Compositions of TriAx antibodies and methods of making and using same

Abstract

A multispecific antibody having an N-terminus and a C-terminus comprising a first monomer comprising a VL domain from the N-terminus to the C-terminus, a first linker and a first Fc domain, a second monomer comprising a VH domain from the N-terminus to the C-terminus, a second linker and a second Fc domain, and at least a first binding domain, and the first binding domain is linked to the N-terminus or C-terminus of a multispecific antibody wherein the first monomer and the second monomer are paired by an interaction between the VL domain and the VH domain, and wherein the multispecific antibody is stabilized by a disulfide bond between a first linker and a second linker.

Description

[0001] This application is a divisional application of Chinese invention patent application (application number: 2020800835298; invention title: composition of TriAx antibody and preparation and use method thereof).

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 944,230, filed December 5, 2019, under 35U.SC119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0004] This disclosure generally relates to the technical field of cancer immunotherapy, and more specifically to compositions of modified antibodies having multiple antigen-binding specificities. Background Technology

[0005] Unless otherwise stated herein, the materials described in this section are not prior art to the claims of this application and are not acknowledged as prior art by virtue of their inclusion in this section.

[0006] Despite recent advances in drug discovery and clinical imaging, cancer remains one of the deadliest diseases in humans. Our understanding of how tumors begin, how they survive stress, how they settle / metastasize to distant organs and sites, and how they become resistant to drugs remains limited. The American Cancer Society estimates that there were 1.6 million new cancer cases in the United States in 2014, and there is no approved curative treatment for most major types of cancer.

[0007] Gastrointestinal (GI) cancers (colorectal cancer, stomach cancer, pancreatic cancer, esophageal cancer, bile duct cancer, and liver cancer) are leading causes of morbidity and mortality worldwide. Colorectal cancer alone accounts for approximately 10% of all cancer diagnoses and is the second leading cause of cancer death worldwide. In China, liver and stomach cancers are among the deadliest malignancies globally, accounting for over half of all diagnoses and causing >1.42 million deaths annually worldwide. This is believed to be attributable to viral / bacterial endemic diseases (hepatitis B virus [HBV] and Helicobacter pylori infection), chemical poisoning, environmental pollution, and food contamination. There are no effective treatments. Therefore, the development of potential drugs for these aggressive cancers requires novel biomarkers and therapeutic targets. Molecularly targeted agents proven to eliminate or inhibit the growth of these cancers would have significant clinical value and substantial market impact. If the disease is diagnosed early, these tumors can be effectively removed surgically. Unfortunately and very often, most GI cancers are asymptomatic and are detected at a very late stage when they do appear in the clinic. Without effective treatment, these patients either die shortly after diagnosis or relapse after salvage therapy.

[0008] CDH17 is a significant cancer biomarker characterized by its overexpression in both liver and gastric cancers, but not from normal tissues of healthy adults. Anti-CDH17 monoclonal antibodies have shown inhibitory effects on the growth of liver and gastric tumor cells. CDH17 is highly expressed in metastatic cancers, and blocking CDH17 expression and function significantly reduces lung metastases in hepatocellular carcinoma (HCC). These observations suggest that humanized anti-CDH17 antibodies could be developed as targeted therapeutic agents for cancer patients with indications of CDH17 biomarkers in tumor tissues and / or serum samples. While antibody-drug conjugates are promising as antibody therapies, multispecific antibody therapeutics utilize the immune response to cancer and activate T cell-mediated cytotoxicity against cancer cells.

[0009] Bispecific antibodies targeting CD3-positive T cells and CD19-positive B cells have been shown to be effective in treating hematologic malignancies (Labrijn 2019, Yu 2017, Suurs 2019, and Bates 2019). However, attempts to target solid tumors have shown limited success, likely due to a lack of entry into solid tumor cells and appropriate immunomodulatory signals. Antibody-based scaffolds are needed to effectively target multiple tumor antigens and immune cell antigens or products to generate more effective immunotherapeutic agents that better address the complexities of the pro-tumor microenvironment and tumor escape mechanisms. Summary of the Invention

[0010] In one aspect, this application provides multispecific antibodies. The antibodies can be bispecific, trispecific, tetraspecific, or pentaspecific. The antibodies can have a truncated structure.

[0011] In one embodiment, this application provides a multispecific antibody having an N-terminus and a C-terminus, comprising a first monomer, a second monomer, and at least a first binding domain. The first monomer includes a VL domain from the N-terminus to the C-terminus, a first adapter, and a first Fc domain. The second monomer includes a VH domain from the N-terminus to the C-terminus, a second adapter, and a second Fc domain. The first binding domain is connected to either the N-terminus or the C-terminus of the multispecific antibody. The first monomer and the second monomer pair through an interaction between the VL domain and the VH domain, and the multispecific antibody is stabilized by a disulfide bond between the first adapter and the second adapter.

[0012] In one embodiment, the first binding domain is connected to the VH domain at the N-end, the VL domain at the N-end, the first Fc domain at the C-end, or the second Fc domain at the C-end.

[0013] In one embodiment, the multispecific antibody further includes a second binding domain, and the antibody is trispecific. In one embodiment, the first binding domain is connected to the C-terminus of the first Fc domain and the second binding domain is connected to the C-terminus of the second Fc domain. In one embodiment, the first binding domain is connected to the N-terminus of the VH domain and the second binding domain is connected to the C-terminus of the first Fc domain.

[0014] In one embodiment, the multispecific antibody further includes a second binding domain, and the antibody is a trispecific antibody. In one embodiment, the first binding domain and the second binding domain are connected to opposite ends of the antibody. In one embodiment, the first binding domain and the second binding domain are connected to the same end of the antibody. In one embodiment, the first binding domain is connected to the N-terminus at the VH domain and the second binding domain is connected to the N-terminus at the VL domain.

