TriAx antibody compositions and methods of making and using the same

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC 119(e) of the filing date of U.S. Provisional Application Serial No. 62 / 944,230, filed on December 5, 2019, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates generally to the technical field of cancer immunotherapy, and more particularly to compositions of modified antibodies with multiple antigen-binding specificities. Background Art

[0004] Unless otherwise indicated herein, the materials described in this section are not prior art to the claims in this application and are not admitted to be prior art by inclusion in this section.

[0005] Despite recent advances in drug discovery and clinical imaging, cancer remains one of humanity's most deadly diseases. Our understanding of how tumors initiate, survive stress, colonize / metastasize to distant organs and sites, and become drug-resistant remains limited. The American Cancer Society estimates that there were 1.6 million new cancer cases in the United States in 2014, and there are no approved curative treatments for most major types of cancer.

[0006] Gastrointestinal (GI) cancers (colorectal, gastric, pancreatic, esophageal, bile duct, and liver) are the leading causes of morbidity and mortality worldwide. Colorectal cancer (CRC) alone accounts for approximately 10% of all cancer diagnoses and is the second leading cause of cancer death worldwide. In China, liver and stomach cancers are among the most lethal malignancies worldwide, with more than half of all cases diagnosed, resulting in >1.42 million deaths worldwide each year, which 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, potential drug development for these aggressive cancers requires new biomarkers and therapeutic targets. Molecular targeted agents that have been shown to eliminate or inhibit the growth of these cancers would have significant clinical value and significant market impact. If the disease is diagnosed in the early stages, these tumors can be effectively removed by surgery. Unfortunately, and very often, most GI cancers are asymptomatic and are detected at a very late stage when they appear in the clinic. In the absence of effective treatment, these patients die shortly after diagnosis or relapse after salvage therapy.

[0007] CDH17 is a significant cancer biomarker, characterized by its overexpression in both liver cancer and gastric cancer, but not in normal tissues from healthy adults. Anti-CDH17 monoclonal antibodies have shown growth inhibitory effects on liver and gastric tumor cells. CDH17 is highly expressed in metastatic cancer, and blocking CDH17 expression and function can significantly reduce lung metastasis of hepatocellular carcinoma (HCC). These observations indicate that humanized anti-CDH17 antibodies can be developed as target therapeutics for treating cancer patients with indications of CDH17 biomarkers in tumor tissue and / or serum samples. Although antibody drug conjugates are promising as antibody therapies, multispecific antibody therapeutics utilize immune responses to cancer and activate T cell-mediated cytotoxicity to cancer cells.

[0008] Bispecific antibodies that target CD3-positive T cells and CD19-positive B cells have been shown to be effective in treating hematological 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 access to solid tumor cells and appropriate immunomodulatory signals. Antibody-based scaffolds for effectively targeting multiple tumor antigens and immune cell antigens or products are needed to generate more effective immunotherapeutics that better address the complexity of the pro-tumor microenvironment and tumor escape mechanisms. Summary of the Invention

[0009] In one aspect, the present application provides multispecific antibodies. The antibodies can be bispecific, trispecific, tetraspecific, or pentaspecific. The antibodies can have truncated structures.

[0010] In one embodiment, the present 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 comprising a VL domain from the N-terminus to the C-terminus, a first linker, and a first Fc domain, the second monomer comprising a VH domain from the N-terminus to the C-terminus, a second linker, and a second Fc domain, and the first binding domain is connected to the N-terminus or C-terminus of the multispecific antibody, wherein the first monomer and the second monomer are paired through the interaction between the VL domain and the VH domain, and wherein the multispecific antibody is stabilized by a disulfide bond between the first linker and the second linker.

[0011] In one embodiment, the first binding domain is linked to a VH domain at the N-terminus, a VL domain at the N-terminus, a first Fc domain at the C-terminus, or a second Fc domain at the C-terminus.

[0012] In one embodiment, the multispecific antibody further comprises 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.

[0013] In one embodiment, the multispecific antibody further comprises 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- end at the VH domain and the second binding domain is connected to the N- end at the VL domain.

[0014] In one embodiment, the multispecific antibody further comprises a third binding domain, and the antibody is a tetraspecific antibody. In one embodiment, the first binding domain is linked to the N-terminus at the VH domain, the second binding domain is linked to the N-terminus at the VL domain, and the third binding domain is linked to the C-terminus at the first Fc domain or the C-terminus at the second Fc domain.

[0015] In one embodiment, the multispecific antibody further comprises a fourth binding domain, and the antibody is pentaspecific. In one embodiment, the third binding domain is linked to the C-terminus of the first Fc domain, and the fourth binding domain is linked to the C-terminus of the second Fc domain.

[0016] All binding domains can have binding affinities for different antigens. Alternatively, some binding domains can have binding affinities for the same antigen as another binding domain. In one embodiment, the first binding and the second binding are identical. In one embodiment, the first binding and the second binding are different. In one embodiment, the first binding domain, the second binding domain, and the third binding domain are different from each other. In one embodiment, the first binding domain, the second binding domain, and the third binding domain are different from each other, and wherein the fourth binding domain is identical to one of the first binding domain, the second binding domain, and the third binding domain.

[0017] Each first binding domain may be independently selected from the group consisting of a scFv domain, a ligand, a single domain nanobody, a binding region of a natural protein, a chemokine and a cytokine.

[0018] In one embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 1, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 2.

