Anti-HER-2 / TROP-2 constructs and uses thereof

Abstract

The present application provides novel bispecific antigen-binding antibodies and antigen-binding fragments thereof that bind to both HER2 and Trop-2. These bispecific anti-HER2 / Trop-2 antibodies comprise a first antigen-binding domain that specifically binds to human HER2 or Trop-2 and a second antigen-binding domain that specifically binds to human Trop-2 or HER2. The bispecific antibodies have high binding affinity to HER2 and Trop-2 and can be internalized by cells expressing HER2 and / or Trop-2. The bispecific antibody drug conjugates (ADCs) can inhibit the growth of tumor cells expressing HER2 and / or Trop-2 in vitro and in vivo. The bispecific antibodies can be used to diagnose, prognose, and treat HER2 and / or Trop-2-associated human diseases (e.g., cancer, infectious diseases, autoimmune diseases, asthma, transplant rejection, and inflammatory disorders).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 140,638, filed January 22, 2021, the entire contents of which are incorporated by reference for all purposes.

[0002] The present disclosure relates to anti-HER2 / Trop2 constructs and uses thereof.

[0003] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated by reference in their entirety into this specification: Sequence Listing in Computer Readable Form (CRF) (Filename: 227362000440SEQLIST.TXT, Recorded: January 24, 2022, Size: 104,030 bytes). [Background technology]

[0004] Today, cancer remains a leading cause of death worldwide, despite the development of numerous advanced diagnostic and therapeutic methods. A major barrier to successful cancer treatment and prevention lies in the fact that many cancers remain unresponsive to current chemotherapy and immunotherapy interventions, and many patients experience recurrence or death even after aggressive treatment.

[0005] Cancer immunotherapy is enjoying a renaissance, a field that has advanced rapidly in recent years, resulting in several new cancer treatments. Numerous cancer immunotherapy strategies have been the focus of extensive research and clinical evaluation, including, but not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints); treatment with bispecific T cell-engaging antibodies (BiTEs®) such as blinatumomab; treatment with administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatment with dendritic cell vaccines, or tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TILs); and treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic).

[0006] HER2 (also known as CD340 or ERBB2), a member of the human epidermal growth factor receptor family, is aberrantly expressed in certain malignancies, including breast, gastric, and lung cancers, mainly due to HER2 genomic amplification. Overexpression of HER2 is associated with an increased risk of recurrence and poor prognosis in these cancers. In HER2-positive malignancies, this protein may play an important role in cell proliferation by stimulating downstream RAS-RAF-ERK and PI3K-PTEN-AKT signaling.

[0007] Trophoblast cell surface antigen-2 (Trop-2), also known as tumor-associated calcium signal transducer 2 (TACSTD1), membrane component chromosome 1 surface marker 1 (M1S1), gastrointestinal antigen 733-1 (GA733-1), and epithelial glycoprotein-1 (EGP-1), belongs to the TACSTD family, which includes at least two type I membrane proteins. Trop-2 was first discovered as a cell surface marker for trophoblast cells, but subsequent studies have suggested that Trop-2 is overexpressed in various cancer types.

[0008] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entireties. Summary of the Invention

[0009] The present application provides, in one aspect, new constructs (e.g., bispecific antibodies or ADCs described herein) that specifically bind to both HER2 and Trop-2. In some embodiments, these constructs include a full-length antibody targeting one of HER2 and Trop-2 fused to an scFv targeting the other of HER2 and Trop2. See, e.g., Figures 1-5. In some embodiments, the constructs are particularly advantageous in their ability to robustly bind to cancer cells expressing HER2 and / or Trop2 at higher levels than the parent antibody (see, e.g., exemplary bispecific antibodies HT002 and TH002, as shown in Figure 6A). In some embodiments, the constructs are particularly advantageous in their ability to robustly bind to cancer cells expressing high levels of HER2 and / or Trop-2 (see, e.g., exemplary bispecific antibodies HT002 and TH001b, as shown in Figure 7). In some embodiments, the constructs are particularly advantageous in their ability to achieve a sustained and / or complete anti-tumor response (see, eg, FIG. 12).

[0010] In another aspect, the application provides methods of using the constructs described herein, for example, to treat cancer. In some embodiments, the cancer is HER2 positive and / or Trop-2 positive. In some embodiments, the cancer is HER2 low expressing and / or Trop-2 low expressing. In some embodiments, the cancer is HER2 high / medium expressing and / or Trop-2 high / medium expressing.

[0011] Bispecific antibodies are antibodies that contain two different antigen-binding sites on a monoclonal antibody that can bind to two different antigens or two different sites on one antigen. In addition to enhancing tumor cell killing by simultaneously blocking two different signaling pathways, bispecific antibodies may also enhance binding specificity by interacting with two different cell surface antigens instead of one. Bispecific antibodies are currently considered the next generation of effective molecules for cancer therapy. They can minimize regulatory and commercial issues that arise from the administration of multiple therapeutic molecules. They may also have new activities that are not present in the mixture of parent antibodies. Several bispecific antibodies are on the market and many are in clinical development.

[0012] The present application provides novel bispecific antibodies and antigen-binding fragments that specifically bind and neutralize both HER2 and Trop-2 proteins. The present application further provides ADCs comprising the novel bispecific antibodies and antigen-binding fragments linked to effector molecules. Accordingly, the present application relates to novel compositions and methods for diagnosing, prognosing, and treating conditions that would benefit from modulating HER2 and Trop-2 (e.g., cancer, persistent infections, autoimmune diseases, asthma, transplant rejection, and inflammatory disorders) using the novel bispecific anti-HER2 / Trop-2 antibodies, antigen-binding fragments, and ADCs described herein.

[0013] In one aspect, the present application provides constructs comprising isolated bispecific antibodies and antigen-binding fragments thereof specifically designed to bind and neutralize both HER2 and Trop-2. In various embodiments, the bispecific antibodies and antigen-binding fragments thereof are designed as shown in Figures 1-5.

[0014] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain and a second immunoglobulin antigen binding domain, where the first immunoglobulin antigen binding domain specifically binds to a HER2 antibody binding site and the second immunoglobulin antigen binding domain specifically binds to a Trop-2 antibody binding site. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain and a second immunoglobulin antigen binding domain, where the first immunoglobulin antigen binding domain specifically binds to a Trop-2 antibody binding site and the second immunoglobulin antigen binding domain binds to a HER2 antibody binding site.

[0015] In various embodiments, the first immunoglobulin antigen-binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises an scFv antibody fragment and the second antigen-binding domain comprises a V H and V L In various embodiments, the first antigen-binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises V H and V L and the second immunoglobulin antigen-binding domain comprises an scFv antibody fragment.