[0015] In one embodiment, the multispecific antibody further includes a third binding domain, and the antibody is a tetraspecific antibody. In one embodiment, the first binding domain is connected to the N-terminus of the VH domain, the second binding domain is connected to the N-terminus of the VL domain, and the third binding domain is connected to the C-terminus of either the first Fc domain or the second Fc domain.

[0016] In one embodiment, the multispecific antibody further includes a fourth binding domain, and the antibody is five-specific. In one embodiment, a third binding domain is attached to the C-terminus of the first Fc domain, and a fourth binding domain is attached to the C-terminus of the second Fc domain.

[0017] All binding domains may have binding affinity for different antigens. Alternatively, some binding domains may have binding affinity for the same antigen as another binding domain. In one embodiment, the first and second binding domains are the same. In one embodiment, the first and second binding domains are different. In one embodiment, the first, second, and third binding domains are different from each other. In one embodiment, the first, second, and third binding domains are different from each other, and a fourth binding domain is the same as one of the first, second, and third binding domains.

[0018] Each first binding domain can be independently selected from the group consisting of scFv domains, ligands, single-domain nanobodies, binding regions of natural proteins, chemokines, and cytokines.

[0019] In one embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO.1, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO.2.

[0020] In one embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 1, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 3. In another embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 1, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 4. In yet another embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 5, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 6.

[0021] In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 7, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 8. In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 9, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 10. In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 11, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 2. In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 12, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 4. In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 12, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 2.

[0022] In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 14, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 15.

[0023] In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 14, and a second monomer comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 16.

[0024] In one embodiment, the binding domain can be attached to a multispecific antibody via a linker. In one embodiment, the linker comprises a proline-rich amino acid sequence. In one embodiment, the linker may comprise at least 20%, 30%, or 50% proline residues. In one embodiment, the linker may comprise about 2 to about 31 amino acids.

[0025] On the other hand, this application provides isolated nucleic acid sequences encoding multispecific antibodies as disclosed herein.

[0026] In another aspect, this application provides expression vectors that include isolated nucleic acid sequences as disclosed herein.

[0027] In another aspect, this application provides host cells comprising isolated nucleic acid sequences as disclosed herein.

[0028] In another aspect, this application provides a method for generating multispecific antibodies as disclosed herein. In one embodiment, the method includes culturing host cells to express a DNA sequence encoding a multispecific antibody, and purifying the multispecific antibody.

[0029] In another aspect, this application provides a method for preparing multispecific antibodies. In one embodiment, the method includes the steps of culturing host cells under conditions that generate the multispecific antibodies and recovering the antibodies.

[0030] In another aspect, this application provides immunoconjugates. In one embodiment, the immunoconjugate comprises a multispecific antibody and a cytotoxic agent. In another embodiment, the immunoconjugate comprises a multispecific antibody and an imaging agent.

[0031] In another aspect, this application provides pharmaceutical compositions. In one embodiment, the pharmaceutical composition comprises a multispecific antibody and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may further comprise a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof. In one embodiment, the pharmaceutical composition may comprise an immunoconjugate as disclosed above and a pharmaceutically acceptable carrier.

[0032] In another aspect, this application provides a method for treating or preventing cancer in a subject. In one embodiment, the method includes administering to the subject a pharmaceutical composition comprising a purified multispecific antibody disclosed herein. In one embodiment, a method for treating a subject with cancer includes administering to the subject an effective amount of the multispecific antibody disclosed herein. In one embodiment, the method may further include administering in combination an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. The subject may be a human.

[0033] In another aspect, this application provides a solution comprising an effective concentration of the multispecific antibody disclosed herein, wherein the solution is the plasma of a subject. Attached Figure Description

[0034] Embodiments according to this disclosure can now be described with reference to the accompanying drawings, wherein the same reference numerals denote the same elements.

[0035] Figure 1 The conformations of a class of multispecific antibodies collectively referred to as TriAx are described, including but not limited to TriAx-A, TriAx-C, TriAx-D, TriAx-E, TriAx-I, and TriAx-J antibodies;

[0036] Figure 2 The generation, heterodimerization, and purification of TriAx-A antibody are illustrated;

[0037] Figure 3 The generation and binding specificity of TriAx-C antibody were demonstrated;

[0038] Figure 4 The thermal stability of the TriAx antibody was demonstrated;

[0039] Figure 5 This demonstrates the cytotoxicity of redirected T cells by targeting TROP2 with the TriAx-A antibody;

[0040] Figure 6 This demonstrates the retargeting T cell cytotoxicity achieved by targeting FAP with the TriAx-A antibody;

[0041] Figure 7A low-affinity anti-CD3 binding domain with amino acid substitutions was described;

[0042] Figure 8 TriAx-A containing L4 was shown to exhibit reduced T cell affinity and activation;

[0043] Figure 9 This demonstrates that TriAx-A containing L4 mediated cytotoxicity comparable to TriAx-A without the L4 mutation;

[0044] Figure 10 The specificity of T cell cytotoxicity binding and the retargeting of T cell cytotoxicity were demonstrated by the TriAx-E antibody.

[0045] Figure 11 The spatial effects of additional binding domains on TriAx core functions, such as anti-CD3 binding affinity, are described.

[0046] Figure 12 The stabilized scFv(LocV) binding domain in the TriAx antibody was described; and

[0047] Figure 13 Stable, low-antigenic linkers in TriAx antibodies are described. Detailed Implementation

[0048] In the following detailed description, reference is made to the accompanying drawings, which form a part of this document. In the drawings, similar symbols generally denote similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended 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, as generally described herein and illustrated in the drawings, aspects of this disclosure can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are explicitly contemplated herein.