[0019] In one embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 1, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 3. In one embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 1, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 4. In one embodiment, the bispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 5, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 6.

[0020] In one embodiment, a trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.7, and a second monomer c comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.8. In one embodiment, a trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.9, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.10. In one embodiment, a trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.11, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.2. In one embodiment, a trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.12, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.4. In one embodiment, a trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.12, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO.2.

[0021] In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 14, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 15.

[0022] In one embodiment, the trispecific antibody may have a first monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 14, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO. 16.

[0023] In one embodiment, the binding domain can be attached to the multispecific antibody via a linker. In one embodiment, the linker comprises a proline-rich amino acid sequence. In one embodiment, the linker can comprise at least 20%, 30%, or 50% proline residues. In one embodiment, the linker can comprise from about 2 to about 31 amino acids.

[0024] In another aspect, the present application provides isolated nucleic acid sequences encoding the multispecific antibodies as disclosed herein.

[0025] In yet another aspect, the present application provides an expression vector comprising the isolated nucleic acid sequence as disclosed herein.

[0026] In yet another aspect, the present application provides a host cell comprising the isolated nucleic acid sequence as disclosed herein.

[0027] In another aspect, the present application provides a method for producing a multispecific antibody as disclosed herein. In one embodiment, the method comprises culturing a host cell to express a DNA sequence encoding a multispecific antibody, and purifying the multispecific antibody.

[0028] In another aspect, the present application provides a method for preparing a multispecific antibody. In one embodiment, the method includes the steps of culturing a host cell under conditions that produce the multispecific antibody and recovering the antibody.

[0029] In yet another aspect, the present application provides an immunoconjugate. In one embodiment, the immunoconjugate comprises a multispecific antibody and a cytotoxic agent. In one embodiment, the immunoconjugate comprises a multispecific antibody and an imaging agent.

[0030] In yet another aspect, the present 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.

[0031] In yet another aspect, the present application provides a method for treating or preventing cancer in a subject. In one embodiment, the method comprises administering to the subject a pharmaceutical composition comprising a purified multispecific antibody disclosed herein. In one embodiment, the method for treating a subject suffering from cancer comprises administering to the subject an effective amount of a multispecific antibody disclosed herein. In one embodiment, the method may further comprise co-administering 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 human.

[0032] In yet another aspect, the present application provides a solution comprising an effective concentration of a multispecific antibody disclosed herein, wherein the solution is plasma from a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments according to the present disclosure may now be described with reference to the drawings, wherein like reference numerals refer to like elements throughout.

[0034] Figure 1 depicts the configuration of a class of multispecific antibodies collectively referred to as TriAx, including but not limited to TriAx-A, TriAx-C, TriAx-D, TriAx-E, TriAx-I, and TriAx-J antibodies;

[0035] FIG2 shows the production, heterodimerization, and purification of the TriAx-A antibody;

[0036] FIG3 shows the generation and binding specificity of the TriAx-C antibody;

[0037] FIG4 shows the thermal stability of TriAx antibodies;

[0038] FIG5 shows redirected T cell cytotoxicity by targeting TROP2 by TriAx-A antibody;

[0039] FIG6 shows redirected T cell cytotoxicity by targeting FAP with the TriAx-A antibody;

[0040] FIG7 depicts a low affinity anti-CD3 binding domain with amino acid substitutions;

[0041] FIG8 shows that TriAx-A containing L4 exhibits reduced T cell affinity and activation;

[0042] FIG9 shows that TriAx-A containing L4 mediates cytotoxicity comparable to TriAx-A without L4 mutation;

[0043] FIG10 demonstrates T cell cytotoxic binding specificity and redirected T cell cytotoxicity by TriAx-E antibodies;

[0044] FIG11 depicts the steric effects of additional binding domains on TriAx core functions, such as anti-CD3 binding affinity;

[0045] Figure 12 depicts the stabilized scFv (LocV) binding domain in the TriAx antibody; and

[0046] FIG13 depicts stable low antigenicity linkers in TriAx antibodies. DETAILED DESCRIPTION

[0047] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally indicate similar parts, unless the 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 various aspects of the present disclosure, as generally described herein and illustrated in the drawings, may be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0048] In order to enable immunotherapeutics to treat cancer more effectively, especially solid tumors, combination therapeutics are extremely important, which combine multiple target specificities and / or mechanisms of action that exceed typical bispecific antibodies. It is essentially necessary for the effective treatment of cancer and more frequently to achieve complete and lasting responses using combination therapeutics as described herein. Specifically, a scaffold with certain characteristics is needed to create a combination therapeutic with favorable mechanisms of action, manufacturing, pharmacokinetics, and low antigenic properties relative to approved bispecific antibodies. Many bispecific antibodies based on whole antibodies can have a larger mass relative to the trispecific antibodies described herein. Although antibodies with smaller mass based on antibody fragments may have greater tumor penetration, they generally have relatively poor pharmacokinetic properties, such as the FDA-approved bispecific antibody Blincyto without Fc region. In addition, many bispecific antibodies have mutations in the constant domain of the Ig structure based on knob-into-hole, which may contribute to anti-drug antibody (ADA) responses. In this article, a group of modified antibodies described herein as triaxial or TriAx antibodies do not require mutations in any constant domain, but have multiple antigen binding specificities.