[0016] In various embodiments, the first or second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody further comprises a V H and V further comprising a light chain constant region or a fragment thereof. LIn various embodiments, the heavy chain constant region or fragment thereof is an IgG constant region. In various embodiments, the IgG constant region or fragment thereof is an IgG1 constant region. In various embodiments, the LC constant region is a kappa constant region. In various embodiments, the light chain constant region is a lambda constant region. In various embodiments, the first or second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises a heavy chain that can include an Fc domain comprising a CH2 and a CH3 region. In various embodiments, the Fc domain is an IgG1 Fc domain. In various embodiments, the IgG1 Fc domain is a native IgG1 Fc domain.

[0017] In various embodiments, the first or second immunoglobulin antigen-binding domain is a monoclonal antibody. In various embodiments, the first or second immunoglobulin antigen-binding domain is a humanized antibody. In various embodiments, the first or second immunoglobulin antigen-binding domain is a human antibody. In various embodiments, the first or second immunoglobulin antigen-binding domain is a chimeric antibody. In various embodiments, the first or second immunoglobulin antigen-binding domain is an affinity-optimized antibody.

[0018] In various embodiments, the second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody is covalently linked to the carboxy terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a linker interposed between the second immunoglobulin antigen binding domain and the carboxy terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 25. In various embodiments, the second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody is covalently linked to the amino terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a linker interposed between the second immunoglobulin antigen binding domain and the amino terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 25.

[0019] In various embodiments, the V of the scFv H and V L is covalently linked via a peptide linker. In various embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO:24.

[0020] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:3 and the second immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:4. H and V comprising the amino acid sequence set forth in SEQ ID NO:5 L Includes.

[0021] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds Trop-2 and a second immunoglobulin antigen binding domain that specifically binds HER2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:6 and the second immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:7. H and V comprising the amino acid sequence set forth in SEQ ID NO:8 L Includes.

[0022] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO:7. H and V comprising the amino acid sequence set forth in SEQ ID NO:8 L and the second immunoglobulin antigen-binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:9.

[0023] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen-binding domain that specifically binds Trop-2 and a second immunoglobulin antigen-binding domain that specifically binds HER2, wherein the first immunoglobulin antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 10. H and V comprising the amino acid sequence set forth in SEQ ID NO:11 L and the second immunoglobulin antigen-binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:3.

[0024] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:3 and the second immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:10. H and V comprising the amino acid sequence set forth in SEQ ID NO:11 L Includes.

[0025] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), wherein the HC comprises the amino acid sequence of SEQ ID NO: 12 and the LC comprises the amino acid sequence of SEQ ID NO: 13.

[0026] In various embodiments, the bispecific antibody has a concentration of at least about 1×10 -6 M, at least about 1 × 10 -7 M, at least about 1 × 10 -8 M, at least about 1 × 10 -9 M, at least about 1 × 10 -10 M, at least about 1 × 10 -11 M, or at least about 1 × 10 -12 Dissociation constant of M (K D ) which binds to HER-2 and Trop-2 proteins.

[0027] In another aspect, the application provides an isolated immunoconjugate or fusion protein comprising a bispecific HER2 / Trop-2 binding antibody conjugated, linked (or otherwise stably associated) to an effector molecule. In various embodiments, the effector molecule is an immunotoxin, cytokine, chemokine, drug moiety, therapeutic agent, or chemotherapeutic agent. In various embodiments, the application provides an antibody drug conjugate (ADC) having the formula: Ab-(LD)n, where Ab is a bispecific HER2 / Trop-2 binding antibody, L is a linker, D is a drug moiety, and n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0028] In another aspect, the bispecific antibodies disclosed herein may be covalently linked (or otherwise stably associated) with additional functional moieties, such as a label or a moiety that confers desirable pharmacokinetic properties. In various embodiments, the label is selected from the group consisting of a fluorescent label, a radioactive label, and a label having a characteristic nuclear magnetic resonance signature.

[0029] In another aspect, the present application relates to a pharmaceutical composition comprising an isolated bispecific antibody or antigen-binding fragment or ADC of the present application in admixture with a pharma- ceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises an isolated human antibody in admixture with a pharma- ceutically acceptable carrier. In various embodiments, the pharmaceutical composition is formulated for administration via a route selected from the group consisting of subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or infusion.

[0030] In another aspect, the application relates to the use of the bispecific antibodies or antigen-binding fragments thereof, or ADCs of the application for diagnosing, prognosing, and treating human diseases (e.g., cancer, infectious diseases, autoimmune diseases, asthma, transplant rejection, and inflammatory disorders).

[0031] In various embodiments, the present application relates to a method of treating a subject suffering from a HER-2- or Trop-2-associated disorder, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or an ADC of the present application. In various embodiments, the cancer is a cancer associated with elevated expression of HER-2 or Trop-2. In various embodiments, the cancer is selected from the group consisting of colorectal cancer (CRC), renal cancer, non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, liver cancer, head and neck cancer, and glioma. In various embodiments, the subject previously responded to treatment with an anti-cancer therapy but relapsed upon cessation of treatment (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer.

[0032] In another aspect, the present application relates to a combination therapy designed to treat cancer or an infectious disease in a subject, comprising: a) administering to the subject a therapeutically effective amount of an isolated antibody or antigen-binding fragment of the present application; and b) administering one or more additional therapies selected from the group consisting of immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, vaccination protocols, stem cell transplantation, and immune cell therapy (CAR-T, CAR-NK), wherein the combination therapy increases cell killing of tumor cells, i.e., there is a synergistic effect between the isolated antibody or antigen-binding fragment and the additional therapy when administered together.

[0033] In another aspect, the present application provides methods for detecting the presence of human HER2 and Trop-2 antigens in a sample in vitro or in vivo, e.g., for diagnosing human HER2 and Trop-2 associated diseases.

