Compositions of TriAx antibodies, methods of making and using same

Abstract

1. A multispecific antibody having an N-terminus and a C-terminus, comprising: a first monomer comprising, from the N-terminus to the 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 at least a first binding domain attached to either the N-terminus or the C-terminus of the multispecific antibody, wherein the first and second monomers are paired by 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.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national stage application of International Application No. PCT / US2020 / 063461, filed December 4, 2020, and claims priority to U.S. Provisional Application No. 62 / 944,230, filed December 5, 2019 (35 U.S.C. 119(e)), the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing The ASCII text file of the Sequence Listing, entitled "ARTI906PCT_ST25," is 84kb in size and was created and submitted electronically via EFS-Web on December 4, 2020, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to the field of cancer immunotherapy, and in particular to compositions of modified antibodies with multiple antigen-binding specificities. [Background technology]

[0004] Unless otherwise stated herein, the material described in this section is not prior art to the claims of this application and is 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 the deadliest diseases affecting humans. Our understanding of how tumors initiate, survive under stress, colonize / metastasize to distant organs and sites, and become resistant to drugs is still limited. The American Cancer Society estimates that there were 1.6 million new cases of cancer in the United States in 2014, and there are no approved treatments for most major types of cancer.

[0006] Gastrointestinal (GI) cancers (colorectal, stomach, pancreas, esophagus, bile duct, and liver) are a major cause of morbidity and mortality worldwide. Colorectal cancer (CRC) alone accounts for approximately 10% of all cancer diagnoses and is the second leading cause of cancer deaths worldwide. Liver and stomach cancers are among the world's deadliest malignancies, with over half of diagnosed cases in China, causing more than 1.42 million deaths annually worldwide. These cancers are thought to be attributable to endemic viral / bacterial diseases (hepatitis B virus [HBV] and Helicobacter pylori infections), chemical poisoning, environmental pollution, and food contamination. However, effective treatments are lacking. Therefore, new biomarkers and therapeutic targets are needed to develop potential drugs against these aggressive cancers. Approved molecular-targeted drugs that can eliminate or inhibit the growth of these cancers have significant clinical value and significant market impact. These tumors can be effectively removed by surgery if diagnosed early; unfortunately, most GI cancers are asymptomatic and often present at a very advanced stage by the time patients present to the hospital. Without effective treatment, these patients risk dying soon after diagnosis or relapsing after salvage therapy.

[0007] CDH17 is a prominent cancer biomarker characterized by its overexpression in both liver and gastric cancers but not in normal tissues of healthy adults. Anti-CDH17 monoclonal antibodies exhibit growth inhibitory effects on liver and gastric tumor cells. CDH17 is highly expressed in metastatic cancers, and blocking CDH17 expression and function can significantly reduce lung metastasis of hepatocellular carcinoma (HCC). These observations suggest that humanized anti-CDH17 antibodies could be developed as targeted therapeutic agents for treating cancer patients with CDH17 biomarker signatures in tumor tissues and / or serum samples.

[0008] While antibody-drug conjugates hold promise as antibody therapies, multispecific antibody therapies harness the immune response against cancer to activate T cell-mediated cytotoxicity against cancer cells.

[0009] Bispecific antibodies targeting CD3+ T cells and CD19+ B cells have proven effective in treating hematological malignancies (Labrijn 2019, Yu 2017, Suurs 2019, and Bates 2019). However, attempts to target solid tumors have been largely unsuccessful, likely due to a lack of access to solid tumor cells and appropriate immunomodulatory signals. Antibody-based scaffolds are needed to efficiently target multiple tumor antigens and immune cell antigens or products to develop more effective immunotherapies that better address the complexities of the pre-tumor microenvironment and tumor evasion mechanisms. Summary of the Invention

[0010] In one aspect, the present invention provides a multispecific antibody. The antibody may be bispecific, trispecific, tetraspecific, or pentaspecific. The antibody may have a truncated structure.

[0011] In one embodiment, the present invention provides a multispecific antibody having an N-terminus and a C-terminus, comprising: a first monomer comprising, from the N-terminus to the 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 at least a first binding domain attached to the N-terminus or the C-terminus of the multispecific antibody, wherein the first and second monomers are paired by 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.

[0012] In one embodiment, it is linked to the N-terminal VH domain, the N-terminal VL domain, the C-terminal first Fc domain, or the C-terminal second Fc domain.

[0013] 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 attached to the C-terminus of the first Fc domain and the second binding domain is attached to the C-terminus of the second Fc domain. In one embodiment, the first binding domain is attached to the N-terminus of the VH domain and the second binding domain is attached to the C-terminus of the first Fc domain.

[0014] 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 attached to opposite ends of the antibody. In one embodiment, the first binding domain and the second binding domain are attached to the same end of the antibody. In one embodiment, the first binding domain is attached to the N-terminus of the VH domain, and the second binding domain is attached to the N-terminus of the VL domain.

[0015] 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 attached to the N-terminus of the VH domain, the second binding domain is attached to the N-terminus of the VL domain, and the third binding domain is attached to the C-terminus of the first Fc domain or the C-terminus of the second Fc domain.

[0016] 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 attached C-terminally to the first Fc domain, and the fourth binding domain is attached C-terminally to the second Fc domain.

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

[0018] Each first binding domain may be independently selected from the group consisting of an scFv domain, a ligand, a single domain nanobody, a binding region of a natural protein, a chemokine, and a cytokine.

