17 ELISA) was retained (FIG. 11C). Stable cell pools of hlOGl, TZsh5Gl and h3sh5Gl were generated by stable transfection of CHO-S cells and reached yields of l-3g / L (FIG. 12A). Higher yield was expected after single cloning. %monomer was assayed by SEC-HPLC showing high purity of hlOGl and 15-20% aggregation of the two scFv-IgG molecules (FIG. 12B). The developability of the BsAbs was considered acceptable overall.

[0071] TABLES

[0072] Table 1. Cross-Reactivity of anti-CDH17 monospecific antibodies.

[0073] Table 2. Characterization of anti-CDH17 monospecific antibodies.

[0074] Table 3A. Characterization of anti-CDH17 bispecific antibodies.

[0075] Table 3B. Characterization of anti-CDH17 bispecific antibodies.

[0076] SEQUENCE LISTING

[0077] *CDR regions in amino acid sequences are underlined.

[0078] >Sequence ID NO 1: h9E9, light chain

[0079] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSFLSASVGDRVTITCRASQDVGTAVAWYQQKPGQAPKVLIYWA

[0080] STLHTGVPSRFSGSGSGTDFTLTISNLQSEDLATYFCQQYSRYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS

[0081] GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0082] >Sequence ID NO 2: h3sh9E9Gl, heavy chain

[0083] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSpYYMYWVRQAPGKGLEWVASIS FDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSG

[0084] GGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGV

[0085] PDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAPGGGGSQVQLVQSGPELKKPGA

[0086] SVKISCKASGYTFSDYYINWVRQAPGQGLEWIGWIYPGNNNNEYAEKFRDRVTLTRDTSASTAYMELSSLTSE

[0087] DTAVYFCARWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[0088] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0089] >Sequence ID NO 3: h5, light chain MRLPAQLLGLLMLWVSGSSGEIVLTQSPATLSLSPGERATLSCRASQSIRNYLHWYQQKPGEAPRLLIYYASQSI

[0090] SGIPARFSGSGSGTDFTLTISSLETEDFAMYYCQHSNSWPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

[0091] >Sequence ID NO 4: hlO, light chain

[0092] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTST

[0093] LDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

[0094] >Sequence ID NO 5: h3sh5Gl, heavy chain

[0095] MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSpYYMYWVRQAPGKGLEWVASI SFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGS GGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKGAPGGGGSQVQLVQSGAEVKK PGASVKVSCKVSAYAFSSSWMNWVRQAPGKGLEWMGRIYPRDGDTNYNGKFKGRVTMTADTSTDTAYM

[0096] ELSSLRSEDTAVYYCAREGDGYYWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

[0097] TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

[0098] >Sequence ID NO 6: CXshlOGl, heavy chain

[0099] MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFSFTNYGVHWVRQAPGKGLEWVSVI WSGGNTDYNTSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIYYASESISGIPARFS GSGSGTDFTLTISSLEPEDFAVYYCQQNNNWPTTFGQGTKLEIKGAPGGGGSEVQLVESGGGLVQPGGSLRL SCAASGFTFSSYAMSWVRQTPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDT

[0100] AVYYCSSYTNLGAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0101] >Sequence ID NO 7: TZshlOGl, heavy chain

[0102] MEFGLSWVFLVALLRGVQCDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASF LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQ LVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSK NTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSGAPGGGGSEVQLVESGGGLVQPGG

[0103] SLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRA EDTAVYYCSSYTNLGAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] >Sequence ID NO 8: hlOG4h8v4, heavy chain

[0105] MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ.TPGKGLEWVAVI DSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP

[0106] REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLGKPAGGGGSDIQMTQSPSSLSASVGDRVTITCRASENIDNYL AWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEI KGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINS DGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSS

[0107] >Sequence ID NO 9: h3scFv

[0108] QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQ TPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCFQGSHVPLTFGAGTKLELK

[0109] >Sequence ID NO 10: CXscFv EVQLVESGGGLVQPGGSLRLSCAASGFSFTNYGVHWVRQAPGKGLEWVSVIWSGGNTDYNTSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSP ATLSLSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIYYASESISGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQNNNWPTTFGQGTKLEIK

[0110] >Sequence ID NO 11: TZscFv

[0111] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTL TISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGSEVQLVESGGGLVQPGGSLRLSC AASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVY YCSRWGGDGFYAMDVWGQGTLVTVSS

[0112] >Sequence ID NO 12: h8v4scFv DIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTL TISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA ASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCVRCSYYSY DY F D Y WG QGTLVTVSS

[0113] >Sequence ID NO 13: h8v4scFv (S122D)

[0114] DIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLT ISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAAS GFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCV RCSYYDYDYFDYWGQGTLVTVSS

[0115] >Sequence ID NO 14: h8v4scFv (D124E)

[0116] DIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTL TISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA

[0117] ASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVY YCVRCSYYSYEYFDYWGQGTLVTVSS

[0118] >Sequence ID NO 15: h3 light chain CDR1

[0119] RSSQSIVHSNGNTYLE

[0120] >Sequence ID NO 16: h3 light chain CDR2

[0121] KVSNRFS

[0122] >Sequence ID NO 17: h3 light chain CDR3

[0123] FQGSHVPLT

[0124] >Sequence ID NO 18: h3 heavy chain CDR1 DYYMY

[0125] >Sequence ID NO 19: h3 heavy chain CDR2

[0126] SISFDGTYTYYTDRVKG

[0127] >Sequence ID NO 20: h3 heavy chain CDR3

[0128] DRPAWFPY

[0129] >Sequence ID NO 21: h5 light chain CDR1

[0130] QSIRNY

[0131] >Sequence ID NO 22: h5 light chain CDR2 YAS

[0132] >Sequence ID NO 23: h5 light chain CDR3

[0133] QHSNSWPLT

[0134] >Sequence ID NO 24: h5 heavy chain CDR1

[0135] AYAFSSSW

[0136] >Sequence ID NO 25: h5 heavy chain CDR2

[0137] IYPRDGDT

[0138] >Sequence ID NO 26: h5 heavy chain CDR3

[0139] AREGDGYYWYFDV >Sequence ID NO 27: hlO light chain CDR1

[0140] QDISGY

[0141] >Sequence ID NO 28: hlO light chain CDR2

[0142] TTS

[0143] >Sequence ID NO 29: hlO light chain CDR3

[0144] LQYASSPFT

[0145] >Sequence ID NO 30: hlO heavy chain CDR1

[0146] GFTFSSYA

[0147] >Sequence ID NO 31: hlO heavy chain CDR2

[0148] IDSNGGST >Sequence ID NO 32: hlO heavy chain CDR3

[0149] SSYTNLGAY

[0150] >Sequence ID NO 33: h8 light chain CDR1

[0151] ENIDNY

[0152] >Sequence ID NO 34: h8 light chain CDR2 AAT

[0153] >Sequence ID NO 35: h8 light chain CDR3

[0154] QHYYSNQLT

[0155] >Sequence ID NO 36: h8 heavy chain CDR1

[0156] GFTFSTYT

[0157] >Sequence ID NO 37: h8 heavy chain CDR2

[0158] INSDGYNI

[0159] >Sequence ID NO 38: h8 heavy chain CDR3

[0160] ARCSYYSYDYFDY

Claims

MULTISPECIFIC ANTIBODY-DRUG CONJUGATES (ADC) TARGETING CDH17-EXPRESSING TUMORS AND METHOD OF MAKING AND USING THEREOFCLAIMSWhat is claimed is:

1. An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof, comprising a bispecific tetravalent antibody comprising two heavy chains (HCs) and two light chains (LCs) each having an N-terminal and a C-terminal, and a cytotoxin conjugated to the bispecific tetravalent antibody through a linker, wherein each HC comprises a VH domain and a single-chain variable fragment (scFv) domain, wherein each LC comprises a VL domain and the VH domain forming a Fab region, and wherein the bispecific tetravalent antibody has a first binding specificity to human Cadherin-17 (CDH17) and a second binding specificity to a tumor-associated antigen independently selected from EGFR, HER2, TROP2, a fragment or a derivative thereof.