[0049] All forms of TriAx antibodies contain a characteristic core structure that includes a single pair of VH and VL (Fv) that defines the first 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 region, such as an scFv. A second linker can be added to the 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 domain for optimal ligand binding. TriAx antibodies can be composed entirely of human, humanized, and low-antigenicity linker sequences to reduce the risk of ADA responses.

[0050] TriAx antibodies are designed to bind to two or more effector cell receptors to induce two or more mechanisms of anti-tumor activity, such as T- or NK-mediated cytotoxicity (CD3, NKG2D), tumor cell phagocytosis (FcR, CR3, CR4, AXL, CD13, CD206) or apoptosis (DR5, death receptor 5), immune cell stimulation (CD40, OX40), immune checkpoint inhibition (PD-L1, TIGIT, PD1, CTLA4), or the transition of tumor-associated macrophages (TAMs) from an immunosuppressive to an inflammatory phenotype (CD206, TREM-2).

[0051] As shown in Figure 1, the TriAx platform allows the generation of TriAx-A, TriAx-C, TriAx-D, TriAx-E, TriAx-I and TriAx-J antibodies. Multispecific antibodies, i.e., bispecific antibodies, trispecific antibodies, tetraspecific antibodies and pentaspecific antibodies can be generated according to these formats. The TriAx antibody core is characterized in that the Fv region of the paired VL and VH is directly connected to the Fc domain in the absence of CH1, and the core can be formed and stabilized by pairing two asymmetric monomers, i.e., LC and HC monomers, via a disulfide bridge (Ig hinge or other linker). The Fv-Fc core has at least one additional linker connected to the antigen-binding domain that is bound to the target antigen / ligand. TriAx-A is a bispecific antibody format in which a scFv domain is added that is covalently linked to the N-terminal of VH. TriAx-C is a trispecific antibody format in which a scFv domain is added that is covalently linked to the N-terminal of VH and a second scFv domain that is covalently linked to the C-terminal of the CH3 domain. TriAx-D is a trispecific antibody format with two different scFv domains added to its C-terminus. TriAx-E is a trispecific antibody format with two different scFv domains added to its N-terminus and linked to VL and VH, respectively. TriAx-I is a tetraspecific antibody format with a third scFv domain added to the C-terminus of TriAx-E; and TriAx-J is a pentaspecific antibody format with a fourth scFv domain added to the C-terminus of TriAx-I. Other multispecific antibody formats do not have the TriAx core structure that directs and covalently stabilizes the multispecific heterodimer format, 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 this context, the following examples illustrate that TriAx antibodies can not only be generated, but also function with the designed efficiency and stability. The generation of these TriAx antibodies showed a higher percentage of correctly formed heterodimers relative to other modified antibodies, such as knob-into-hole antibody types.

[0052] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural unless the context is inappropriate.

[0053] The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions with multiple epitope specificities, and antibody fragments, such as Fab, F(ab')2 and Fv, as long as they exhibit desired biological activity. In certain embodiments, the antibody can be a monoclonal antibody, a chimeric antibody, a single-chain antibody, a multispecific antibody, a multi-effective antibody, a human antibody, and a humanized antibody. Examples of active antibody fragments that are bound 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 above-mentioned antibodies and fragments. In certain embodiments, the antibody can include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, i.e., molecules that contain immunospecific binding sites to antigens. Immunoglobulins can be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or categories (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses of immunoglobulin molecules. In one embodiment, the antibody can be a full antibody and any antigen-binding fragment derived from a full antibody. A typical antibody refers to a heterotetrameric protein that generally includes two heavy (H) chains and two light (L) chains. Each heavy chain is composed of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain portion is composed of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementary determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) is generally composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxyl terminus. Within the variable regions of the heavy and light chains, there is a binding region that interacts with the antigen.

[0054] As used herein, the term "multispecific" antibody refers to an antibody having at least two binding sites, each of which has binding affinity for an epitope of an antigen. As used herein, the term "bispecific, trispecific, tetraspecific, or pentaspecific" antibody refers to an antibody having two, three, four, five, or six antigen-binding sites.

[0055] The term "humanized antibody" refers to an engineered antibody type having a CDR derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one (or more) human immunoglobulin. In addition, framework support residues can be changed to maintain binding affinity. The method for obtaining a "humanized antibody" is 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, a "humanized antibody" can be obtained by genetic engineering methods, which can produce affinity-matured human-like polyclonal antibodies in large animals such as, for example, rabbits (see U.S. Patent number 7,129,084).

[0056] The term "antigen" refers to an entity or fragment thereof that can induce an immune response in an organism, particularly an animal, more particularly a mammal including humans. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0057] The term "epitope", also called "antigenic determinant", is the part of an antigen that is recognized by the immune system, particularly antibodies, B cells or T cells, and is the specific part of an antigen that binds to an antibody.

[0058] The term "immunogenic" refers to a substance that triggers or enhances the production of antibodies, T cells, or other reactive immune cells against an immunogenic agent in a human or animal and contributes to an immune response. An immune response occurs when an individual produces enough antibodies, T cells, and other reactive immune cells against the administered immunogenic composition of the present application to alleviate or alleviate the condition to be treated.

[0059] As used herein, the term "tumor antigen" refers to an antigen molecule produced in tumor cells. Tumor antigens can trigger an immune response in a host. In one embodiment, tumor cells express tumor antigens, including but not limited to tumor-specific antigens (TSAs), neoantigens, and tumor-associated antigens (TAAs).