[0019] In one embodiment, the bispecific antibody may comprise 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.

[0020] 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.

[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:7, and a second monomer comprising an amino acid sequence having at least 98% sequence identity to 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 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, the 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, the 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, the 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.

[0022] In one embodiment, the tetraspecific 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.

[0023] In one embodiment, the pentaspecific 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.

[0024] In one embodiment, the binding domain may 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 may comprise at least 20%, 30%, or 50% proline residues. In one embodiment, the linker may comprise from about 2 to about 31 amino acids.

[0025] In another aspect, the present invention provides an isolated nucleic acid sequence encoding the multispecific antibody.

[0026] In a further aspect, the present invention provides an expression vector comprising the isolated nucleic acid sequence.

[0027] In a further aspect, the invention provides a host cell comprising the isolated nucleic acid sequence.

[0028] In a further aspect, the present invention provides a method for producing said multispecific antibody, in one embodiment said method comprising the steps of culturing a host cell to express DNA sequences encoding said multispecific antibody, and culturing said multispecific antibody.

[0029] In a further aspect, the present invention provides a method for producing said multispecific antibody, hi one embodiment, said method comprising culturing host cells under conditions such that said multispecific antibody is produced, and recovering said antibody.

[0030] In a further aspect, the present invention 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.

[0031] In a further aspect, the present invention provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises the 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 the immunoconjugate and a pharmaceutically acceptable carrier.

[0032] In a further aspect, the present invention 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 the purified multispecific antibody. In one embodiment, a method for treating a subject with cancer comprises administering to the subject a therapeutically effective amount of the multispecific antibody. In one embodiment, the method may further comprise co-administering a therapeutically 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.

[0033] In a further aspect, the present invention provides a solution comprising an effective concentration of said multispecific antibody, said solution being plasma of a subject. [Brief explanation of the drawings]

[0034] Embodiments according to the present disclosure will now be described with reference to the drawings, in which like elements are designated with like numerals.

[0035] [Figure 1] The composition of a class of multispecific antibodies collectively known as tri-axial antibodies (abbreviation: TriAx) (including, but not limited to, TriAx-A, TriAx-C, TriAx-D, TriAx-E, TriAx-I, and TriAx-J antibodies) is shown. [Figure 2] 1 shows the production, heterodimerization and purification of TriAx-A antibody. [Figure 3] 1 shows the production and binding specificity of TriAx-C antibodies. [Figure 4] Demonstrates the thermostability of the TriAx antibody. [Figure 5] Figure 1 shows the cytotoxicity of redirected T cells by the TriAx-A antibody targeting TROP2. [Figure 6] Figure 1 shows the cytotoxicity of redirected T cells by TriAx-A antibody targeting FAP. [Figure 7] 1 shows a low affinity anti-CD3 binding domain with amino acid substitutions. [Figure 8] 1 shows that TriAx-A with L4 exhibits reduced T cell affinity and activation. [Figure 9] 1 shows that TriAx-A with L4 mediates cytotoxicity comparable to TriAx-A without the L4 mutation. [Figure 10] The binding specificity and redirected T cell cytotoxicity of the TriAx-E antibody are demonstrated. [Figure 11] 1 shows the steric effect of additional binding domains on the function of the TriAx core, such as anti-CD3 binding affinity. [Figure 12] 1 shows the stabilized scFv(LocV) binding domain in the TriAx antibody. [Figure 13] 1 shows a stabilized, low-antigenic linker in the TriAx antibody. DETAILED DESCRIPTION OF THE INVENTION

[0036] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in various configurations expressly contemplated herein.

[0037] To enable more effective cancer treatment, especially immunotherapy for solid tumors, combination therapies incorporating multiple target specificities and / or mechanisms of action beyond typical bispecific antibodies are paramount. Therapies that are inherently combination therapies, such as those described above, are necessary to effectively treat cancer and achieve more frequent, complete, and durable responses. Specifically, scaffolds with specific properties are needed to develop combination therapeutics with more favorable mechanisms of action, manufacturing, pharmacokinetics, and low antigenicity compared to approved bispecific antibodies. Many bispecific antibodies based on whole antibodies may have a larger mass than the trispecific antibodies described herein. Smaller mass antibodies based on antibody fragments (e.g., the FDA-approved bispecific antibody Blincyto without an Fc region) are more likely to penetrate tumors and generally have relatively poor pharmacokinetic properties. Furthermore, many bispecific antibodies are based on knobs-into-hole (knobs-into-hole) structures, which have mutations within the constant domain of the Ig structure, potentially contributing to anti-drug antibody (ADA) responses. In this case, a group of modified antibodies, described herein as Tri-axial or TriAx antibodies, possess multiple antigen-binding specificities without the need for mutations in the constant domains.

[0038] All TriAx antibody formats contain a characteristic core structure comprising a single pair of VH and VL (Fv) domains that defines the initial antigen-binding specificity while precisely driving heterodimerization of two Fc-containing monomers. This core structure is stabilized by multiple disulfide bonds at the C-terminus of the Fv region. At a minimum, a third linker ("triaxial" core) is used to add at least one additional antigen-binding domain, such as an scFv. A second linker can be added, such as a second scFv, to enhance tumor cell binding specificity or modulate the immune response. These "TriAx" antibodies may be further modified with engineered proline-rich rigid peptide linkers to position the binding domains for optimal ligand binding. TriAx antibodies may also be constructed with fully human, humanized, and low-antigenicity linker sequences to reduce the risk of ADA responses.