[0034] In another aspect, an isolated nucleic acid is provided that comprises a polynucleotide sequence encoding an antibody or antigen-binding fragment disclosed herein. Also provided is an isolated cell that comprises an expression vector of the present application. In various embodiments, the cell is a host cell that comprises the expression vector of the present application. In various embodiments, the cell is a hybridoma, where the chromosome of the cell comprises the nucleic acid of the present application. Additionally provided is a method of making an antibody or antigen-binding fragment of the present application, comprising culturing or incubating a cell under conditions that allow the cell to express the antibody or antigen-binding fragment of the present application. [Brief description of the drawings]

[0035] [Figure 1] A representative bispecific antibody (designated HT2 (HT002)) is shown, comprising anti-Trop-2 antibody A1,2X4 to which an N-terminal Herceptin scFv is attached via a linker. [Diagram 2] A representative bispecific antibody (designated TH2 (TH002)) containing the anti-HER2 antibody Herceptin to which the N-terminal A1,2X4 scFv is attached via a linker is shown. [Diagram 3] A representative bispecific antibody (designated HB1 (HT001a)) containing the anti-HER2 antibody Herceptin attached via a linker to a C-terminal hRS7 scFv is shown. [Figure 4] A representative bispecific antibody (designated BH1 (TH001a)) is shown, comprising the anti-Trop-2 antibody hRS7 to which a C-terminal Herceptin scFv is attached via a linker. [Diagram 5] A representative bispecific antibody (designated BH2(TH001b)) is shown, comprising the anti-Trop-2 antibody hRS7 to which the N-terminal Herceptin scFv is attached via a linker. [Figure 6A] FIG. 1 shows a line graph depicting the results of evaluation of the binding affinity of bispecific antibodies HT2 (HT002) and TH2 (TH002) by flow cytometry analysis (FACS). [Figure 6B]FIG. 1 shows a line graph depicting the results of evaluation of the binding affinity of bispecific antibody HB1 (HT001a) by flow cytometry analysis (FACS). [Figure 6C] FIG. 1 shows a line graph depicting the results of evaluation of the binding affinity of bispecific antibodies BH1 (TH001a) and BH2 (TH001b) by flow cytometry analysis (FACS). [Figure 7] 1 shows the results of flow cytometry analysis of the internalization of bispecific antibodies TH2 (TH002), BH1 (TH001a), and BH2 (TH001b). [Figure 8] 1 shows the conjugation procedure between the DM payload and reduced mAb. [Figure 9A] 1 shows the in vitro cytotoxic activity of the bispecific ADC in tumor cells with different levels of HER2 and Trop-2 expression. [Figure 9B] 1 shows the in vitro cytotoxic activity of the bispecific ADC in tumor cells with different levels of HER2 and Trop-2 expression. [Figure 9C] 1 shows the in vitro cytotoxic activity of the bispecific ADC in tumor cells with different levels of HER2 and Trop-2 expression. [Figure 10] AB show the in vivo efficacy of HT002-BI-P203 in a BxPC3 xenograft model. [Figure 11] AB show the in vivo efficacy of HT002-BI-P203 in the N87 xenograft model. [Figure 12] Shows the in vivo efficacy of HT002-MMAE in a BxPC3 xenograft model. [Figure 13] 1 shows the inhibitory effect of HT002-MMAE on JIMT-1 breast cancer cells in an in vitro cytotoxicity assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] In one aspect, the present application relates to novel isolated bispecific antibodies and antigen-binding fragments thereof that specifically bind and neutralize both human HER2 and Trop-2. In another aspect, the present application relates to the use of novel bispecific anti-HER2 / Trop-2 antibodies and antigen-binding fragments thereof to diagnose, prognose, and treat conditions that would benefit from modulating HER2 and Trop-2 (e.g., cancer, persistent infections, autoimmune diseases, asthma, transplant rejection, and inflammatory disorders). Also provided are nucleic acid molecules comprising sequences of polynucleotides encoding all or a portion of a polypeptide that binds to HER2 and Trop-2, as well as derivatives and fragments thereof, e.g., nucleic acids encoding all or a portion of an anti-HER2 / Trop-2 antibody, antibody fragment, or antibody derivative. Also provided are vectors and plasmids comprising such nucleic acids, as well as cells or cell lines comprising such nucleic acids and / or vectors and plasmids. Also provided are methods for making, identifying or isolating antigen binding proteins that bind human HER2 and Trop-2, such as anti-HER2 / Trop-2 antibodies, methods for determining whether an antigen binding protein binds Trop-2, methods for making compositions, such as pharmaceutical compositions, that include antigen binding proteins that bind human HER2 and Trop-2, and methods for administering to a subject an antibody or antigen binding fragment thereof that binds HER2 and Trop-2, e.g., for treating a condition mediated by HER2 and Trop-2.

[0037] In another embodiment, the present application also relates to antibody drug conjugates (ADCs) derived from bispecific anti-HER2 / Trop-2 antibodies. Also provided are methods for producing ADCs derived from such bispecific antibodies. Also provided are methods for using ADCs derived from bispecific antibodies, for example, for treating cancer in a subject in need thereof.

[0038] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include singular terms. In general, the nomenclature used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are commonly used and well known in the art. Unless otherwise stated, the methods and techniques of this application are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are commonly used and well known in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0039] Polynucleotide and polypeptide sequences are designated using standard one-letter or three-letter abbreviations. Unless otherwise noted, polypeptide sequences have the amino terminus on the left and the carboxy terminus on the right, and single-stranded and double-stranded nucleic acid sequences have the 5' terminus on the left and the 3' terminus on the right. A particular portion of a polypeptide can be designated by the amino acid residue number, such as amino acids 80-119, or by the actual residue at that site, such as Ser80-Ser119. A particular polypeptide or polynucleotide sequence can also be described based on how it differs from a reference sequence. Polynucleotide and polypeptide sequences of particular light and heavy chain variable domains are designated L1 ("light chain variable domain 1") and H1 ("heavy chain variable domain 1"). Antibodies comprising light and heavy chains are designated by combining the name of the light chain variable domain with the name of the heavy chain variable domain. For example, "L4H7" refers to an antibody comprising, for example, a light chain variable domain of L4 and a heavy chain variable domain of H7.

[0040] The term "antigen-binding molecule" as used herein, in its broadest sense, refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, antibody fragments, and scaffold antigen-binding proteins. An "antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks the reference molecule from binding to the antigen by 50% or more in a competitive assay, and conversely, refers to a reference molecule that blocks the antigen-binding molecule from binding to the antigen by 50% or more in a competitive assay.

[0041] As used herein, the term "antigen-binding site" refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding site" refers to a portion of an antibody that contains the region that specifically binds to and is complementary to part or all of an antigen. If the antigen is large, the antigen-binding molecule may only bind to a specific portion of the antigen, which portion is called an epitope. An antigen-binding site may be provided, for example, by one or more variable domains (also called variable regions). Preferably, an antigen-binding site is provided by an antibody light chain variable region (V L ) and antibody heavy chain variable region (VH ).

[0042] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a stretch of contiguous amino acids or a conformation consisting of distinct regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding moiety binds and forms an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Proteins useful as antigens herein can be proteins in any naturally occurring form from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise noted. In various embodiments, the antigen is a human protein.

[0043] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments provided that they exhibit the desired antigen-binding activity.