[0060] As used herein, the term "specifically binds to" or "specifically binds to" or "specifically for" a particular antigen or epitope refers to binding that is measurably different from non-specific interactions. Specific binding can be measured by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that has no binding activity. Specific binding can be determined by comparison to a control molecule that is similar to the target. Specific binding for a particular antigen or epitope can be determined 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 , alternatively at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 In some embodiments, a multispecific 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 than that of the antigen or epitope relative to a control molecule. In addition, specific binding to a particular antigen or epitope can be exhibited by an antibody that has a KD or Ka for the antigen or epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater than that of the epitope relative to the control, where KD or Ka refers to the off-rate of the specific antibody-antigen interaction.

[0061] Examples

[0062] The present 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.

[0063] Example 1: Characteristics of TriAx Antibodies

[0064] TriAx antibodies are heterodimers characterized by an Fv-Fc core structure that includes, from N- to C-terminus, an Fv region, a modified Ig hinge, and an Ig Fc region, as shown in Figure 1. The additional binding domain can be a scFv, scFab, Fab, single-domain VH, or a native protein fragment.

[0065] The TriAx core component includes two linkers (e.g., glycine-rich linkers fused to a truncated Ig hinge) that covalently link the VH and VL chains of the Fv to the CH2-CH3 monomer. This flexible glycine-rich linker and the Ig hinge can promote efficient VH-VL pairing. The TriAx binding domain can be attached via a flexible glycine-rich linker (such as PAGGGGS) or a more rigid proline-rich linker (such as PAGPPP). The linker is typically 4 to 7 residues in length. The TriAx Fc can be composed 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 a glycine-rich linker such as GAPGGGG or PAGGGGS. The hinge residues at positions 234 and 235 (G1 numbering) can be LL, FL, or AA to modulate the extent of FcR binding (Saunders 2019). The CH2 and CH3 domains can both be IgG1 or IgG4 or a combination, such as G1 CH2 and G4 CH3. TriAx molecules can have substituted CH3 to create a knob-into-hole (Merchant 1998).

[0066] Built on the central TriAx Fv-Fc core structure, TriAx-A is a bivalent antibody format with a single scFv linked to the VH or VL of an Fv, such as the h10Ta, h5Ta, h8Ta, and hB2Ta antibodies, whose structural features are listed in Table 1. TriAx-C is a trivalent antibody format 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 an scFv 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 format 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 format with one scFv added to the N-terminus of each VH and VL, plus one scFv added to the C-terminus of each CH2-CH3 monomer. TriAx-J is a pentavalent format with one scFv added to the N-terminus of each VH and VL and one scFv added to the C-terminus of each CH2-CH3 monomer.

[0067] Example 2: TriAx-A Antibody

[0068] h8Ta is a TriAx-A bispecific antibody targeting TROP2 and CD3 (SEQ ID NO. 1 and 4, also see Table 1). TROP2 is a transmembrane glycoprotein that is dysregulated in all cancer types independent of the baseline level of TROP2 expression. TROP2 is an ideal candidate for targeted therapeutics. Several antibody therapeutics targeting TROP2 in early clinical trials have demonstrated safety and clinical benefits in the treatment of triple-negative breast cancer, platinum-resistant urothelial carcinoma, and small cell lung cancer.

[0069] h8Ta was produced in HEK293 cells by co-transfection of plasmids encoding the heavy (core Fv VH) and light (core Fv Vk) chains with PEI. Three days after transfection, culture medium samples were subjected to SDS-PAGE, as shown in Figure 2; culture medium from transfected cells (lanes 1 and 2), and culture medium from mock transfection (lane 3). After a one-step Protein-A purification, the h8Ta antibody was subjected to SDS-PAGE under non-reducing and reducing conditions, as shown in lanes 4 and 5 of Figure 2, respectively. Under non-reducing conditions, the TriAx-A antibody migrated as a protein of approximately 116 kDa, consistent with its calculated heterodimer size. Under reducing conditions, the heavy chain was approximately 70 kDa, and the light chain was approximately 44 kDa, consistent with its calculated size. The efficiency of heterodimerization was approximately 90% or greater. No other significant TriAx products or fragments were detected compared to mock control culture medium. These TriAx-As can have linkers and Fc sequences of varying lengths and compositions to modify FcR binding and circulation half-life.

[0070] Example 3: TriAx-C Antibody

[0071] Two TriAx-C antibodies with binding specificity for the phagocyte 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 both significant cancer biomarkers, characterized by their overexpression in various forms of solid tumors including stomach, colon, pancreas, liver and liver. CDH17 is highly expressed in metastatic cancer, and blocking CDH17 expression and function can significantly reduce lung metastasis of hepatocellular carcinoma (HCC). Both anti-CDH17 monoclonal antibodies and anti-CDH17 / CD3 bispecific antibodies showed growth inhibitory effects on liver and gastric tumor cells (see the applicant's application WO / 2019 / 222428, the entire text of which is incorporated herein). CR3, or complement receptor 3, is a heterodimer of α (CD11b) and β (CD18) transmembrane glycoproteins. The I-domain of 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 the opsonization process, C3d is deposited on the surface of target cells, where it serves as a ligand for macrophage CR3 for phagocytosis. Binding to CR3 via its ligands, such as C3d or activating antibodies, can direct the primary phagocyte receptor to tumor cells and induce widespread 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, which then present C3d for macrophage CR3-dependent phagocytosis. TriAx-C antibodies can also bind to FcR, which can further activate and enhance macrophage CR3 tumor cell phagocytosis. These TriAx-C antibodies can broadly target different tumor types and achieve greater efficacy and safety compared to targeting CD47 or CD24 phagocyte checkpoints.