[0039] TriAx antibodies are designed to bind to two or more effector cell receptors to induce two or more mechanisms of anti-tumor activity (e.g., 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 conversion of tumor-associated macrophages (TAMs) from an immunosuppressive to a pro-inflammatory phenotype (CD206, TREM-2)).

[0040] The TriAx platform enables the production of TriAx-A, TriAx-C, TriAx-D, TriAx-E, TriAx-I, and TriAx-J antibodies, as shown in Figure 1. Multispecific antibodies, i.e., bispecific, trispecific, tetraspecific, and pentaspecific antibodies, can be produced according to these formats. The TriAx antibody core is characterized by paired VL and VH Fv regions directly linked to an Fc domain in the absence of a CH1. This core can be formed and stabilized by pairing two asymmetric monomers (LC and HC monomers) via disulfide bridges (Ig hinge or other linkers) (Table 1). This Fv-Fc core has at least one additional linker attached to the antigen-binding domain that binds to the target antigen / ligand. TriAx-A is a bispecific antibody format in which one scFv domain is added covalently to the N-terminus of the VH. TriAx-C is a trispecific antibody format with one scFv domain covalently attached to the N-terminus of the VH and a second scFv domain covalently attached to the C-terminus of the CH3 domain. TriAx-D is a trispecific antibody format with two different scFv domains attached to the C-terminus. TriAx-E is a trispecific antibody format with two different scFv domains attached to the N-terminus, each attached to the VL and VH. TriAx-I is a tetraspecific antibody format with a third scFv domain attached to the C-terminus of Trix-E. TriAx-J is a pentaspecific antibody format with a fourth scFv domain attached to the C-terminus of Trix-I.Other multispecific antibody formats lack this TriAx core structure, which directs and covalently stabilizes this multispecific heterodimeric architecture (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). The following examples demonstrate that TriAx antibodies can not only be produced, but also function with the efficiency and stability for which they were designed. The production of these TriAx antibodies suggests that the percentage of correctly formed heterodimers is higher than with other modified antibodies, such as knob-into-hole antibodies.

[0041] As used herein, the terms "a," "an," and "the" are defined to mean "one or more," and include the plural referents unless the context is inappropriate.

[0042] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (agonist and antagonist antibodies), antibody compositions with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, chimeric, single-chain, multispecific, multipotent, human, and humanized antibodies. Examples of active antibody fragments that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, as well as the products of 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 immunoreactive portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds to an antigen. Immunoglobulins may be immunoglobulin molecules of any type (IgG, IgM, IgD, IgE, IgA, and IgY), class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one embodiment, antibodies may be whole antibodies and any antigen-binding fragments derived from whole antibodies. A typical antibody generally refers to a heterotetrameric protein consisting of two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable domain (V H Each light chain portion is composed of a light chain variable domain (V L V H Area and V L The region can be further divided into domains of hypervariable complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs). H or V L ) is usually composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding regions that interact with antigens.

[0043] As used herein, the term "multispecific" antibody refers to an antibody having at least two binding sites, each with binding affinity for an epitope of an antigen. The terms "bispecific, trispecific, tetraspecific, or pentaspecific" antibody refer to an antibody having two, three, four, five, or six antigen-binding sites.

[0044] The term "humanized antibody" refers to an engineered antibody having CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one or more human immunoglobulins. Additionally, framework support residues can be altered to retain binding affinity. Methods for obtaining "humanized antibodies" are known to those skilled in the art (Queen et al., Proc. Natl Acad Sci USA, 1989; Hodgson et al., Bio / Technology, 1991). In one embodiment, "humanized antibodies" can be obtained by genetic engineering methods capable of producing affinity-matured, human-like polyclonal antibodies in large animals (e.g., rabbits) (US Pat. No. 7,129,084).

[0045] The term "antigen" refers to an entity or fragment thereof that is capable of inducing an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

[0046] The term "epitope", also known as "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 the antigen to which an antibody binds.

[0047] The term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against the immunogenic substance and contributes to a human or animal 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 the invention to alleviate or ameliorate the disorder being treated.

[0048] As used herein, the term "tumor antigen" refers to an antigenic molecule produced by a tumor cell. A tumor antigen can elicit an immune response in a host. In one embodiment, a tumor cell expresses a tumor antigen, including, but not limited to, tumor-specific antigens (TSAs), neoantigens, and tumor-associated antigens (TAAs).

[0049] As used herein, the terms "binds specifically," "specific binding," or "specific" for a particular antigen or epitope refer to binding that is measurably different from non-specific interactions. Specific binding can be measured by determining binding of a molecule compared to binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. Specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or epitope is at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 Specific binding to a particular antigen or epitope can be demonstrated by an antibody having a KD for the antigen or epitope of M or greater. Here, KD refers to the dissociation rate of a particular antibody-antigen interaction. In some embodiments, a multispecific antibody that specifically binds to an antigen has a KD for the antigen or epitope that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, or 10,000-fold or greater compared to a control molecule. Specific binding to a particular antigen or epitope can also be demonstrated by an antibody having a K A or K B for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, or 10,000-fold greater than the control. K A or K B refers to the association rate of a particular antibody-antigen interaction.

[0050] Example 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 specified. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same or similar results.