2. The antibody-drug conjugate (ADC) of Claim 1, wherein the Fab domain has the binding specificity to CDH17.

3. The antibody-drug conjugate (ADC) of Claim 1, wherein the scFv domain has the binding specificity to CDH17.

4. The antibody-drug conjugate (ADC) of Claim 1, wherein the Fab domain has the binding specificity to EGFR, HER2, or TROP2.

5. The antibody-drug conjugate (ADC) of Claim 1, wherein the scFv domain has the binding specificity to EGFR, HER2, or TROP2.

6. The antibody-drug conjugate (ADC) of Claim 1, wherein the Fab domain has the binding specificity to CDH17 and the scFv domain has the binding specificity to EGFR, HER2, or TROP2, or wherein the scFv domain has the binding specificity to CDH17, and the Fab domain has the binding specificity to EGFR, HER2, or TROP2.

7. The antibody-drug conjugate (ADC) of Claim 1, wherein the scFv is connected to the HC at its N-terminal, or wherein the scFv is connected to the HC at its C-terminal.

8. The antibody-drug conjugate of claim 1, wherein the VH domain comprises the amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2, 5, 6, 7, or 8.

9. The antibody-drug conjugate of claim 1, wherein the VL domain comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 1, 3, or 4.

10. The antibody-drug conjugate of claim 1, wherein the VH domain comprises 3 CDRs selected from:SEQ ID NO. 18, 19 and 20,SEQ ID NO. 24, 25 and 26,SEQ ID NO. 30, 31, and 32, andSEQ ID NO. 36, 37, and 38.

11. The antibody-drug conjugate of claim 1, wherein the VL domain comprises 3 CDRs selected from:SEQ ID NO. 15, 16, and 17,SEQ ID NO. 21, 22, and 23,SEQ ID NO. 27, 28, and 29, andSEQ ID NO. 33, 34, and 35.

12. The antibody-drug conjugate of claim 1, wherein the scFv domain comprises an amino acid having SEQ ID NO. 9, 10, 11, 12, 13, or 14.

13. The antibody-drug conjugate of claim 1, wherein the scFv domain comprises CDRs from SEQ ID NO. 9, 10, 11, 12, 13, or 14.

14. The antibody-drug conjugate of claim 1, wherein the cytotoxin comprises an anti-microtubule agent, a topoisomerase inhibitor, an RNA polymerase II inhibitor, a photosensitizing agent, a DNA alkylating agent, or a combination thereof.

15. The antibody-drug conjugate of claim 1, wherein the cytotoxin is an anti-microtubule agent selected from the group consisting of a maytansinoid, an auristatin, and a tubulysin.

16. The antibody-drug conjugate of claim 1, wherein the cytotoxin is an auristatin selected from a group consisting of monomethyl auristatin E (MMAE).

17. The antibody-drug conjugate of claim 1, wherein the linker comprises a maleimidocaproyl (MC) group, a valine-citruline p-aminobenzylcarbamate (VC-PAB) unit, or a combination thereof.

18. A method for making the antibody-drug conjugate of claim 1, comprising conjugating the bispecific tetravalent antibody with the cytotoxin through the linker.

19. A pharmaceutical composition, comprising the antibody-drug conjugate of Claim 1 and a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of Claim 19, further comprising of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapy agent, an enzyme, a radionuclide, or a combination thereof.

21. A method for treating or preventing cancer in a subject, said method comprising administering to the subject a pharmaceutical composition comprising the antibody-drug conjugate of claim 1.

22. The method of Claim 21, further comprises co-administering an effective amount of a therapeutic agent, wherein the therapeutic agent comprises an antibody, a chemotherapy agent, an enzyme, a radionuclide, or a combination thereof.

23. The method of Claim 21, wherein the cancer is a cancer of gastric, esophageal, colorectal, or breast.

25. The method of Claim 21, wherein the subject is a human.

26. A solution, comprising the antibody-drug conjugate of Claim 1, wherein the solution comprises blood.

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