[0049] To enable immunotherapeutic agents to treat cancer, particularly solid tumors, more effectively, combination therapies are crucial, incorporating multiple target specificities and / or mechanisms of action beyond those of typical bispecific antibodies. Essentially, combination therapies as described herein are necessary for effective cancer treatment and more frequent, complete, and durable responses. Specifically, a scaffold with certain properties is required to create combination therapies with favorable mechanisms of action, manufacturing, pharmacokinetics, and low antigenicity compared to approved bispecific antibodies. Many bispecific antibodies based on whole antibodies can have a larger mass than the trispecific antibodies described herein. While antibodies based on antibody fragments with smaller masses may have greater tumor penetration, they often have relatively poorer pharmacokinetic properties, such as the FDA-approved bispecific antibody Blincyto, which lacks an Fc region. Furthermore, many bispecific antibodies based on knock-in-hole mutations within constant domains of the Ig structure may contribute to anti-drug antibody (ADA) responses. In this paper, a group of modified antibodies, described herein as triaxial or TriAx antibodies, do not require mutations in any constant domain but possess multiple antigen-binding specificities.

[0050] All forms of TriAx antibodies contain a characteristic core structure comprising a single pair of VH and VL (Fv) domains defining primary antigen binding specificity, while also correctly driving the heterodimerization of two Fc-containing monomers. This core structure is stabilized by forming multiple disulfide bonds at the C-terminus of the Fv region. Minimally, a third linker (“triaxial” core) is used to add at least one additional antigen-binding domain, such as scFv. A second linker can be added for a second scFv, and so on, to increase tumor cell binding specificity or modulate the immune response. These “TriAx” antibodies can be further modified with engineered proline-rich rigid peptide linkers to position the binding domains for optimal ligand binding. TriAx antibodies can be composed entirely of human, humanized, and low-antigenic linker sequences to reduce the risk of ADA response.

[0051] TriAx antibodies are designed to bind to two or more effector cell receptors to induce two or more antitumor activity mechanisms, such as T or NK-mediated cytotoxicity (CD3, NKG2D), tumor cell phagocytosis (FcR, CR3, CR4, AXL, CD13, CD206) or apoptosis (DR5, i.e., death receptor 5), immune cell stimulation (CD40, OX40), immune checkpoint inhibition (PD-L1, TIGIT, PD1, CTLA4) or the shift of tumor-associated macrophages (TAMs) from immunosuppression to an inflammatory phenotype (CD206, TREM-2).

[0052] like Figure 1As shown, the TriAx platform allows the generation of TriAx-A, TriAx-C, TriAx-D, TriAx-E, TriAx-I, and TriAx-J antibodies. Multispecific antibodies, namely bispecific, trispecific, tetraspecific, and pentaspecific antibodies, can be generated according to these forms. The TriAx antibody core is characterized by the direct linking of paired Fv regions of the VL and VH domains to the Fc domain in the absence of CH1, and this core can be formed and stabilized by pairing two asymmetric monomers, namely LC and HC monomers, via disulfide bridges (Ig hinges or other linkers) (Table 1). The Fv-Fc core has at least one additional linker linked to an antigen-binding domain that binds to the target antigen / ligand. TriAx-A is a bispecific antibody form in which an scFv domain covalently linked to the N-terminus of VH is added. TriAx-C is a trispecific antibody form in which a second scFv domain covalently linked to the N-terminus of VH and a second scFv domain covalently linked to the C-terminus of the CH3 domain are added. TriAx-D is a trispecific antibody formulation with two distinct scFv domains added to its C-terminus. TriAx-E is a trispecific antibody formulation with two distinct scFv domains added to its N-terminus and linked to VL and VH, respectively. TriAx-I is a tetraspecific antibody formulation with a third scFv domain added to the C-terminus of TriAx-E; and TriAx-J is a pentaspecific antibody formulation with a fourth scFv domain added to the C-terminus of TriAx-I. Other multispecific antibody forms lack the TriAx core structure that guides and covalently stabilizes this multispecific heterodimer form, including BiTE, DART-Fc, IgG-scFv, TandAb, DVD-Ig, CrossMab, Duobody, Fab-scFv-Fc, ADAPTIR, ImmTac, TriKE, scFv-scFv-scFv, CODV-Ig, Two-in-one, Tandem-scFv-Fc, scFv-Fc knobs-Into-holes, F(ab')2, and scDiabody-Fc (Labrijn 2019, Yu 2017, Suurs 2019, and Bates 2019). In context, the examples below illustrate that not only can TriAx antibodies be generated, but they also function with the designed efficiency and stability. The generation of these TriAx antibodies demonstrates a higher percentage of correctly formed heterodimers compared to other modified antibodies, such as knob-into-hole antibody types.

[0053] As used in this document, the terms “a”, “an”, and “the” are defined as meaning “one or more” and include plural forms, unless the context is inappropriate.

[0054] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with multi-epitope specificity, and antibody fragments such as Fab, F(ab')2, and Fv, provided they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal antibodies, chimeric antibodies, single-chain antibodies, multispecific antibodies, multipotent antibodies, human antibodies, 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 products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the aforementioned antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, i.e., molecules containing binding sites that specifically bind to antigens. Immunoglobulins may be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecules. In one embodiment, an antibody can be a whole antibody or any antigen-binding fragment derived from a whole antibody. A typical antibody is a heterotetrameric protein that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain. Each light chain portion consists of a light chain partially variable domain (VL) and a light chain partially constant domain. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. Binding regions that interact with the antigen exist within the variable regions of the heavy and light chains.

[0055] The term "multispecific" antibody, as used herein, refers to an antibody having at least two binding sites, each with binding affinity to an epitope of the antigen. The terms "bispecific, trispecific, tetraspecific, or pentaspecific" antibody, as used herein, refer to antibodies having two, three, four, five, or six antigen-binding sites.