[0044] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0045] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, with all or substantially all of its HVRs (e.g., CDRs) corresponding to a non-human antibody and all or substantially all of its FRs corresponding to a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present application are those in which the constant region has been additionally modified or altered from the original antibody to provide properties in accordance with the present application, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0046] A "human" antibody is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0047] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical and / or bind to the same epitope, excluding variant antibodies that may, for example, contain naturally occurring mutations or arise during production of a monoclonal antibody preparation. Such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations that typically include a variety of antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0048] As used herein, the term "monospecific" refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.

[0049] The term "bispecific" refers to an antibody that has antibody heavy chain variable domains (VHVs) that bind to at least two distinct antigenic determinants, e.g., different antigens or different epitopes on the same antigen. H ) and antibody light chain variable domain (V L ) is specifically capable of binding to two binding sites formed by a pair of antigens or epitopes, respectively. Such bispecific antibodies are in a 1+1 format. Other bispecific antibody formats are the 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or the 2+2 format (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, bispecific antibodies contain two antigen binding sites, each of which is specific for a different antigenic determinant.

[0050] The term "valent" as used within the present application refers to the presence of a particular number of binding sites in an antigen-binding molecule. Thus, the terms "bivalent", "tetravalent", and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. A bispecific antibody according to the present application is at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In various embodiments, an antibody of the present application has two or more binding sites and is bispecific. That is, an antibody can be bispecific even if there are more than two binding sites (i.e., the antibody is trivalent or multivalent). In particular, the present application relates to bispecific bivalent antibodies having one binding site that specifically binds to each antigen.

[0051] The terms "full-length antibody," "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody. A "native antibody" refers to a naturally occurring immunoglobulin molecule having a variety of structures. For example, native IgG class antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. Each heavy chain contains, from the N-terminus to the C-terminus, a variable region (V), also called a variable heavy domain or heavy chain variable domain. H ), followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, each light chain has, from N-terminus to C-terminus, a variable region (V), also called a variable light domain or light chain variable domain. L ) followed by a light chain constant domain (CL), also called the light chain constant region. The heavy chain of an antibody can be assigned to one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subtypes, e.g., gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), gamma 4 (IgG4), alpha 1 (IgA1) and alpha 2 (IgA2). The light chain of an antibody can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0052] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen that the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies formed from antibody fragments and single domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994), and also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments that contain two antigen-binding sites that can be bivalent or bispecific, see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In addition, antibody fragments can be made of V H Domain characteristics, i.e., V Lcapable of assembling together with the V domain into a functional antigen-binding site; L Domain characteristics, i.e., V H Antibody fragments include single-chain polypeptides having domains capable of assembling together to form a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0053] Papain digestion of an intact antibody yields two identical antigen-binding fragments, called "Fab" fragments, each of which contains the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to the V of the light chain (CL). L The light chain fragment contains the V domain and the constant domain, and the heavy chain V H The term "Fab'" refers to an antibody fragment that contains the heavy chain CH1 domain and the first constant domain (CH1). Fab' fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) in the constant domain bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that contains two antigen-binding sites (two Fab fragments) and part of the Fc region.

[0054] A "single-chain Fab fragment" or "scFab" is a fragment of an antibody heavy chain variable domain (V H ), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, the antibody domains and the linker being arranged in the following order from the N-terminus to the C-terminus: H -CH1-Linker-V L -CL, b)V L -CL-Linker-V H -CH1, c)VH -CL-Linker-V L -CH1 or d)V L -CH1-Linker-V H -CL, where the linker is at least 30 amino acids, preferably 32-50 amino acids. The single-chain Fab fragments are stabilized via a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules can be further stabilized by the creation of an interchain disulfide bond by the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering).

[0055] A "single-chain variable fragment (scFv)" is a fragment of an antibody heavy chain (V H ) and light chain (V L ) is a fusion protein in which the variable regions of V and VL are connected by a short linker peptide of 10 to about 25 amino acids. The linker usually contains a lot of glycine for flexibility and serine or threonine for solubility, and H N-terminus and V L The C-terminus of the VFv antibody can be connected to the C-terminus of the VFv antibody or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments can be H Domain characteristics, i.e., V L capable of assembling together with the V domain into a functional antigen-binding molecule; L Domain characteristics, i.e., V H It comprises a single polypeptide chain having domains capable of assembling into a functional antigen-binding molecule, thereby providing the antigen-binding properties of a full-length antibody.

[0056] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In particular, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain is always presented with the C-terminal lysine, although variants that do not include the C-terminal lysine are also included in the present application.

[0057] The IgG Fc region comprises the IgG CH2 and IgG CH3 domains. The "CH2 domain" of the human IgG Fc region typically spans from an amino acid residue at or around position 231 to an amino acid residue at or around position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues from the C-terminus of the Fc region to the CH2 domain (i.e., from an amino acid residue at or around position 341 to an amino acid residue at or around position 447 of IgG). The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain in which a "protrusion" ("knob") is introduced in one chain and a corresponding "cavity" ("hole") is introduced in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0058] "Region equivalent to the Fc region of immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of immunoglobulin, as well as variants with modifications that cause substitution, addition, or deletion, but do not substantially reduce the ability of immunoglobulin to mediate effector function (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of immunoglobulin without substantially impairing biological function. Such variants can be selected according to general rules known in the art to minimize the effect on activity (see, for example, Bowie, JU et al., Science 247:1306-10 (1990)).

[0059] The term "effector function" refers to biological activities attributable to the Fc region of an antibody and vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0060] An "activating Fc receptor" is an Fc receptor that, upon binding to the Fc region of an antibody, initiates a signaling event that stimulates a receptor-bearing cell to carry out an effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637, version 141).

[0061] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, a blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, the bispecific antibodies of the present application block signaling through HER2 and Trop-2.

[0062] As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) technology (BIAcore instrument-based analysis) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0063] The term "affinity" or "binding affinity" as used herein refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A particular method for measuring affinity is surface plasmon resonance (SPR). As used herein, the term "high affinity" of an antibody refers to an antibody that has a binding affinity of 10 to a target antigen. -9 M or less, and more specifically, Kd of 10 -10 The term "low affinity" for an antibody refers to an antibody having a Kd of 10 M or less. -8 The term "reduced binding" as used herein refers to an antibody having a Kd of equal to or greater than M. The term "reduced binding" as used herein refers to a decrease in affinity for the respective interaction, for example as measured by SPR. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0064] The terms "bispecific antibody comprising a first immunoglobulin antigen-binding domain that specifically binds HER2 and a second immunoglobulin antigen-binding domain that specifically binds Trop-2", "bispecific antibody that specifically binds HER2 and Trop-2", "bispecific antigen-binding molecule specific for HER2 and Trop-2" are used interchangeably herein and refer to bispecific antibodies capable of binding to HER2 and Trop-2 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting HER2 and Trop-2.