[0051] Example 1: Characteristics of TriAx antibodies TriAx antibodies are heterodimers (Figure 1) characterized by an Fv-Fc core structure composed from N- to C-terminus of an Fv region, a modified Ig hinge, and an Ig Fc region. The additional binding domains may be scFv, scFab, Fab, single domain VH, or native protein fragments.

[0052] The TriAx core component contains two linkers (e.g., a glycine-rich linker fused to a truncated Ig hinge) that covalently link both the VH and VL chains of the Fv to the CH2-CH3 monomer. A flexible glycine-rich linker fused to the Ig hinge can promote efficient VH-VL pairing. The TriAx binding domains can be linked by a flexible glycine-rich linker (e.g., PAGGGGS) or a more rigid proline-rich linker (e.g., PAGPPP). The linker is typically 4 to 7 residues in length. The TriAx Fc may be composed of an IgG1 hinge or an IgG4 hinge with an S228P substitution. The first seven N-terminal amino acids, EPKSCDK, of the IgG1 hinge may be replaced with a glycine-rich linker (e.g., GAPGGGG or PAGGGGS). The hinge residues at positions 234 and 235 (G1 numbering) can be LL, FL, or AA to modulate FcR binding (Saunders 2019). The CH2 and CH3 domains can be all IgG1, all IgG4, or a combination, such as a G1 CH2 and a G4 CH3. TriAx molecules can also have CH3s with substitutions to create knobs-into-holes (Merchant 1998).

[0053] TriAx-A, constructed based on the central TriAx Fv-Fc core structure, is a bivalent antibody format in which a single scFv is linked to the VH or VL of the Fv (e.g., h10Ta, h5Ta, h8Ta, and hB2Ta antibodies). Its structural characteristics are shown 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 either the CH2-CH3 monomer (e.g., hC3dh10Tc antibody). Its structural characteristics are shown in Table 1. TriAx-D is a trivalent antibody in which an scFv is linked to the C-terminus of each CH3 of the Fv-Fc core (e.g., h8C3dTa antibody). Its structural characteristics are shown 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. The structural features and sequence numbers of exemplary TriAx-E antibodies (e.g., h10Te, h8Te, and h8h10Te) are shown in Table 1. TriAx-I is a tetravalent antibody format in which one scFv is added to the N-terminus of each of VH and VL, and one scFv is added to the C-terminus of either of the CH2-CH3 monomers. TriAx-J is a pentavalent format in which one scFv is added to the N-terminus of each of VH and VL and to the C-terminus of each of the CH2-CH3 monomers.

[0054] Example 2: TriAx-A antibody h8Ta is a TriAx-A bispecific antibody that targets both TROP2 and CD3 (SEQ ID NOS: 1 and 4; see also Table 1). TROP2 is a transmembrane protein that is deregulated in all types of cancer, despite baseline levels of TROP2 expression. TROP2 is an ideal candidate for targeted therapy. In several early-phase clinical trials, antibody therapeutics targeting TROP2 have demonstrated safety and clinical efficacy in the treatment of triple-negative breast cancer, platinum-resistant urothelial carcinoma, and small-cell lung cancer.

[0055] h8Ta was produced in HEK293 cells by PEI cotransfection of plasmids for the heavy chain (core Fv VH) and light chain (core Fv Vk). Three days after transfection, media samples were subjected to SDS-PAGE (Figure 2; transfected cell media (lanes 1 and 2), mock-transfected media (lane 3)). After one-step protein A purification, the h8Ta antibody was subjected to SDS-PAGE under non-reducing and reducing conditions (lanes 4 and 5, Figure 2, respectively). Under non-reducing conditions, this TriAx-A antibody migrates as a protein of approximately 116 kDa, consistent with the calculated heterodimer size. Under reducing conditions, the heavy chain is approximately 70 kDa and the light chain is approximately 44 kDa, consistent with the calculated sizes. Heterodimerization efficiency was approximately 90% or greater. No other significant TriAx products or fragments were detectable compared to mock control media. These TriAx-As can have linkers of different lengths, compositions and Fc sequences to alter FcR binding and circulating half-life.

[0056] Example 3: TriAx-C antibody Two TriAx-C antibodies were generated with binding specificity for the phagocyte receptor CR3. h10Cd3Tc is a TriAx-C trispecific antibody (SEQ ID NOs: 7 and 8) targeting CDH17, CR3, and CD3. h8C3dTd is a TriAx-D trispecific antibody (SEQ ID NOs: 9 and 10) targeting TROP2, CR3, and CD3. TROP2 and CDH17 are both important cancer biomarkers characterized by overexpression in various forms of solid tumors, including gastric cancer, colon cancer, pancreatic cancer, and liver cancer. CDH17 is highly expressed in metastatic cancers, 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 exhibit growth inhibitory effects on liver and gastric tumor cells (see applicant's application WO / 2019 / 222428, incorporated herein in its entirety). CR3, or complement receptor 3, is a heterodimer of α (CD11b) and β (CD18) transmembrane proteins. The I domain containing the 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 target cell surface and serves as a macrophage CR3 ligand for phagocytosis. Binding to CR3 via its ligand (e.g., C3d) or an activating antibody can target the major phagocytic receptor to tumor cells, broadly inducing tumor cell phagocytosis and proinflammatory macrophage polarization. TriAx-C antibodies (e.g., h8C3dTd and h10C3dTc) can bind TROP2 and / or CDH17 to tumor cells and present C3d for macrophage CR3-dependent phagocytosis. TriAx-C antibodies can also bind FcR, thereby further activating and enhancing phagocytosis of CR3 tumor cells by macrophages. These TriAx-C antibodies can broadly target various tumor types and achieve greater efficacy and safety than those targeting the CD47 or CD24 phagocytic checkpoints.