[0056] The term "humanized antibody" refers to an engineered antibody type that has a CDR derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulins. Furthermore, the framework support residues can be modified to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art (see Queen et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1989; Hodgson et al., Bio / Technology, 1991). In one embodiment, "humanized antibodies" can be obtained through genetic engineering methods that enable the production of affinity-mature human-like polyclonal antibodies in large animals such as, for example, rabbits (see U.S. Patent No. 7,129,084).

[0057] The term "antigen" refers to an entity or fragment thereof that can induce an immune response in organisms, particularly animals, and more particularly in mammals, including humans. This term includes immunogens and the regions responsible for antigenicity or antigenic determinants.

[0058] The term "epitope," also known as "antigenic determinant," is an antigenic portion that is recognized by the immune system, particularly antibodies, B cells, or T cells, and is a specific portion of the antigen that binds to antibodies.

[0059] The term "immunogenicity" refers to a substance in humans or animals that induces or enhances the production of antibodies, T cells, or other reactive immune cells against an immunogenic agent and contributes to an immune response. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells against the administered immunogenic composition of this application to alleviate or reduce the condition to be treated.

[0060] As used herein, the term "tumor antigen" refers to antigen molecules produced in tumor cells. Tumor antigens can elicit an immune response in the host. In one embodiment, tumor cells express tumor antigens, including but not limited to tumor-specific antigens (TSA), neoantigens, and tumor-associated antigens (TAA).

[0061] As used herein, the terms "specifically bind to," "specifically bind to," or "specifically target" a specific antigen or epitope refer to a binding that is measurably distinct from nonspecific interactions. Specific binding can be measured by determining the binding of a molecule compared to a control molecule, typically a molecule with a similar structure that does not possess binding activity. Specific binding can be determined by comparison with a control molecule similar to the target. Specific binding to a specific antigen or epitope can be achieved by having at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6M, at least about 10 -7 M, at least about 10 - 8 M, at least about 10 -9 At least about 10 alternatives -10 M, at least about 10 -11 M, at least about 10 -12 Antibodies with a KD of M or greater than that of the antigen or epitope are expressed, where KD refers to the dissociation rate of a specific antibody-antigen interaction. In some embodiments, multispecific antibodies that specifically bind to the antigen will have a KD of 20, 50, 100, 500, 1000, 5,000, 10,000, or more times greater than that of the antigen or epitope for the control molecule. Furthermore, antibodies with a KA or Ka that is at least 20, 50, 100, 500, 1000, 5,000, 10,000, or more times greater than that of the epitope than the control may exhibit specific binding to a specific antigen or epitope, where KA or Ka refers to the binding rate of a specific antibody-antigen interaction.

[0062] Example

[0063] This disclosure is 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 stated. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same or similar results.

[0064] Example 1: Characteristics of TriAx Antibodies

[0065] TriAx antibodies are heterodimers characterized by an Fv-Fc core structure comprising an Fv region, a modified Ig hinge, and an IgFc region from the N- to C-terminus. Figure 1 As shown. Additional binding domains can be scFv, scFab, Fab, single-domain VH, or native protein fragments.

[0066] The TriAx core components include two linkers (e.g., glycine-rich linkers fused to a truncated Ig hinge) that covalently link the VH and VL chains of Fv to the CH2-CH3 monomer. These flexible, glycine-rich linkers, along with the Ig hinge, facilitate efficient VH-VL pairing. The TriAx binding domain can be attached via flexible glycine-rich linkers (such as PAGGGGS) or more rigid proline-rich linkers (such as PAGPPP). The linker length is typically 4 to 7 residues. The TriAx Fc can consist of an IgG1 hinge or an IgG4 hinge with an S228P substitution. The first 7 N-terminal amino acids of the IgG1 hinge, EPKSCDK, can be replaced with glycine-rich linkers such as GAPGGGG or PAGGGGS. The hinge residues at positions 234 and 235 (G1 number) can be LL, FL, or AA to modulate the degree of FcR binding (Saunders 2019). Both the CH2 and CH3 domains can be IgG1 or IgG4 or a combination thereof, such as G1 CH2 and G4 CH3. TriAx molecules can have substituted CH3 to produce a knock-into-hole (Merchant 1998).

[0067] Built upon a central TriAx Fv-Fc core structure, TriAx-A is a bivalent antibody form with a single scFv linked to the VH or VL of the Fv, such as h10Ta, h5Ta, h8Ta, and hB2Ta antibodies, whose structural features are listed in Table 1. TriAx-C is a trivalent antibody form in which one scFv is added to the N-terminus of the VH or VL, and a second scFv or protein-binding domain is added to the C-terminus of the CH2-CH3 monomer, such as the hC3dh10Tc antibody, whose structural features are listed in Table 1. TriAx-D is a trivalent antibody with scFvs linked to the C-terminus of each CH3 of the Fv-Fc core, such as the h8C3dTa antibody, whose structural features are listed in Table 1. TriAx-E is a trivalent antibody form in which two scFvs are linked to the VH and VL of the Fv-Fc core, respectively. The structural features and sequence IDs of examples of TriAx-E antibodies such as h10Te, h8Te, and h8h10Te are listed in Table 1. TriAx-I is a tetravalent antibody form in which an scFv is added to the N-terminus of each VH and VL, plus an scFv added to the C-terminus of any CH2-CH3 monomer. TriAx-J is a pentavalent form in which an scFv is added to the N-terminus of each VH and VL and the C-terminus of each CH2-CH3 monomer.