[0065] The terms "anti-HER2 antibody" and "antibody comprising an antigen-binding site that binds to HER2" refer to an antibody capable of binding to HER2, particularly HER2 polypeptides expressed on cell surfaces, with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting HER2. In one embodiment, the extent to which an anti-HER2 antibody binds to unrelated non-HER2 proteins is less than about 10% of the binding of the antibody to HER2, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or a surface plasmon resonance assay using a biosensor system such as a Biacore® system. In certain embodiments, an antigen-binding molecule that binds to human HER2 has a binding affinity of, for example, 10% or less for binding to human HER2. -8 M~10 -13 It has a binding affinity KD value of M. The term "anti-HER2 antibody" also encompasses bispecific antibodies capable of binding to HER2 and a second antigen.

[0066] The terms "anti-Trop-2 antibody" and "antibody comprising an antigen-binding site that binds to Trop-2" refer to an antibody capable of binding to Trop-2, particularly a Trop-2 polypeptide expressed on a cell surface, with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting Trop-2. In one embodiment, the extent to which an anti-Trop-2 antibody binds to an unrelated, non-Trop-2 protein is less than about 10% of the binding of the antibody to Trop-2, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or a surface plasmon resonance assay using a biosensor system such as a Biacore® system. In certain embodiments, an antigen-binding molecule that binds to human Trop-2 has an affinity of, for example, 10% for binding to human Trop-2. -8 M~10 -13 It has a KD value for binding affinity of M. The term "anti-Trop-2 antibody" also encompasses bispecific antibodies capable of binding to Trop-2 and a second antigen.

[0067] The term "recombinant human antibody", as used herein, is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, produced or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in various embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), such that the V and V regions of the recombinant antibody may be modified. H and V L The amino acid sequence of the region is similar to that of the human germline VH and V L These recombinant procedures are all well known to those skilled in the art and are intended to provide a method for the preparation of recombinant human antibody sequences which are derived from and related to the sequences, but which may not naturally occur in the human antibody germline repertoire in vivo.

[0068] The term "epitope" as used herein includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitope determinants generally consist of chemically active molecular surface groups such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all of the interaction points between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points occur across amino acid residues on the protein that are separated from each other. Once a desired epitope on an antigen has been determined, it is possible to generate antibodies against that epitope, for example, using the techniques described in this disclosure. Alternatively, in the drug discovery process, antibody generation and characterization can reveal information about the desired epitope. From this information, it is possible to competitively screen for antibodies that bind to the same epitope. An approach to achieve this is to perform cross-competition studies to find antibodies that bind competitively with one another, for example antibodies that compete for binding to an antigen.

[0069] Antigen-binding proteins, including antibodies, are at least 1×10 -6 M, or at least 1 × 10 -7 M, or at least 1 × 10 -8 M, or at least 1 × 10 -9 M, or at least 1 × 10 -10 M, or at least 1 × 10 -11 Dissociation constant of M (K DAn antigen-binding protein "specifically binds" to an antigen if it binds to the antigen with high binding affinity as determined by a binding affinity (Kb) value, or the corresponding Kb value defined below. An antigen-binding protein that specifically binds to a human antigen of interest may also be capable of binding to the same antigen of interest from other species with the same or different affinity. As used herein, "K D The term "antibody-antigen interaction" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0070] The term "immunoconjugate" or "fusion protein" as used herein refers to a molecule comprising an antibody or antigen-binding fragment thereof conjugated (or linked) directly or indirectly to an effector molecule. The term "fusion" as used herein refers to components linked by peptide bonds, either directly or through one or more peptide linkers. In various embodiments, the effector molecule is an immunotoxin, cytokine, chemokine, drug moiety, therapeutic agent, or chemotherapeutic agent. The antibody or antigen-binding fragment thereof may be conjugated to the effector molecule via a peptide linker. The immunoconjugate and / or fusion protein retains the immunoreactivity of the antibody or antigen-binding fragment, e.g., the antigen-binding ability of the antibody or antigen-binding fragment after conjugation is approximately the same as or only slightly reduced as before conjugation. As used herein, the immunoconjugate may also be referred to as an antibody-drug conjugate (ADC). Immunoconjugates and / or fusion proteins are sometimes called "chimeric molecules" because they are originally prepared from two molecules with distinct functions, such as an antibody and an effector molecule.

[0071] "Linker" refers to a molecule that connects two other molecules, either through covalent, ionic, van der Waals, or hydrogen bonds, for example, a nucleic acid molecule that connects two non-complementary sequences by hybridizing to one complementary sequence at its 5' end and another complementary sequence at its 3' end. "Cleavable linker" refers to a linker that can be degraded or otherwise cleaved to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers can also be cleaved by environmental triggers, such as, for example, changes in temperature, pH, salt concentration, and the like.

[0072] The term "peptide linker" as used herein refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Preferably, a non-immunogenic linker peptide is used, such as (G4S) n , (SG4) n or a G4 (SG4)n peptide linker, where "n" is generally a number from 1 to 10, typically from 2 to 4.

[0073] The term "immunogenicity" as used herein refers to the ability of an antibody or antigen-binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient, including, for example, a human anti-mouse antibody (HAMA) response. A HAMA response is initiated when a subject's T cells mount an immune response to an administered antibody. The T cells then recruit B cells to produce specific "anti-antibody" antibodies.

[0074] As used herein, the term "immune cell" refers to any cell of the hematopoietic lineage that is involved in regulating the immune response to an antigen (e.g., a self-antigen). In various embodiments, the immune cell is, for example, a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, a Langerhans cell, or a Kuffer cell.

[0075] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, a "peptide", "polypeptide" and "protein" can be a chain of amino acids whose alpha carbons are linked through peptide bonds. Thus, the terminal amino acid at one end of the chain (e.g., the amino terminus) has a free amino group and the terminal amino acid at the other end of the chain (e.g., the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free α-amino group on the amino terminal amino acid of a peptide, or the α-amino group (imino group when involved in a peptide bond) of an amino acid at any other position within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminal of a peptide, or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include essentially any polyamino acid, including, but not limited to, peptidomimetics such as amino acids connected by ethers as opposed to amide bonds.

[0076] The term "recombinant polypeptide," as used herein, is intended to include all polypeptides, including fusion molecules that are prepared, expressed, produced, derived or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell.