[0057] In addition to the anti-CD3 Fv and anti-CDH17 scFv domains, h10Cd3Tc contains 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, the level of antibody expression was determined using Octet (BLI). The 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). Production medium samples were subjected to SDS-PAGE. As shown in Figure 3A, the molecular weight of h10C3dTc was approximately 180 kDa, larger than the calculated approximately 150 kDa. Compared to mock control cultures, no other significant TriAx products or fragments were detected. Because both the heavy and light chain monomers of h10Cd3Tc are roughly the same size, homodimer formation cannot be easily distinguished by standard SDS-PAGE. When concentrations were all adjusted to 5 μg / ml in ELISA to quantify binding to the immobilized CR3 I domain, peak I domain binding occurred at a plasmid ratio of 6:1 (Figure 3B). Due to the low binding affinity of C3d for the I domain (approximately 400 nM), the OD values ​​in ELISA were low, but peak binding was approximately 7-fold greater than that of control cultures from mock-transfected HEK293 cells. Binding of h10C3dTc to CDH17 and CD3 was determined by ELISA. In ELISA, the sample antibody binds to soluble CD3, followed by immobilized CDH17, followed by HRP conjugates that bind to recombinant CD3. As shown in Figure 3C, peak binding was obtained at a plasmid ratio of 4:1, indicating optimal heterodimerization can occur when the plasmids are cotransfected into HEK293 cells at a specific ratio. The binding activity shown in ELISA demonstrates that trispecific TriAx-C antibodies can be produced. These TriAx-C antibodies can have linkers of different lengths, compositions, and Fc sequences to alter FcR binding and circulating half-life.

[0058] Example 4: Thermal stability of TriAx antibodies The thermal stability of the TriAx antibody was measured by thermal shift assay using SYPRO orange (King et al. 2011). TriAx-A antibodies (e.g., h8Ta (SEQ ID NOs: 1 and 4), hB2Ta (SEQ ID NOs: 1 and 5), and hA12Ta (SEQ ID NOs: 1 and 6) (see Table 1)) were analyzed. These TriAx-A antibodies (in PBS) were centrifuged in a microfuge for 10 minutes 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 an optically clear 96-well plate for assay in a qPCR instrument. The temperature was increased from 25 °C to 99 °C at a rate of 1 °C / min, and measurements were taken at an excitation wavelength of 470 nm and an emission wavelength of 586 nm, with each measurement held for 1 min. Figure 4 shows the 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.

[0059] Example 5: Redirected T cell cytotoxicity of TriAx-A antibodies targeting TROP2 and CD3 To evaluate the functionality of TriAx platform antibodies, we assessed the cytotoxicity of redirected T cells using the TriAx-A bispecific antibody h8Ta (Example 2). Three luciferase-expressing GI tumor cell lines, DLD1 (colorectal cancer), SW480 (colorectal cancer), and AGS (gastric cancer), were used in a 24-hour assay at an E / T ratio of 4. Dead cells were then removed by washing, and live cells were quantified using Bio-Glo (Promega) and a multimode plate reader. As shown in Figure 5 (top panel), h8Ta was used in flow cytofluorometry analysis to detect TROP2 expression in the three tumor cell lines (Figures 5A, 5B, and 5C). In the absence of T cells, cell viability did not decrease across a range of h8Ta antibody concentrations. In the presence of T cells, the EC50 values ​​for h8Ta antibody-dependent tumor cell killing were 0.8 pM for DLD, 2 pM for AGS, and 11 pM for SW480 (Figure 5 (bottom panel), 5D, 5E, and 5F). The lower EC50 value of SW480 appears to correlate with its lower level of TROP2 expression. Thus, this bispecific TriAx-A antibody can mediate potent sub-pM tumor cell killing.

[0060] Example 6: Redirected T cell cytotoxicity of TriAx-A antibodies targeting FAP and CD3 Fibroblast activation protein α (FAP) is a 97 kDa, type II cell surface glycoprotein belonging to the serine protease family. In colorectal cancer (CRC) metastasis, fibroblast activation protein α (FAPα) plays an important role. It has been reported that FAPα was expressed in cancer-associated fibroblasts in all CRC samples examined, but not in normal colon, hyperplastic polyp, or adenoma samples.

[0061] The TriAx-A bispecific antibody hB2Ta (SEQ ID NOs: 5 and 6) was generated to target both CD3 (via Fv) and FAP (via scFv). To determine the cytotoxic activity of its redirected T cells, FAP mRNA was electroporated into DLD1 cells (DLD1-FAP), which also express luciferase. The following day, microtiter plate cytotoxicity assays were initiated with or without expanded T cells (E / T: 4). After adding the antibody to a mixture of DLD1 or DLD1-FAP at a range of experimental concentrations, the assays were incubated for 24 hours, after which they were washed, Bio-Glo substrate was added, and luciferase activity was measured using a multimode plate reader. As shown in Figure 6A, h1B2Ta mediated concentration-dependent tumor cell cytotoxicity (EC50 = 41 pM) in the presence of T cells and DLD1-FAP, whereas no such cytotoxicity was detected or demonstrated in the absence of FAP-expressing tumor cells or T cells. FAP expression in DLD1 cells was determined by flow cytofluorometry at the start of the assay (Figure 6B). The percentage of DLD1 cells expressing FAP at 68% (MFI = 4,256) appears to correlate with approximately 70% of maximum cytotoxicity. Therefore, TriAx antibodies with binding specificity to FAP can effectively kill tumor cells with low FAP expression.