[0068] Example 2, TriAx-A antibody

[0069] h8Ta is a TriAx-A bispecific antibody targeting TROP2 and CD3 (SEQ ID NO. 1 and 4, see also Table 1). TROP2 is a transmembrane glycoprotein that is dysregulated in all cancer types independent of baseline TROP2 expression levels. TROP2 is an ideal candidate for targeted therapeutics. Several TROP2-targeting antibody therapies in early clinical trials have demonstrated safety and clinical benefit in the treatment of triple-negative breast cancer, platinum-resistant urothelial carcinoma, and small cell lung cancer.

[0070] h8Ta was generated in HEK293 cells by co-transfection of heavy (core Fv VH) and light (core Fv Vk) chains of plasmids with PEI. On day 3 post-transfection, SDS-PAGE was performed on the culture medium samples, as shown... Figure 2 As shown; transfected cell culture medium (lanes 1 and 2), and simulated transfection culture medium (lane 3). After one-step protein-A purification, h8Ta antibody was subjected to SDS-PAGE under non-reducing and reducing conditions, as shown in the figures. Figure 2 Lanes 4 and 5 are shown. Under non-reducing conditions, this TriAx-A antibody migrates as a ~116 kDa protein, consistent with its calculated heterodimer size. Under reducing conditions, the heavy chain is ~70 kDa and the light chain is ~44 kDa, consistent with its calculated size. The heterodimerization efficiency is approximately 90% or higher. No other significant TriAx products or fragments were detected compared to the simulated control medium. These TriAx-A antibodies can have linker and Fc sequences of varying lengths and compositions to modify FcR binding and cycling half-life.

[0071] Example 3: TriAx-C antibody

[0072] Two TriAx-C antibodies with binding specificity to the phagocytic receptor CR3 were generated. h10Cd3Tc is a TriAx-C trispecific antibody targeting CDH17, CR3, and CD3 (SEQ ID NO. 7 and 8). h8C3dTd is a TriAx-D trispecific antibody targeting TROP2, CR3, and CD3 (SEQ ID 9 and 10). TROP2 and CDH17 are significant cancer biomarkers, characterized by their overexpression in various forms of solid tumors, including the stomach, colon, pancreas, liver, and hepatomegaly. CDH17 is highly expressed in metastatic cancers, and blocking CDH17 expression and function significantly reduces lung metastases in hepatocellular carcinoma (HCC). Both the anti-CDH17 monoclonal antibody and the anti-CDH17 / CD3 bispecific antibody have shown growth inhibitory effects on liver and gastric tumor cells (see the applicant's application WO / 2019 / 222428, the entire contents of which are incorporated herein by reference). CR3, or complement receptor 3, is a heterodimer of α (CD11b) and β (CD18) transmembrane glycoproteins. The I-domain containing the α-integrin binds to the β2 chain (ITGB2) to form a leukocyte-specific integrin called macrophage receptor 1 ('Mac-1') or inactivated C3b (iC3b) receptor 3. During opsonization, C3d is deposited on the surface of target cells, where it acts as a macrophage CR3 ligand for phagocytosis. Binding to CR3 via its ligands, such as C3d or activating antibodies, can direct the major phagocytic receptor to tumor cells and broadly induce tumor cell phagocytosis and pro-inflammatory macrophage polarization. In this context, TriAx-C antibodies, such as h8C3dTd and h10C3dTc, can bind to TROP2, CDH17, or both on tumor cells, and then present C3d for macrophage CR3-dependent phagocytosis. TriAx-C antibodies can also bind to FcR, which can further activate and enhance the phagocytic activity of macrophages at the CR3 level. These TriAx-C antibodies can broadly target different tumor types, achieving greater efficacy and safety compared to targeting CD47 or CD24 phagocytic checkpoints.

[0073] In addition to the anti-CD3 Fv and anti-CDH17 scFv domains, h10Cd3Tc includes C3d as a CR3-binding domain. When expressed in HEK293 cells, the heavy chain (core Fv Vh) and light chain (core Fv Vk) of h10Cd3Tc were co-transfected at ratios of 1:1, 4:1, 6:1, and 12:1 (Vh:Vk). Antibody expression levels were determined by Octet (BLI) 3 days post-transfection. Production levels were 104 μg / ml (1:1), 27.3 μg / ml (4:1), 22 μg / ml (6:1), 21.7 μg / ml (8:1), and 14.6 μg / ml (12:1). SDS-PAGE was performed on the production medium samples. Figure 3 As shown in Figure A, the molecular weight of h10C3dTc is approximately 180 kDa, slightly larger than the calculated weight of ~150 kDa. No other significant TriAx products or fragments were detected compared to the simulated control medium. Because the heavy and light chain monomers of h10Cd3Tc are of similar size, the formation of homodimers could not be easily distinguished by standard SDS-PAGE. When the concentration was adjusted to 5 μg / ml in ELISA to quantitatively bind to the immobilized CR3 I domain, peak binding to the I-domain was achieved at a plasmid ratio of 6:1. Figure 3 B). Due to the low affinity (~400 nM) of C3d binding to the I domain, the OD value in ELISA was low. However, the peak binding was approximately 7 times higher than that from control medium derived from simulated transfection of HEK293. The binding of h10C3dTc to CDH17 and CD3 was determined in ELISA, where the sample antibody bound to soluble form of CD3, followed by immobilization of CDH17, and subsequently to an HRP conjugate bound to recombinant CD3. Figure 3 As shown in Figure C, a plasmid ratio of 4:1 leads to peak binding, indicating that optimal heterodimerization may occur when plasmids are co-transfected into HEK293 cells at a specific ratio. The binding activity indicated in the ELISA can generate trispecific TriAx-C antibodies. These TriAx-C antibodies can have linkers and Fc sequences of varying lengths and compositions to modify FcR binding and cycling half-life.