[0077] Polypeptides of the present disclosure include polypeptides that have been modified by any means and for any reason, for example, to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) alter binding affinity, and (5) impart or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made to the native sequence (e.g., in portions of the polypeptide other than the domain(s) that form intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with a functionally similar amino acid. The following six groups of amino acids are each conservative substitutions for one another: Alanine (A), Serine (S), and Threonine (T) Aspartic acid (D) and glutamic acid (E) Asparagine (N) and Glutamine (Q) Arginine (R) and Lysine (K) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0078] "Non-conservative amino acid substitutions" refer to the replacement of a member of one of these classes with a member of another class. According to various embodiments, in making such changes, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0079] The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indices or scores and still maintain similar biological activity. When making changes based on hydropathic indices, in various embodiments, substitutions of amino acids with hydropathic indices within ±2 are included. In various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0080] It is also understood in the art that substitutions of like amino acids can be made effectively on the basis of hydrophilicity, particularly when the biologically functional proteins or peptides so produced are intended for use in the immunological embodiments disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of the adjacent amino acids, correlates with immunogenicity and antigenicity, i.e., a biological property of the protein.

[0081] These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0), lysine (+3.0), aspartic acid (+3.0.+-.1), glutamic acid (+3.0.+-.1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (-0.4), proline (-0.5.+-.1), alanine (-0.5), histidine (-0.5), cysteine ​​(-1.0), methionine (-1.3), valine (-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3), phenylalanine (-2.5), and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included. Exemplary amino acid substitutions are set forth in Table 1. [Table 1]

[0082] The terms "polypeptide fragment" and "truncated polypeptide" as used herein refer to a polypeptide having an amino-terminal and / or carboxy-terminal deletion compared to the corresponding full-length protein. In various embodiments, a fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids in length. In various embodiments, a fragment can also be, for example, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 450, up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 50, up to 25, up to 10, or up to 5 amino acids in length. A fragment may further comprise one or more additional amino acids at either or both of its termini, such as a sequence of amino acids derived from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0083] The terms "polypeptide variant" and "polypeptide mutant" as used herein refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted and / or substituted relative to another polypeptide sequence. In various embodiments, the number of inserted, deleted or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length. Variants of the present disclosure include fusion proteins.

[0084] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugated to another chemical moiety, e.g., polyethylene glycol, albumin (e.g., human serum albumin), etc., phosphorylated, and glycosylated.

[0085] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences, or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm, meaning that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the percent identity is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0086] The term "% sequence homology" is used interchangeably herein with the term "% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences, or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology determined by a defined algorithm, and thus, a homolog of a given sequence has more than 80% sequence homology over the length of the given sequence. In various embodiments, the % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence. In various embodiments, the percent homology is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0087] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, the BLAST suite of programs at the NCBI website, which are publicly available on the Internet, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (with particular reference to the published default settings, i.e., w=4, t=17 parameters) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. Sequence searches are typically performed using the BLASTP program, where a given amino acid sequence is evaluated against amino acid sequences in GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against amino acid sequences in GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an opening gap penalty of 11.0 and an extension gap penalty of 1.0, and utilize the BLOSUM-62 matrix. See ibid.

[0088] In addition to calculating percent sequence identity, BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993).One measure of similarity provided by BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences may occur by chance.For example, if the minimum total probability in the comparison of test nucleic acid and reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001, the nucleic acid is considered to be similar to the reference sequence.

[0089] The term "substantial similarity" or "substantially similar" in the context of a polypeptide sequence indicates that a polypeptide region has a sequence that is at least 70%, typically at least 80%, more typically at least 85%, or at least 90% or at least 95% sequence similarity to a reference sequence. For example, a polypeptide is substantially similar to a second polypeptide, e.g., where the two peptides differ by one or more conservative substitution(s).

[0090] "Polynucleotide" refers to a polymeric compound composed of nucleotide units. Polynucleotides include natural nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include those that contain non-naturally occurring bases, nucleotides that are linked to other nucleotides other than naturally occurring phosphodiester bonds, or bases that are linked by linkages other than phosphodiester bonds. Nucleotide analogs thus include, for example, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally no longer than about 50 nucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood that this also includes RNA sequences in which "U" is replaced by "T" (i.e., A, U, G, C).

[0091] In this specification, conventional notation is used to describe polynucleotide sequences, with the left-hand end of a single-stranded polynucleotide sequence being the 5'-end and the left-hand direction of a double-stranded polynucleotide sequence being referred to as the 5'-direction. The 5' to 3' addition of nucleotides to the nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand", the sequence on the DNA strand having the same sequence as the mRNA transcribed from that DNA and located 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence", and the sequence on the DNA strand having the same sequence as the RNA and located 3' to the 3' end of the RNA transcript is referred to as the "downstream sequence".

[0092] "Complementary" refers to the topological compatibility or matching of the interaction surfaces of two polynucleotides. Thus, two molecules can be described as complementary, and the characteristics of the contact surfaces are also complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.

[0093] "Specifically hybridize" or "specific hybridization" or "selectively hybridize" refers to a nucleic acid molecule that preferentially binds, duplexes, or hybridizes to a particular nucleotide sequence under stringent conditions when the sequence is present in DNA or RNA of various mixtures (e.g., whole cells). The term "stringent conditions" refers to conditions under which a probe hybridizes preferentially to its target subsequence and hybridizes to a lesser extent or not at all to other sequences. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence-dependent and are different under various environmental parameters. Extensive guidance on nucleic acid hybridization can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, NY; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0094] Generally, highly stringent hybridization and washing conditions are selected to be about 5° C. lower than the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. Tm is the temperature (at a defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Highly stringent conditions are selected to be equal to the Tm of a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than about 100 complementary residues on a filter in a Southern or Northern blot is 50% formalin with 1 mg heparin at 42° C., with hybridization performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent washing conditions is 0.2×SSC wash at 65° C. for 15 minutes. For a description of SSC buffer, see Sambrook et al. A low stringency wash can be performed prior to the high stringency wash to remove background probe signal. For example, an exemplary medium stringency wash for duplexes of greater than about 100 nucleotides is 1x SSC, 45°C, 15 minutes. For example, an exemplary low stringency wash for duplexes of greater than about 100 nucleotides is 4-6x SSC, 40°C, 15 minutes. In general, a signal-to-noise ratio of 2x (or more) than that observed with an unrelated probe in a particular hybridization assay indicates detection of specific hybridization.

[0095] "Primer" refers to a polynucleotide that can specifically hybridize to a designated polynucleotide template and provide a point of initiation of synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and a polymerizing agent such as DNA polymerase. Primers are typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, although a wide variety of synthetic and natural primers are useful for many applications. Primers are complementary to the template they are designed to hybridize with and serve as a site for initiating synthesis, but need not reflect the exact sequence of the template. In such cases, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled, for example, with chromogenic, radioactive, or fluorescent moieties, and used as detectable moieties.