[0072] In addition to 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). Three days after transfection, antibody expression levels were determined by Octet (BLI). Production levels were 104 ug / ml (1:1), 27.3 ug / ml (4:1), 22 ug / ml (6:1), 21.7 ug / ml (8:1), and 14.6 ug / ml (12:1). Production culture medium samples were subjected to SDS-PAGE. As shown in Figure 3A, the molecular weight of h10C3dTc is approximately 180 kDa, which is slightly larger than the calculated weight of ~150 kDa. No other significant TriAx products or fragments were detected compared to mock control culture medium. Because the heavy and light chain monomers of h10Cd3Tc are of similar size, homodimer formation cannot be easily distinguished by standard SDS-PAGE. When all concentrations were adjusted to 5 μg / ml to quantify binding to the immobilized CR3 I domain in an ELISA, I-domain binding peaked at a plasmid ratio of 6:1 (Figure 3B). Due to the low affinity of C3d binding to the I domain (~400 nM), the OD values ​​in the ELISA were low. However, peak binding was approximately 7-fold higher than in control culture medium from mock-transfected HEK293 cells. Binding of h10C3dTc to CDH17 and CD3 was determined in an ELISA in which the sample antibody bound to a soluble form of CD3, followed by immobilized CDH17, and then an HRP conjugate bound to recombinant CD3. As shown in Figure 3C, a 4:1 plasmid ratio resulted in peak binding, indicating that optimal heterodimerization can occur when the plasmids are co-transfected into HEK293 cells at certain ratios. The binding activity demonstrated in ELISA indicates that trispecific TriAx-C antibodies can be generated. These TriAx-C antibodies can have linker and Fc sequences of varying lengths and compositions to modify FcR binding and circulation half-life.

[0073] Example 4: Thermal stability of TriAx antibodies

[0074] The thermal stability of TriAx antibodies was determined using SYPRO Orange in a thermal shift assay (Kinget et al. 2011). TriAx-A antibodies such as h8Ta (SEQ IDs 1 and 4), hB2Ta (SEQ IDs 1 and 5), and hA12Ta (SEQ IDs 1 and 6) (see Table 1) were analyzed. These TriAx-A antibodies in PBS were centrifuged in a microcentrifuge for 10 minutes and the concentration was adjusted to 5uM. A mixture of TriAx-A (50ul) and SYPRO Orange (1ul) (125X in PBS; final 2.5X) was transferred to an optically clear 96-well plate for measurement in a qPCR instrument. The temperature was increased from 25°C to 99°C at a rate of 1°C per minute, held for one minute for each measurement, with excitation at 470nm and emission at 586nm. Figure 4 shows initial unfolding peaks at 66°C (hB2Ta), 68°C (hA12Ta), and 72°C (h8Ta), indicating that the TriAx platform antibodies are sufficiently stable for further development.

[0075] Example 5: Redirected T cell cytotoxicity by TriAx-A antibodies targeting TROP2 and CD3

[0076] To evaluate the function of the TriAx platform antibody h8Ta, the redirected T cell cytotoxicity of the TriAx-A bispecific antibody (see Example 2) was evaluated. Three luciferase-expressing GI tumor cell lines, DLD1 (colorectal cancer), SW480 (colorectal cancer), and AGS (gastric cancer), were used for a 24-hour assay with an E:T ratio of 4. After washing to remove dead cells, live cells were quantified using Bio-Glo (Promega) and a multi-mode plate reader. As shown in Figure 5 (top), h8Ta was used in flow cytofluorimetry analysis to detect the expression of TROP2 in all three tumor cell lines (Figures 5A, 5B, and 5C). In the absence of T cells, cell activity did not decrease over the concentration range of the h8Ta antibody. In the presence of T cells, EC50 values ​​for h8Ta antibody-dependent tumor cell killing were determined: 0.8 pM for DLD, 2 pM for AGS, and 11 pM for SW480 ( Figure 5 , lower panel, Figures 5D, 5E, and 5F ). ​​The lower EC50 value for SW480 appears to correlate with its lower level of TROP2 expression. Therefore, this bispecific TriAx-A antibody can mediate potent sub-pM tumor cell killing.

[0077] Example 6: Redirected T cell cytotoxicity by TriAx-A antibodies targeting FAP and CD3

[0078] Fibroblast activation protein alpha (FAP) is a 97 kDa type II cell surface glycoprotein that belongs to the serine protease family. Fibroblast activation protein-alpha (FAPα) plays a key role in colorectal cancer (CRC) metastasis. FAPα was reported to be expressed in cancer-associated fibroblasts in all CRC samples examined, but not in normal colon, hyperplastic polyps, or adenoma samples.