[0074] Example 4: Thermal stability of TriAx antibodies

[0075] Thermostability of TriAx antibodies was determined using SYPRO Orange in a heat transfer assay (Kinget et al., 2011). TriAx-A antibodies such as h8Ta (SEQ ID 1 and 4), hB2Ta (SEQ ID 1 and 5), and hA12Ta (SEQ ID 1 and 6) (see Table 1) were analyzed. These TriAx-A antibodies in PBS were centrifuged for 10 min in a microcentrifuge and the concentration was adjusted to 5 μM. A mixture of TriAx-A (50 μL) and SYPRO Orange (1 μL) (125X in PBS; final 2.5X) was transferred to optically clear 96-well plates for assays in a qPCR instrument. The temperature was increased from 25 °C to 99 °C at a rate of 1 °C per minute and held for one minute for each measurement, with excitation at 470 nm and emission at 586 nm. Figure 4 The initial unfolding peaks at 66℃ (hB2Ta), 68℃ (hA12Ta), and 72℃ (h8Ta) are shown, indicating that the TriAx platform antibody is sufficiently stable for further development.

[0076] Example 5: Redirected T-cell cytotoxicity of TriAx-A antibodies targeting TROP2 and CD3

[0077] To evaluate the function of the TriAx platform antibody h8Ta, the retargeting T-cell cytotoxicity of the TriAx-A bispecific antibody (see Example 2) was assessed. Three luciferase-expressing GI tumor cell lines—DLD1 (colorectal cancer), SW480 (colorectal cancer), and AGS (gastric cancer)—were used for 24-hour assays, with an E:T ratio of 4. After washing to remove dead cells, viable cells were quantified using Bio-Glo (Promega) and a multi-mode plate reader. Figure 5 As shown in the figure above, h8Ta was used to detect TROP2 expression in all three tumor cell lines in flow cytofluorimetry analysis. Figure 5 A, Figure 5 B and Figure 5 C). In the absence of T cells, cell viability was not reduced within the h8Ta antibody concentration range. In the presence of T cells, the EC50 values ​​for h8Ta antibody-dependent tumor cell killing were determined as follows: DLD 0.8 pM, AGS 2 pM, SW480 11 pM. Figure 5 The image below, Figure 5 D、 Figure 5 E and Figure 5 F). The lower EC50 value of SW480 appears to be associated with its lower level of TROP2 expression. Therefore, this bispecific TriAx-A antibody may mediate effective killing of subpM tumor cells.

[0078] Example 6: Redirected T-cell cytotoxicity of TriAx-A antibodies targeting FAP and CD3

[0079] Fibroblast activation protein α (FAPα) is a 97 kDa type II cell surface glycoprotein belonging to the serine protease family. FAPα plays a crucial role in colorectal cancer (CRC) metastasis. It has been reported that FAPα is expressed in cancer-associated fibroblasts in all CRC samples examined, but not in normal colon, hyperplastic polyp, or adenoma samples.

[0080] TriAx-A bispecific antibodies hB2Ta (SEQ ID 5 and 6) were generated to target CD3 (via Fv) and FAP (via scFv). To determine its redirected T cell cytotoxic activity, FAP mRNA was electroporated into DLD1 cells that also expressed luciferase (DLD1-FAP). The following day, microtiter plate cytotoxicity assays were initiated with and without expanded T cells (E:T = 4). The assay was incubated for 24 hours after adding the antibody to a mixture with DLD1 or DLD1-FAP at the experimental concentration range, followed by washing, addition of Bio-Glo substrate, and a multi-mode plate reader for measuring luciferase activity. Figure 6 As shown in Figure A, h1B2Ta mediated concentration-dependent tumor cell cytotoxicity in the presence of T cells and DLD1-FAP (EC50 = 41 pM), while such cytotoxicity was undetectable or unconfirmed in the absence of FAP-expressing tumor cells or T cells. FAP expression in DLD1 cells was determined by flow cytometry at the start of the assay. Figure 6 B). The percentage of DLD1 cells expressing FAP at 68% (MFI = 4,256) appears to be associated with ~70% maximum cytotoxicity. Therefore, TriAx antibodies with binding specificity to FAP effectively kill tumor cells with low FAP expression.

[0081] Example 7, L4, low-affinity anti-CD3 binding domain

[0082] To reduce the risk of extratumor T cell signaling and T cell and lymphoid tissue sedimentation, the low-affinity monovalent CD3-binding region L4 (SEQ ID 13) was introduced into TriAx platform antibodies. The CDRs of UCHT1 Vh and Vk (Shalaby 1992) were partially or completely replaced with human germline sequences to generate low-affinity and low-antigenic anti-CD3 variants. Replacing Vk CDR1 with the IGKV1-33*01 germline sequence produced the lower-affinity mutant L4, which is incorporated into the core Fv of several TriAx antibodies. Therefore, the amino acid sequence of this anti-CD3 variant includes the UCTH1CDR sequence, except for CDRL1 substitution, R24Q, and R30S, as shown below. Figure 7 As shown.

[0083] Example 8: T cell affinity and activation mediated by TriAx antibody with L4.

[0084] To assess their affinity for and activation of T cells, TriAx antibodies containing L4 or its parent FvVk CDR1 (“wt”), namely h10Ta-L4 (SEQ ID 1 and 2) and h10Ta-wt, were identified by flow cytometry. Figure 8 The affinity of antibodies binding to peripheral blood T cells was determined within the concentration range shown in A. MFI was plotted and affinity (EC50) was determined using GraphPad PRISM. Three independent affinity determinations and their mean are indicated. Results indicated that modified CDRL1 (320 nM) in L4 resulted in a 5-fold decrease in T cell affinity relative to the parental Fv (60 nM). T cell signaling was determined using a T cell line with an NFAT-inducible promoter for luciferase expression (Jurkat Promega Kit NFAT J1621). h10Ta antibodies bound to both CDH17 (on DLD1) and CD3. The indicated antibody concentration range and 100 × 10⁶ cells per microtiter well (96-well plate) were used over 24 hours according to the manufacturer's protocol. 3 Jurkat reporter cells and 30×10 3 Signal transduction was determined in DLD1 cells. Luciferase expression / activity was measured using a multimodal plate reader. Figure 8 As shown in B, T cell signaling in h10Ta-L4 was reduced by 2-fold compared to h10Ta-wt with parental Fv. Controls did not include antibodies or CD3, CD28, CD2 agonists (Immunocult; stem cells) to induce maximum stimulation.