[0062] Example 7: L4 (low affinity anti-CD3 binding domain) To reduce the risk of T cell signaling to normal tissues (off-tumor T cell signaling) and sinking to T cells and lymphoid tissues, the low-affinity monovalent CD3-binding region L4 (SEQ ID NO: 13) was introduced into the TriAx platform antibody. The CDRs of UCHT1 Vh and Vk (Shalaby 1992) were partially or completely replaced with human germline sequences to generate low-affinity and low-antigenicity anti-CD3 variants. Substitution of VkCDR1 with IGKV1-33*01 germline sequences yielded the low-affinity variant L4, which was incorporated into the core Fv of several TriAx antibodies. The amino acid sequences of the anti-CD3 variants thus contain the UCTH1 CDR sequences, except for the CDRL1 substitutions, R24Q and R30S, as shown in Figure 7.

[0063] Example 8: T cell affinity and activation mediated by TriAx antibodies with L4 To assess T cell affinity and activation potential, TriAx antibodies bearing L4 or its parent Fv Vk CDR1(wt) (i.e., h10Ta-L4 (SEQ ID NOs: 1 and 2) and h10Ta-wt) were assayed by flow cytofluorometry. For antibody binding to peripheral blood T cells, T cell affinity was measured across a range of concentrations, as shown in Figure 8A. MFI was plotted, and affinity (EC50) was determined using GraphPad PRISM. Three independent affinity measurements and their average are shown. The results show that the modified CDRL1 in L4 (320 nM) causes a 5-fold decrease in T cell affinity compared to the parent 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). Both h10Ta antibodies bound to CDH17 (on DLD1) and CD3. Signaling was determined according to the manufacturer's protocol (prescribed antibody concentration ranges, as well as 100 x 10 Jurkat receptor cells per microtiter well (96-well plate)). 3 and DLD1 cells 30x10 3(The cells were treated with IgG.) Luciferase expression / activity was measured using a multimode plate reader. As shown in Figure 8B, T cell signaling with h10Ta-L4 was two-fold reduced compared with h10Ta-wt with the parent Fv. Controls did not contain antibodies or CD3, CD28, or CD2 agonists (Immunocult; StemCell) to induce maximal stimulation.

[0064] Example 9: Tumor cell cytotoxicity mediated by TriAx antibodies bearing L4 The cytotoxicity of redirected T cells was determined for both h10Ta-L4 and h10Ta-wt. Additionally, h10Ta-L4c, derived from h10Ta-L4 by incorporating Vk ACys43 and Vh Q114C substitutions to form stable interdomain disulfides, was included in this study. T cell killing against the luciferase-expressing colon cancer cell line DLD1 and gastric cancer cell line AGS was determined over a range of antibody concentrations in a 24-h assay with an E / T ratio of 4. Dead cells were then removed by washing, and 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 (h10Ta-wt), 0.4 pM (h10Ta-L4), and 1.2 pM (h10Ta-L4c). The EC50 for killing AGS was 1.4 pM (h10Ta-wt), 2 pM (h10Ta-L4), and 4.7 pM (h10Ta-L4c). These results, shown in Figure 9, indicate that the lower affinity of L4 for CD3 did not significantly reduce cytotoxic activity, as determined in this assay. For the TriAx antibody with L4c, the results showed a slight decrease in cytotoxic activity compared to the antibody with the parent L4, suggesting that the interdomain disulfide can exert a negative position effect by altering the position of CDR residues involved in CDH17 binding.

[0065] Example 10: TriAx-E antibody h8h10Te (SEQ ID NOs: 12 and 2) is a TriAx-E trispecific antibody containing both 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 cytofluorometry. Figure 10 shows that h8h10Te specifically bound to HEK293 transfectants expressing transmembrane forms of either CDH17 or Trop2. h8h10Te also specifically bound to Jurkat cells expressing CD3. Thus, a TriAx-E trispecific antibody can be generated that can bind to all three target antigens. The functionality of TriAx-E is shown in Figure 10. h8h10Te supports effective redirected T cell killing against DLD1 tumor cells (24 hour cytotoxicity assay E / T ratio 4).

[0066] Example 11: Steric Effects of Multispecific TriAx Antibodies If the TriAx core structure contains an anti-CD3 binding domain at the Fv position, adding one or more binding domains, such as an scFv domain, may affect the antibody's efficacy in binding to cellular CD3. In this regard, the TriAx-A antibody h10Ta (SEQ ID NOs: 1 and 2) and the TriAx-E antibody h10Te, which have identical anti-CD3 Fv regions (wt), were used for comparison. CD3 binding activity was determined by flow cytometry using 5 μg / ml of each TriAx antibody. Figure 11 shows that binding of the TriAx-E antibody was approximately 2- to 6-fold reduced (median fluorescence intensity; MFI) compared to binding of the TriAx-A antibody. The reduced binding to the anti-CD3 Fv, as in the TriAx-E core, may be due to steric inhibition. TriAx formats in which scFvs are attached to the core Fv Vh and Vk, as in TriAx-E (e.g., TriAx-I and TriAx-J), may also exhibit reduced binding to CD3 or other core Fv specificities. Compared to certain other formats, administration of these TriAx antibodies can reduce sinks to T cells or lymphoid tissues and increase biodistribution to tumor tissues. Therefore, this structure can provide better local activity in the tumor microenvironment (TME), superior efficacy, and safety. The TriAx-A and TriAx-C formats have a smaller N-terminal mass due to the single N-terminal scFv, but can bind to tumor antigens more efficiently than TriAx-E or typical whole antibodies.