[0079] The TriAx-A bispecific antibody hB2Ta (SEQ ID 5 and 6) was 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 (DLD1-FAP) that also express luciferase. The next day, microtiter plate cytotoxicity assays were started with and without amplified T cells (E:T of 4). After adding the antibody at an experimental concentration range to the mixture with DLD1 or DLD1-FAP, the assay was incubated for 24 hours, then washed, and Bio-Glo substrate and a multimode plate reader were added to measure luciferase activity. As shown in Figure 6A, in the presence of T cells and DLD1-FAP (EC50 = 41pM), h1B2Ta mediated concentration-dependent tumor cell cytotoxicity, while in the absence of FAP-expressing tumor cells or T cells, such cytotoxicity was not detected or confirmed. FAP expression in DLD1 cells was determined by flow cytometry at the start of the assay (Figure 6B). A 68% percentage of DLD1 cells expressing FAP (MFI = 4,256) appeared to correlate with a maximum cytotoxicity of ~70%. Thus, TriAx antibodies with binding specificity for FAP effectively killed tumor cells with low FAP expression.

[0080] Example 7, L4, low affinity anti-CD3 binding domain

[0081] To reduce the risk of extratumoral T cell signaling and T cell and lymphoid tissue deposition, a low-affinity monovalent CD3 binding region, L4 (SEQ ID 13), was introduced into the TriAx platform antibodies. The CDRs of the UCHT1 Vh and Vk sequences (Shalaby 1992) were partially or completely replaced with human germline sequences to generate low-affinity and low-antigenicity anti-CD3 variants. Substitution of the Vk CDR1 with the IGKV1-33*01 germline sequence generated a lower-affinity mutant L4, which was incorporated into the core Fv of several TriAx antibodies. Thus, the amino acid sequence of this anti-CD3 variant includes the UCTH1 CDR sequence, except for the CDRL1 substitutions, R24Q and R30S, as shown in Figure 7.

[0082] Example 8. T cell affinity and activation mediated by TriAx antibodies with L4

[0083] To assess their ability to bind to and activate T cells, TriAx antibodies with L4 or its parental Fv Vk CDR1 ("wt"), i.e., h10Ta-L4 (SEQ ID 1 and 2) and h10Ta-wt, were determined by flow cytometry. The affinity of the antibodies bound to peripheral blood T cells for T cells was determined within the concentration range shown in Figure 8A. MFI was plotted and affinity (EC50) was determined using GraphPad PRISM. Three independent affinity determinations and their average values ​​are indicated. The results indicate that the 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 (Jurkat Promega kit NFAT J1621) with an NFAT inducible promoter for luciferase expression. The h10Ta antibodies all engage CDH17 (on DLD1) and CD3. The indicated antibody concentration ranges and 100 × 10 per microtiter well (96-well plate) were used according to the manufacturer's protocol for 24 h. 3 Jurkat reporter cells and 30 × 10 3 Signaling was determined in DLD1 cells. Luciferase expression / activity was measured using a multimode plate reader. As shown in Figure 8B, h10Ta-L4 showed a 2-fold reduction in T cell signaling compared to h10Ta-wt with the parental Fv. Controls did not include antibodies or CD3, CD28, or CD2 agonists (Immunocult; Stem Cell) to induce maximal stimulation.

[0084] Example 9: Tumor Cell Cytotoxicity Mediated by TriAx Antibodies with L4

[0085] The redirected T cell cytotoxicity of h10Ta-L4 and h10Ta-wt was determined. In addition, h10Ta-L4c was included in the test, which was derived from h10Ta-L4 by carrying Vk ACys43 and Vh Q114C substitutions to produce stable interdomain disulfides. In a 24-hour assay, luciferase-expressing T cells of the colon cancer cell line DLD1 and the gastric cancer cell line AGS were determined over a range of antibody concentrations at an E:T ratio of 4. After washing to remove dead cells, live cells were quantified using Bio-Glo (Promega) and a multimode plate reader. In the presence of T cells, the EC50 for killing DLD1 was 0.5 pM for h10Ta-wt, 0.4 pM for h10Ta-L4, and 1.2 pM for h10Ta-L4c. The EC50 for AGS killing was 1.4 pM for h10Ta-wt, 2 pM for h10Ta-L4, and 4.7 pM for h10Ta-L4c. These results, presented in Figure 9, demonstrate that the lower affinity for CD3 in L4 did not significantly reduce cytotoxic activity as determined in this assay. Regarding the TriAx antibody with L4c, the results indicate that cytotoxic activity was slightly reduced relative to the antibody with parental L4, suggesting that the interdomain disulfide can exert a negative positional effect by altering the position of CDR residues involved in CDH17 binding.

[0086] Example 10: TriAx-E Antibody

[0087] h8h10Te (SEQ ID 12 and 2) is a TriAx-E trispecific antibody that includes anti-TROP2 (h8) and CDH17 (h10) scFv binding domains at the N-terminus of the anti-CD3 core Fv (Table 1). Binding of the h8h10Te antibody (with the parent anti-CD3 Fv) to all three antigens was demonstrated by flow cytometry. Figure 10 shows that h8h10Te specifically binds to HEK293 transfectants expressing transmembrane forms of CDH17 or TROP2. h8h10Te also specifically binds to Jurkat cells expressing CD3. Thus, a TriAx-E trispecific antibody can be generated and is capable of binding to all three target antigens. TriAx-E function is depicted in Figure 10. h8h10Te supports efficient redirected T cell killing of DLD1 tumor cells in a 24-hour cytotoxicity assay using an E:T ratio of 4.