[0085] Example 9: Tumor cell cytotoxicity mediated by TriAx antibodies with L4

[0086] The retargeted T-cell cytotoxicity of h10Ta-L4 and h10Ta-wt was determined. Additionally, h10Ta-L4c, derived from h10Ta-L4 by carrying Vk ACys43 and Vh Q114C substitutions to produce a stable interdomain disulfide, was included in the assay. T-cell killing of luciferase-expressing colon cancer cell line DLD1 and gastric cancer cell line AGS was determined within antibody concentration ranges at an E:T ratio of 4 in a 24-hour assay. Viable cells were quantified using a Bio-Glo (Promega) and multimodal plate reader after washing to remove dead cells. The EC50 for killing DLD1 in the presence of T cells was 0.5 pM for h10Ta-wt, 0.4 pM for h10Ta-L4, and 1.2 pM for h10Ta-L4c. The EC50 for AGS kill is 1.4 pM for h10Ta-wt, 2 pM for h10Ta-L4, and 4.7 pM for h10Ta-L4c. Figure 9 These results demonstrate that the lower affinity of L4 for CD3 does not significantly reduce cytotoxic activity, as determined in this assay. Regarding TriAx antibodies with L4c, the results indicate a slight decrease in cytotoxic activity compared to antibodies with parental L4, suggesting that interdomain disulfides can exert a negative positional effect by altering the position of CDR residues involved in CDH17 binding.

[0087] Example 10, TriAx-E antibody

[0088] h8h10Te (SEQ ID 12 and 2) are TriAx-E trispecific antibodies containing anti-TROP2 (h8) and CDH17 (h10)scFv binding domains at the N-terminus of the anti-CD3 core Fv (Table 1). Flow cytometry analysis confirmed the binding of the h8h10Te antibody (with parental anti-CD3 Fv) to all three antigens. Figure 10 The study demonstrated that h8h10Te specifically binds to HEK293 transfectants expressing transmembrane forms of CDH17 or Trop2. h8h10Te also specifically binds to Jurkat cells expressing CD3. Therefore, a TriAx-E trispecific antibody can be generated and is able to bind to all three target antigens. Figure 10 The TriAx-E function is described in [the document]. h8h10Te supports the effective redirection of T-cell killing in DLD1 tumor cells using a 24-hour cytotoxicity assay with an E:T ratio of 4.

[0089] Example 11: Spatial Effects of Multispecific TriAx Antibodies

[0090] When the anti-CD3 binding domain is located at the Fv position of the TriAx core structure, the addition of one or more binding domains, such as the scFv domain, can affect the antibody's potency in binding to cellular CD3. In this regard, comparisons were made using TriAx-A antibody h10Ta (SEQ ID 1 and 2) and TriAx-E antibody h10Te, both possessing the same anti-CD3 Fv region (wt). The activity of each TriAx antibody in binding to CD3 was determined by flow cytometry using 5 μg / ml. Figure 11 The binding of TriAx-E antibody was shown to be approximately 2 to 6-fold reduced compared to TriAx-A antibody binding (median fluorescence intensity; MFI). This reduced binding to anti-CD3 Fv, as the TriAx-E core, is likely a result of steric inhibition. Similar to TriAx-E, TriAx forms of scFvs linked to core Fvs Vh and Vk, such as TriAx-I and TriAx-J, also exhibited reduced specificity for binding to CD3 or other core Fvs. Administration of these TriAx antibodies resulted in less T-cell or lymphoid tissue sedimentation and greater tumor tissue biodistribution compared to some other forms. Therefore, this structure could provide greater tumor microenvironment (TME) localization activity, greater potency, and safety. However, compared to TriAx-E or typical whole antibodies, TriAx-A or TriAx-C forms, with a single N-terminal scFv and therefore a smaller N-terminal mass, may be able to bind to tumor antigens more efficiently.

[0091] Example 12: Stable scFv (LocV) in TriAx antibodies

[0092] Stable versions of TriAx-A scFv specifically targeting TROP2 (h8v5 and h8v6) or CDH17 (h10v3) are generated by reverse mutation of framework residues in humanized versions h8v4 and h10v2 to enhance the Vh-Vk interface (h8v5, h8v6, and h10v3), and by creating a second disulfide bond (h8v6) by substituting two residues with cysteine ​​within the Vh domain (Ewert 2004, McConnell 2012, Weatherill 2012). Figure 12As shown, these humanized and humanized stable variants were expressed in HEK293 cells using Fc:G1G4G1 (A and C) or Fc:G1G4 (B and D). Binding to CDH17 and TROP2 (which were expressed in HEK293 cells via standard PEI transfection) was determined by flow cytometry using 5 μg / ml of each TriAx. The binding levels (MFI) of h8v4, v5, and v6 were similar (A and B). The binding of h10v2 and v3 was also similar (C and D). The data indicate that the substitutions generated for scFv stabilization did not have a significant negative structural effect. On the contrary, the stabilized scFv (LocV) improved the binding of TriAx antibodies to tumor antigens.