[0096] "Probe" when used in reference to a polynucleotide refers to a polynucleotide that can specifically hybridize to a designated sequence of another polynucleotide. A probe specifically hybridizes to a complementary polynucleotide of a target, but does not necessarily reflect the exact complementary sequence of the template. In such a case, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. A probe can be labeled, for example, with a chromogenic, radioactive, or fluorescent moiety and used as a detectable moiety. A probe can also be a primer, if it provides a point of initiation for the synthesis of a complementary polynucleotide.

[0097] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to the vector into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which allows additional DNA segments to be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors that contain a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell, and are thereby replicated along with the host genome. An "expression vector" is a type of vector that can induce the expression of a selected polynucleotide.

[0098] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can exert its effect, for example, directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., a polypeptide that binds to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence.

[0099] A "host cell" is a cell that can be used to express a polynucleotide of the present disclosure. A host cell may be a prokaryote, such as E. coli, or a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, and the nucleic acid can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the nucleic acid is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because some modifications may occur in subsequent generations due, for example, to mutations or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0100] The term "isolated molecule" (where the molecule is, for example, a polypeptide or polynucleotide) refers to a molecule that, by virtue of its origin or source, is (1) not associated with naturally associated components with which it is associated in its native state, (2) substantially free of other molecules from the same species, (3) expressed by cells from a different species, or (4) not naturally occurring. Thus, a chemically synthesized molecule or a molecule expressed in a cellular system different from the cell from which it is naturally derived is "isolated" from naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assessed by a number of means well known in the art. For example, the purity of a polypeptide sample can be assessed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide, using techniques well known in the art. For certain purposes, higher resolution can also be obtained by using HPLC or other purification means well known in the art.

[0101] A protein or polypeptide may be "substantially pure," "substantially homogeneous," or "substantially purified" if at least about 60%-75% of a sample represents a single type of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein is typically about 50%, 60%, 70%, 80% or 90% W / W of a protein sample, more commonly about 95%, and preferably greater than 99% pure. Protein purity or homogeneity can be demonstrated by a number of means well known in the art, such as by subjecting a protein sample to polyacrylamide gel electrophoresis, followed by staining the gel with stains well known in the art to visualize a single polypeptide band. For certain purposes, higher resolution may also be obtained by using HPLC or other purification means well known in the art.

[0102] The term "label" or "labeled" as used herein refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as the incorporation of a radioactively labeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include the following: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochrome P450 ... Examples of suitable agonists include, but are not limited to, cysteine ​​B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0103] As used herein, the term "immunotherapy" includes, but is not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody drug conjugates; treatment with agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as PD-1, PD-L1, OX-40, CD137, GITR, LAG3, TIM-3, and VISTA; treatment with bispecific T cell engaging antibodies (BiTEs®), such as blinatumomab; treatment involving administration of biological response modifiers, such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines, such as sipuleucel-T; treatment with dendritic cell vaccines, or tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TILs); adoptively transferred anti-tumor T cells (ex in vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immune activators such as the Toll-like receptor (TLR) agonists CpG and imiquimod.

[0104] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharma- ceutically acceptable carrier. "Pharmacologically effective amount" refers to an amount of an agent effective to produce an intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as phosphate buffered saline, 5% dextrose in water, and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salts" are salts that can be formulated into compounds for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0105] The term "growth suppression" or "growth inhibition" of cells expressing HER2 or Trop-2, as used herein, refers to the ability of an anti-HER2 / Trop-2 binding molecule, e.g., a HER2 / Trop-2 binding molecule comprising an anti-HER2 / Trop-2 antibody or antigen-binding fragment thereof, to statistically significantly reduce the proliferation of cells expressing HER2 or Trop-2 compared to proliferation in the absence of the anti-HER2 / Trop-2 binding molecule. In one embodiment, the proliferation of cells expressing HER2 or Trop-2 (e.g., cancer cells) may be reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100%, when the cells are contacted with an anti-HER2 / Trop-2 binding molecule, such as an anti-HER2 / Trop-2 antibody or an antigen-binding fragment thereof, compared to the proliferation measured in the absence of the anti-HER2 / Trop-2 binding molecule (control condition). Cell proliferation can be measured according to various methods known in the art, such as by counting the number of viable cells, identifying the presence of a marker of growth markers, measuring the incorporation of molecules (e.g., radiolabeled molecules such as H-thymidine), measuring the size (e.g., volume or weight) of the tumor, etc. In certain embodiments, growth inhibition refers to a reduction in the number, size, or distribution of metastases.

[0106] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention that seeks to alter the natural course of a disease in the individual being treated, and may be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of symptoms of the disease, disorder, or condition. Furthermore, references to "treatment" herein include references to curative, palliative, and prophylactic treatment.

[0107] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease, e.g., improve, alleviate, relieve, and / or delay one or more of its symptoms. In the context of cancer or other undesired cell proliferation, an effective amount includes an amount sufficient to (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit to some extent, suppress, slow, preferably stop, the invasion of cancer cells into surrounding organs, (iv) inhibit (i.e., slow to some extent, preferably stop) the metastasis of tumors, (v) inhibit the growth of tumors, (vi) prevent or delay the onset and / or recurrence of tumors, and / or (vii) alleviate to some extent one or more of the symptoms associated with cancer. An effective amount may be administered in one or more administrations.

[0108] The phrases "administering" or "causing to be administered" refer to the action taken by a medical professional (e.g., physician) or other person managing the medical care of a patient to administer and / or permit the administration of the agent(s) / compound(s) in question to a patient. Causing to be administered may involve determining a diagnosis and / or an appropriate treatment regimen, and / or prescribing a particular agent(s) / compound to a patient. Such prescribing may include, for example, writing a prescription, annotating a medical record, etc. Where administering is described herein, "causing to be administered" is also contemplated.

[0109] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domestic mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). In various embodiments, the patient may be a human (e.g., adult male, adult female, adolescent male, adolescent female, boy, girl) receiving the care of a physician or other medical professional in a hospital, psychiatric facility, outpatient, or other clinical setting. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs, and those with genetic diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers that have been reported to have a high rate of mutations (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0110] "Resistant or refractory cancer" refers to tumor cells or cancer that are unresponsive to previous anti-cancer therapies, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory at the onset of treatment or may become resistant or refractory during treatment. Refractory tumor cells include tumors that are unresponsive at the onset of treatment or tumors that initially respond for a short period of time but become unresponsive to treatment. Refractory tumor cells also include tumors that respond to treatment with anti-cancer therapy but become unresponsive to subsequent courses of therapy. For purposes of this application, refractory tumor cells also encompass tumors that appear to be suppressed by treatment with anti-cancer therapy but recur up to 5 years, and sometimes up to 10 years or more, after cessation of treatment. Anti-cancer therapy may employ chemotherapy agents alone, radiation alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of explanation and not limitation, it is understood that refractory tumor cells can be interchangeable with resistant tumors.