[0088] Example 11: Steric Effects of Multispecific TriAx Antibodies

[0089] When the anti-CD3 binding domain is located in the Fv position of the TriAx core structure, adding one or more binding domains, such as scFv domains, can affect the effectiveness of the antibody binding to cellular CD3. In this regard, TriAx-A antibody h10Ta (SEQ ID 1 and 2) and TriAx-E antibody h10Te with the same anti-CD3 Fv region (wt) were used for comparison. The activity of binding to CD3 was determined by flow cytometry using 5ug / ml of each TriAx antibody. Figure 11 shows that relative to the binding of the TriAx-A antibody, the binding of the TriAx-E antibody was reduced by approximately 2 to 6 times (median fluorescence intensity; MFI). As the TriAx-E core, the reduction in binding to the anti-CD3 Fv may be the result of steric inhibition. Like TriAx-E, TriAx forms of scFv connected to the core Fv Vh and Vk, such as TriAx-I and TriAx-J, can also demonstrate a reduction in binding to CD3 or other core Fv specificities. Administration of these TriAx antibodies can result in less T cell or lymphoid tissue deposition and greater tumor tissue biodistribution relative to certain other formats. Thus, this structure can provide greater tumor microenvironment (TME) localization activity, greater efficacy, and safety. However, relative to TriAx-E or typical whole antibodies, the TriAx-A or TriAx-C formats, which have a single N-terminal scFv and therefore a smaller N-terminal mass, may be able to bind to tumor antigens more effectively.

[0090] Example 12: Stable scFv (LocV) in TriAx Antibodies

[0091] The stable version of TriAx-A scFv specific for TROP2 (h8v5 and h8v6) or CDH17 (h10v3) is by reverse mutation to the framework residues of humanized version h8v4 and h10v2, to enhance Vh-Vk interface (h8v5, h8v6 and h10v3), and by replacing two residues with cysteine ​​in Vh domain to create a second disulfide bond (h8v6) and generate (Ewert 2004, McConnell 2012, Weatherill 2012). As shown in Figure 12, these humanization and humanization stable variants are expressed in HEK293 cells with Fc:G1G4G1 (A and C) or Fc:G1G4 (B and D). With the combination of CDH17 and TROP2 (which is expressed in HEK293 cells by standard PEI-transfection), each TriAx of 5ug / ml is determined by flow cytometry. 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 had no significant negative structural impact. In contrast, the stabilized scFv (LocV) improved the binding of the TriAx antibody to the tumor antigen.

[0092] Example 13: Stable low antigenicity linker

[0093] ARB202 is a bispecific antibody specific for CDH17 and CD3 in the form of IgG-scFv (see applicant's application WO / 2019 / 222428, which is incorporated herein in its entirety) and is 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 Fc to the N-terminus of the anti-CD3 scFv domain. The bispecific antibody (1 mg / ml) was stored in 10 mM histidine buffer (pH 6.0) at 37°C for 56 days. At the indicated time points, the degradation of the samples was analyzed by UPLC. As shown in Figure 13, linker C was given greater stability (77.5%) relative to linker A (47.1%) and linker B (32.5%). In binding and signal transduction assays, the function of the three bispecific antibodies was equal in plasma at 37°C after day 0 and 14 days. Therefore, linker C is able to achieve greater stability without reducing function.

[0094] Example 14: TriAx-I and TriAx-J Antibodies

[0095] h8h10B2Ti (SEQ IDs 14 and 15) is an example of a tetraspecific TriAx-I antibody that includes binding specificities for a tumor-associated antigen, TROP2, CDH17, and FAP (expressed on cancer-associated fibroblasts, or CAFs). This TriAx-I antibody also binds to CD3 to trigger T cell-directed killing of GI cancer cells expressing TROP2, CDH17, or both, thereby reducing the likelihood of tumor escape due to loss of tumor target antigen expression. By binding to FAP, this TriAx-I antibody also targets tumor-associated CAFs. CAFs can be a major cell type in the tumor microenvironment, supporting tumor growth by promoting extracellular matrix remodeling, angiogenesis, and immunosuppression.

[0096] h8h10B2D5Tj (SEQ IDs 14 and 16) is an example of a pentaspecific TriAx-J antibody that, in addition to TROP2, CDH17, FAP, DR5, and CD3, also includes binding specificity for DR5, as does the TriAx-I antibody h8h10B2Ti. DR5, also known as death receptor 5, TRAIL receptor 2, and tumor necrosis factor receptor superfamily member 10B, is a cell surface receptor of the TNF-receptor superfamily that binds 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 engaging DR5 signaling in GI cancer cells.

[0097] 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 certain degree of particularity or with reference to one or more separate embodiments, those skilled in the art may make various changes to the disclosed embodiments without departing from the scope of this application. For example, although the above examples may include binding domains at certain positions, they are provided only by comparison and not by way of limitation. Therefore, the illustrative embodiments of the present application are not intended to be limited to the specific embodiments disclosed. On the contrary, they include all modifications and replacements that fall within the scope of this disclosure. In addition, where appropriate, the aspects of any example described above may be combined with the aspects of any other example described to form other examples having comparable or different characteristics and solving the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments.

[0098] Table 1. TriAx platform antibodies and their SEQ IDs.