[0067] Example 12: Stabilized scFv (LocV) in TriAx antibody Stabilized versions of TriAx-A scFv specific for TROP2 (h8v5 and h8v6) or CDH17 (h10v3) were generated by backmutating framework residues in the humanized versions h8v4 and h10v2, strengthening the Vh-Vk interface (h8v5, h8v6, and h10v3), and substituting two residues in the Vh domain with cysteines to create a second disulfide bond (h8v6) (Ewert 2004, McConnell 2012, Weatherill 2012). As shown in Figure 12, these humanized and stabilized versions were expressed in HEK293 cells as Fc:G1G4G1 (A and C) or Fc:G1G4 (B and D). Binding to CDH17 and TROP2 expressed in HEK293 cells by standard PEI transfection was determined by flow cytofluorometry 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 made to stabilize the scFv did not have a significant negative structural impact. Meanwhile, the stabilized scFv (LocV) improved binding of the TriAx antibody to tumor antigens.

[0068] Example 13: Stabilized Low Antigenic Linkers We compared the stability of three proline-rich linkers, A = PAGPPA, B = PAGPAP, and C = PAGPPP, using ARB202, a bispecific antibody specific for CDH17 and CD3 in an IgG-scFv format (see applicant's WO / 2019 / 222428, incorporated herein in its entirety). The linkers extend from the C-terminus of the Fc domain to the N-terminus of the anti-CD3 scFv domain. Bispecific antibodies (1 mg / ml) were stored at 37°C in 10 mM histidine buffer (pH 6.0) for 56 days. At predetermined time points, samples were analyzed for degradation by UPLC. As shown in Figure 13, linker C had higher stability (77.5%) compared to linker A (47.1%) and linker B (32.5%). In binding and signaling assays, the three bispecific antibodies performed similarly in plasma at 37°C after days 0 and 14. Thus, linker C achieves higher stability without compromising function.

[0069] Example 14: TriAx-I and TriAx-J antibodies h8h10B2Ti (SEQ ID NOs: 14 and 15) is an example of a tetraspecific TriAx-I antibody with binding specificities for the tumor-associated antigens TROP2, CDH17, and FAP (expressed on cancer-associated fibroblasts or CAFs). This TriAx-I antibody also binds CD3, triggering T cell-directed killing of GI cancer cells expressing TROP2 and / or CDH17, thereby reducing the likelihood of tumor escape due to loss of tumor target antigen expression. By binding to FAP, this TriAx-I antibody also directs killing of tumor-associated CAFs. CAFs are a key cell type in the tumor microenvironment and support tumor growth by promoting extracellular matrix remodeling, angiogenesis, and immunosuppression.

[0070] h8h10B2D5Tj (SEQ ID NOs: 14 and 16) is an example of a pentaspecific TriAx-J antibody that, like the TriAx-I antibody h8h10B2Ti, has binding specificity for TROP2, CDH17, FAP, DR5, and CD3, but also has binding specificity for DR5. DR5 (death receptor 5), TRAIL receptor 2, and tumor necrosis factor receptor superfamily member 10B are cell surface receptors of the TNF receptor superfamily that bind to TRAIL and mediate apoptosis. The h8h10B2D5Tj antibody possesses the functions of h8h10B2Ti and also exerts the ability to induce tumor cell apoptosis by participating in DR5 signaling in GI cancer cells.

[0071] The above description and examples provide a complete description of the structure and use of exemplary embodiments. While certain embodiments have been described above with a certain degree of particularity or with reference to one or more individual embodiments, those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the scope of the present application. For example, while the above examples may include binding domains in specific locations, these are provided for comparison purposes only and not as limitations. Therefore, the exemplary embodiments of the present application are not intended to be limited to the specific embodiments disclosed. Rather, they include all modifications and alternatives that fall within the scope of the disclosure. Furthermore, where appropriate, any aspect of the above examples can be combined with any aspect of other examples described to form additional examples having similar or different characteristics and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or to several embodiments.