[0093] Example 13, Stable low-antigenicity adapter

[0094] ARB202 is a bispecific antibody against CDH17 and CD3 in IgG-scFv form (see the applicant's application WO / 2019 / 222428, the full text of which is incorporated herein by reference) used to compare the stability of three proline-rich linkers: A=PAGPPA, B=PAGPAP, and C=PAGPPP. The linker extends from the C-terminus of the Fc to the N-terminus of the anti-CD3 scFv domain. The bispecific antibody (1 mg / ml) was stored at 37°C in 10 mM histidine buffer (pH 6.0) for 56 days. Degradation of samples was analyzed by UPLC at indicated time points. Figure 13 As shown, adapter C (77.5%) conferred greater stability compared to adapter A (47.1%) and adapter B (32.5%). In binding and signal transduction assays, the functions of the three bispecific antibodies were equal in plasma at 37°C after day 0 and day 14. Therefore, adapter C achieves greater stability without diminishing function.

[0095] Example 14, TriAx-I and TriAx-J antibodies

[0096] h8h10B2Ti (SEQ ID 14 and 15) is an example of a quadruple-specific TriAx-I antibody that includes binding specificity to tumor-associated antigens, TROP2, CDH17, and FAP (expressed on cancer-associated fibroblasts or CAFs). This TriAx-I antibody also binds to CD3 to trigger targeted killing of GI cancer cells expressing TROP2, CDH17, or both, by T cells, thereby reducing the likelihood of tumor escape due to loss of expression of tumor target antigens. By binding to FAP, this TriAx-I antibody also targets and kills tumor-associated CAFs. CAFs can be a predominant cell type in the tumor microenvironment, supporting tumor growth by promoting extracellular matrix remodeling, angiogenesis, and immunosuppression.

[0097] h8h10B2D5Tj (SEQ ID 14 and 16) are examples of pentaspecific TriAx-J antibodies that include binding specificity to DR5 in addition to TROP2, CDH17, FAP, DR5, and CD3, as in the TriAx-I antibody h8h10B2Ti. DR5, also known as death receptor 5, TRAIL receptor 2, and member 10B of the tumor necrosis factor receptor superfamily, is a cell surface receptor of the TNF-α superfamily that binds to TRAIL and mediates apoptosis. In this context, the h8h10B2D5Tj antibody acquires the function of h8h10B2Ti and exerts the additional ability to induce tumor cell apoptosis by participating in DR5 signaling in GI cancer cells.

[0098] The foregoing description and examples provide a complete description of the structure and use of exemplary embodiments. Although certain embodiments have been described above with a degree of specificity or by reference to one or more individual embodiments, those skilled in the art can make various changes to the disclosed embodiments without departing from the scope of this application. For example, while the foregoing examples may include combined structural domains in certain locations, they are provided by comparison only and not by limitation. Therefore, the illustrative embodiments of this application are not intended to be limited to the specific embodiments disclosed. Rather, they include all modifications and substitutions falling within the scope of this disclosure. Furthermore, where appropriate, aspects of any of the examples described above can be combined with aspects of any other examples described to form other examples having comparable or different characteristics and solving the same or different problems. Similarly, it should be understood that the benefits and advantages described above may apply to one embodiment or to several embodiments.

[0099] Table 1. TriAx platform antibodies and their SEQ IDs.

[0100]

[0101]

Claims

1. A multispecific antibody having an N-terminus and a C-terminus, comprising: The first monomer includes a VL structural domain extending from the N-end to the C-end, a first connector, and a first Fc structural domain. The second monomer includes a VH structural domain extending from the N-terminus to the C-terminus, a second connector, and a second Fc structural domain. At least a first binding domain, which is linked to the N-terminus or the C-terminus of the multispecific antibody. The first monomer and the second monomer pair through the interaction between the VL domain and the VH domain, and The multispecific antibody is stabilized by a disulfide bond between the first and second adapters.

2. The multispecific antibody according to claim 1, wherein the first binding domain is connected to the VH domain at the N-terminus, the VL domain at the N-terminus, the first Fc domain at the C-terminus, or the second Fc domain at the C-terminus.

3. The multispecific antibody according to claim 1, further comprising at least a second binding domain, wherein the first binding domain and the second binding domain are connected to opposite ends or the same end of the multispecific antibody.

4. The multispecific antibody according to claim 3, further comprising a third binding domain, wherein the first binding domain is connected to the C-terminus at the first Fc domain or the C-terminus at the second Fc domain.

5. The multispecific antibody according to claim 3, further comprising a third binding domain and a fourth binding domain, wherein the third binding domain is connected to the C-terminus at the first Fc domain, and the fourth binding domain is connected to the C-terminus at the second Fc domain.

6. The multispecific antibody according to any one of claims 1-5, wherein the first monomer comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO. 11 or 12, and wherein the second monomer comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO.

2.

7. An isolated nucleic acid sequence encoding the multispecific antibody as described in any one of claims 1 to 6.

8. An expression vector comprising the isolated nucleic acid sequence of claim 7.

9. A host cell comprising the isolated nucleic acid sequence of claim 7 or the expression vector of claim 8.

10. A method for generating a multispecific antibody according to any one of claims 1 to 6, comprising: culturing host cells such that a DNA sequence encoding a multispecific antibody according to any one of claims 1 to 6 is expressed, and purifying the multispecific antibody.

11. An immunoconjugate comprising the multispecific antibody and cytotoxic agent or imaging agent as described in any one of claims 1 to 6.

12. A pharmaceutical composition comprising a multispecific antibody as described in any one of claims 1 to 6 or an immunoconjugate as described in claim 11, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, further comprising a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

14. Use of the pharmaceutical composition of any one of claims 12 or 13 in the preparation of a medicament for treating or preventing cancer in a subject.

15. Use of the pharmaceutical composition and therapeutic agent of any one of claims 12 or 13 in the preparation of a medicament for treating or preventing cancer in a subject, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

Images