[0099]

[0100]

[0101] Sequence Listing

[0102] >Sequence ID 1: LC monomers of h10Ta, h5Ta, h8Ta and hA12Ta

[0103] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0104] >Sequence ID 2: HC monomers of h10Ta, h8h10Te and h10Te

[0105] MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0106] >Sequence ID 3: HC monomer of h5Ta

[0107] MAVLGLLFCLVTFPSCVLSQVQLVQSGAEVKKPGATVKISCKVSAYAFSSSWMNWVQQAPGKGLEWIGRIYPRDGDTNYNGKFKGRVTLTADTSTDTAYMELSSLRSEDTAVYFCAREGDGYYWYFDVWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASQSIRNYLHWYQQKPGQPPKLLIKYASQSISGIPSRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSNSWPLTFGAGTKLELKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0108] >Sequence ID 4: HC monomers of h8Ta and h8Te

[0109] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0110] >Sequence ID 5: LC monomer of hB2Ta

[0111] MEFGLSWVFLVALLRGVQCQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYTMNWVRQAPGQGLEWMGVINPNHGISSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRKISYDYDEGYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLVFFAATRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKLEIKPAGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0112] >Sequence ID 6: HC monomers of hB2Ta and hA12Ta

[0113] MEFGLSWVFLVALLRGVQCEVQLVQSGAEVKKPGATVKISCKVSGFKIQDAYIHWVQQAPGKGLEWMGRIDPANGNSKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCTRALDGYYVGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSNVNYMYWYQQKPGQAPRLLIYDTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPYTFGQGTKLEIKPAGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYTMNWVRQAPGQGLEWMGVINPNHGISSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRKISYDYDEGYAMDYWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0114] >Sequence ID 7: LC monomer of hC3dh10Tc

[0115] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPAGGGGVDAERLKHLIVTPSGAGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAP

[0116] >Sequence ID 8: HC monomer of hC3dh10Tc

[0117] MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0118] >Sequence ID 9: LC monomer of h8C3dTa

[0119] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPAGGGGVDAERLKHLIVTPSGAGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAP

[0120] >Sequence ID 10: HC monomer of h8C3dTa

[0121] MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPAGGGGVDAERLKHLIVTPSGAGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAP

[0122] > Sequence ID 11: LC monomer of h10Te

[0123] MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0124] >Sequence ID 12: HL monomers of h8Te and h8h10Te

[0125] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSPAGGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0126] >Sequence ID 13: L4, germline CDR-L1 with R24Q and R30S

[0127] DIQMTQSPSSLSASVGDRVTITC QASQDISNY LNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

[0128] >Sequence ID 14: LC monomers of h8h10hB2Ti and h8h10hB2D5Ti

[0129] MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSPAGGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYTMNWVRQAPGQGLEWMGVINPNHGISSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRKISYDYDEGYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLVFFAATRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKLEIK

[0130] >Sequence ID 15: HC monomer of h8h10hB2Ti

[0131] MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0132] >Sequence ID 16: HC monomer of h8h10hB2D5Ti

[0133] MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPGGGGSEVQLVQSGGGVERPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRVTISRDNAKNSLYLQMNSLRAEDTAVYYCAKILGAGRGWYFDLWGKGTTVTVSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL

[0134] >Sequence ID 17:

[0135] RASQDIRNY

Claims

1. A multispecific antibody having an N-terminus and a C-terminus, comprising a first monomer comprising, from said N-terminus to said C-terminus, a VL domain, a first linker and a first Fc domain, a second monomer comprising, from the N-terminus to the C-terminus, a VH domain, a second linker, and a second Fc domain, and The first binding domain, wherein the first monomer and the second monomer are paired through the interaction between the VL domain and the VH domain, and The multispecific antibody is stabilized by a disulfide bond between the first linker and the second linker, and the amino acid sequence of the light chain of the multispecific antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

2.

2. The multispecific antibody according to claim 1, further comprising a second binding domain, wherein the amino acid sequence of the light chain and the amino acid sequence of the heavy chain of the multispecific antibody are shown as SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

3. An isolated nucleic acid encoding the multispecific antibody of any one of claims 1 to 2.

4. An expression vector comprising the isolated nucleic acid of claim 3.

5. A host cell comprising the isolated nucleic acid of claim 3. A host cell comprising the expression vector according to claim 4 .

7. A method for producing the multispecific antibody according to any one of claims 1 to 2, comprising: culturing a host cell so that a DNA sequence encoding the multispecific antibody according to any one of claims 1 to 2 is expressed, and The multispecific antibody is purified.

8. A method for preparing the multispecific antibody of any one of claims 1 to 2, comprising culturing a host cell under conditions that produce the multispecific antibody of any one of claims 1 to 2 and recovering the antibody.

9. An immunoconjugate comprising the multispecific antibody according to any one of claims 1 to 2 and a cytotoxic agent.

10. An immunoconjugate comprising the multispecific antibody according to any one of claims 1 to 2 and an imaging agent. 11 . A pharmaceutical composition comprising the multispecific antibody according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claim 11, further comprising a therapeutic agent.

13. The pharmaceutical composition of claim 12, wherein the therapeutic agent is a chemotherapeutic agent or a radionuclide, or a combination thereof. The pharmaceutical composition according to claim 13 , wherein the radionuclide is a radioisotope.

15. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 9 to 10 and a pharmaceutically acceptable carrier. 16 . Use of the multispecific antibody of claim 1 for the preparation of a medicament for treating cancer in a subject, wherein the cancer is gastric cancer, colon cancer, pancreatic cancer, or liver cancer.

17. The use according to claim 16, wherein the medicament is administered in combination with an effective amount of a therapeutic agent.

18. The use according to claim 17, wherein the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

19. The use according to claim 16, wherein the subject is a human.

20. A solution comprising an effective concentration of the multispecific antibody of any one of claims 1 to 2, wherein the solution is plasma from a subject.

Images