[0072] [Table 1] Sequence Listing >Sequence ID NO: 1: LC Monomer for h10Ta, h5Ta, h8Ta, and hA12Ta MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 2: HC Monomer for h10Ta, h8h10Te, and h10Te MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 3: HC Monomer for h5Ta MAVLGLLFCLVTFPSCVLSQVQLVQSGAEVKKPGATVKISCKVSAYAFSSSWMNWVQQAPGKGLEWIGRIYPRDGDTNYNGKFKGRVTLTADTSTDTAYMELSSLRSEDTAVYFCAREGDGYYWYFDVWGQGTMVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASQSIRNYLHWYQQKPGQPPKLLIKYASQSISGIPSRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSNSWPLTFGAGTKLELKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 4: HC Monomer for h8Ta, and h8Te MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 5: LC Monomer for hB2Ta MEFGLSWVFLVALLRGVQCQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYTMNWVRQAPGQGLEWMGVINPNHGISSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRKISYDYDEGYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLVFFAATRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKLEIKPAGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 6: HC Monomer for hB2Ta, and hA12Ta MEFGLSWVFLVALLRGVQCEVQLVQSGAEVKKPGATVKISCKVSGFKIQDAYIHWVQQAPGKGLEWMGRIDPANGNSKYDPKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCTRALDGYYVGMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCSASSNVNYMYWYQQKPGQAPRLLIYDTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPYTFGQGTKLEIKPAGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYTMNWVRQAPGQGLEWMGVINPNHGISSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRKISYDYDEGYAMDYWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 7: LC Monomer for hC3dh10Tc MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPAGGGGVDAERLKHLIVTPSGAGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAP >Sequence ID NO: 8: HC Monomer for hC3dh10Tc MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 9: LC Monomer for h8C3dTa MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPAGGGGVDAERLKHLIVTPSGAGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAP >Sequence ID NO: 10: HC Monomer for h8C3dTa MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPAGGGGVDAERLKHLIVTPSGAGEQNMIGMTPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGYTQQLAFRQPSSAFAAFVKRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVKWLILEKQKPDGVFQEDAPVIHQEMIGGLRNNNEKDMALTAFVLISLQEAKDICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTAKDKNRWEDPGKQLYNVEATSYALLALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQALAQYQKDAP >Sequence ID NO: 11: LC Monomer for h10Te MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 12: HL Monomer for h8Te, and h8h10Te MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSPAGGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 13: L4,Germline CDR-L1 with R24Q and R30S DIQMTQSPSSLSASVGDRVTITC QASQDISNY LNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK >Sequence ID NO: 14: LC Monomer for h8h10hB2Ti, and h8h10hB2D5Ti MRLPAQLLGLLMLWVSGSSGDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKPGKVPKLLIYAATNLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVANINSDGYNIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRCSYYSYDYFDYWGQGTLVTVSSPAGGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYSFTDYTMNWVRQAPGQGLEWMGVINPNHGISSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCVRRKISYDYDEGYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLVFFAATRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKLEIK >Sequence ID NO: 15: HC Monomer for h8h10hB2Ti MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID NO: 16: HC Monomer for h8h10hB2D5Ti MEFGLSWVFLVALLRGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGKAIKRLIYTTSTLDSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYASSPFTFGGGTKVEIKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGAPGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPGGGGSEVQLVQSGGGVERPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRVTISRDNAKNSLYLQMNSLRAEDTAVYYCAKILGAGRGWYFDLWGKGTTVTVSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL >Sequence ID NO: 17: RASQDIRNY

Claims

1. 1. A multispecific antibody having an N-terminus and a C-terminus, a first monomer comprising, from the N-terminus to the 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; at least a first binding domain attached to either the N-terminus or the C-terminus of the multispecific antibody; Including, the first and second monomers pair through interaction between the VL domain and the VH domain; the multispecific antibody is stabilized by a disulfide bond between the first linker and the second linker; and the first monomer comprises the amino acid sequence of SEQ ID NO: 1 or 5 and the second monomer comprises the amino acid sequence of SEQ ID NO: 2, 3, 4 or 6; or 1. A multispecific antibody, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 7, 9, 11 or 12 and the second monomer comprises the amino acid sequence of SEQ ID NO: 8, 10, 2 or 4.

2. the first monomer comprises the amino acid sequence of SEQ ID NO:7; The multispecific antibody of claim 1 , wherein the second monomer comprises the amino acid sequence of SEQ ID NO:

8.

3. the first monomer comprises the amino acid sequence of SEQ ID NO: 12; The multispecific antibody of claim 1 , wherein the second monomer comprises the amino acid sequence of SEQ ID NO:

2.

4. The multispecific antibody of any one of claims 1 to 3, wherein the first binding domain comprises an scFv domain, a ligand, a single domain nanobody, a binding region of a natural protein, a chemokine or a cytokine.

5. An isolated nucleic acid encoding a multispecific antibody according to any one of claims 1 to 3.

6. An expression vector comprising the isolated nucleic acid of claim 5.

7. A host cell comprising the isolated nucleic acid of claim 5.

8. A method for producing a multispecific antibody according to any one of claims 1 to 3, comprising: Culturing a host cell so that a DNA sequence encoding the multispecific antibody of any one of claims 1 to 3 is expressed; purifying the multispecific antibody; A manufacturing method comprising:

9. An immunoconjugate comprising a multispecific antibody according to any one of claims 1 to 3 and a cytotoxic agent or an imaging agent.

10. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

11. 11. The pharmaceutical composition of claim 10, further comprising a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

12. A pharmaceutical composition comprising the immunoconjugate of claim 9 and a pharmaceutically acceptable carrier.

13. 1. A pharmaceutical composition for use in treating a subject with cancer, comprising:

10. The pharmaceutical composition, wherein the treatment comprises administering to a subject a therapeutically effective amount of a multispecific antibody according to any one of claims 1 to 3.

14. 14. The pharmaceutical composition of claim 13, wherein the treatment further comprises co-administering a therapeutically effective amount of a therapeutic agent.

15. The pharmaceutical composition of claim 13 , wherein the subject is a human.

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