[0111] It is understood that aspects and embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the aspects and embodiments.

[0112] Reference herein to "about" a value or parameter includes (and describes) variations on that value or parameter itself. For example, reference to "about X" includes reference to "X."

[0113] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the application described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0114] HER2 antigen HER2 (also known as CD340 or ERBB2), a member of the human epidermal growth factor receptor family, is aberrantly expressed in certain malignancies, including breast, gastric, and lung cancers, mainly due to HER2 genomic amplification. Overexpression of HER2 is associated with increased risk of recurrence and poor prognosis in these cancers. In HER2-positive malignancies, this protein may stimulate downstream RAS-RAF-ERK and PI3K-PTEN-AKT signaling and play an important role in cell proliferation. Therefore, HER2 is a preferred therapeutic target for cancer. Many antibodies targeting HER2 have been developed. For example, anti-HER2 monoclonal antibodies such as trastuzumab and pertuzumab, as well as HER2 tyrosine kinase inhibitors such as lapatinib, have been shown to improve survival and are used as targeted therapies for HER2-positive cancers, including breast and gastric cancers.

[0115] ADCs containing an anti-HER2 monoclonal antibody, a linker, and a cytotoxic payload have also been employed as targeted therapy for HER2-positive malignancies. Trastuzumab emtansine (T-DM1), consisting of a conjugate of the anti-HER2 monoclonal antibody trastuzumab and the tubulin polymerization inhibitor DM1, has already been used to treat HER2-positive breast cancer. In a phase III clinical trial (EMILIA study), T-DM1 was confirmed to improve the survival and quality of life of patients with HER2-positive breast cancer. Furthermore, 44% of patients responded to T-DM1 treatment. However, the efficacy was limited in other cases, and all patients eventually developed resistance to T-DM1. A better understanding of the mechanisms underlying this limitation is needed to improve the treatment of patients with HER2-positive cancer.

[0116] Trastuzumab deruxtecan (DS-8201a) is another HER2-targeted ADC prepared using a potent topoisomerase I inhibitor, an exatecan derivative (DX-8951 derivative, DXd), and a novel linker-payload system. It was effective in xenograft models derived from T-DM1-insensitive patients with high and low HER2 expression. DS-8201a has a strong bystander effect due to its highly membrane-permeable payload, which is beneficial for treating tumors with HER2 heterogeneity that are unresponsive to T-DM1. DS-8201a was recently approved by the FDA for unresectable or previously treated metastatic HER2-positive breast cancer. The approval was based on the phase II DESTINY-Breast01 trial, which included patients with HER2-positive breast cancer who had previously been treated with other HER2 therapies. Overall, 60.9% of patients responded to this novel HER2 ADC. Besides breast cancer, DS-8201a has also shown favorable clinical efficacy in other HER2-positive cancer types, such as lung and gastric cancer. Although a high rate of durable responses has been observed, the clinical benefit of DS-8201a on overall survival remains to be determined.

[0117] As used herein, the term "HER2" includes human HER2 (hHER2), variants, isoforms, and species homologs of hHER2, as well as analogs that share at least one common epitope with hHER2. In various embodiments, the hHER2 polypeptide used herein can include the amino acid sequence set forth in NCBI Reference Sequence: NP_001005862 (SEQ ID NO: 1): MKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYV L IAHNQVRQVPLQRLRIVRGTQLFEDNYALAV L DNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGV LIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFV H TVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRV L QGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVV L GVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKV L GSGAFGTVYKGIWIPDGENVKIPVAIKV L RENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLV H RDLAARNV L VKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMV HHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQ ID NO: 1)

[0118] In various embodiments, the HER2 polypeptide comprises a shared amino acid sequence with the human HER2 sequence of SEQ ID NO: 1, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology observed. Polypeptide variants of HER2 may be described herein with reference to the addition, deletion, or substitution of an amino acid residue present at a given position in the 223 amino acid sequence of SEQ ID NO: 1. Thus, for example, the term "P21W" indicates that the "P" (proline in the standard single letter code) residue at position 21 of SEQ ID NO: 1 has been replaced with a "W" (tryptophan in the standard single letter code).

[0119] Trop-2 antigen Trophoblast cell surface antigen-2 (Trop-2), also known as tumor-associated calcium signal transducer 2 (TACSTD1), membrane component chromosome 1 surface marker 1 (M1S1), gastrointestinal antigen 733-1 (GA733-1), and epithelial glycoprotein-1 (EGP-1), belongs to the TACSTD family, which contains at least two type I membrane proteins. Trop-2 transduces intracellular calcium signals and functions as a cell surface receptor. Trop-2 has 323 amino acids and is composed of one large extracellular domain, one transmembrane domain, and a short cytoplasmic tail.

[0120] Although first discovered as a cell surface marker for trophoblast cells, subsequent studies have revealed that Trop-2 is also expressed at low levels in limited normal tissues, such as nasal, breast, skin, and bronchial epithelial cells. Further studies suggest that Trop-2 is overexpressed in many different cancer types, including oral, head and neck, thyroid, lung, breast, gastric, colon, pancreatic, renal, prostate, ovarian, endometrial, cervical, and glioma cancers. This increased expression is associated with disease progression and poor prognosis in cancer patients. Overexpression of Trop-2 in cancer cells has been shown to stimulate tumor growth both in vitro and in vivo. Similarly, inhibition of Trop-2 expression with siRNA has been shown to suppress tumor cell proliferation. In addition to being important for tumor growth, Trop-2 has also been implicated in metastasis. Trop-2 is involved in at least six major signaling pathways, including the IGF, ErbB3, ERK, MAPK, Notch-Wnt, and Raf pathways. However, its exact role in these pathways and which downstream pathways are important in different cancers and different therapeutic approaches remain to be elucidated.

[0121] Based on its differential expression in tumor and normal tissues, its role in promoting tumor growth and metastasis, and its negative value for prognosis, Trop-2 has been proposed as a promising diagnostic / therapeutic target. Blockade of Trop-2 signaling could be a therapeutic approach for cancer. Antigen-binding fragments (Fabs) targeting Trop-2 have been shown to induce apoptosis and have an inhibitory effect on breast cancer cell proliferation. Although several anti-Trop-2 antibodies have been developed, none have been available for therapeutic use as naked antibodies. Antibody-drug conjugates (ADCs) targeting Trop-2 have been recently developed. Sacituzumab govitecan (IMMU-132) is a conjugate of hRS7, a humanized anti-Trop-2 antibody, and SN-38, an active metabolite of irinotecan. After its development, it was tested in many preclinical trials and was able to inhibit a wide range of tumor growth. It also showed promising efficacy in early-phase clinical trials in patients with non-small cell lung cancer, small cell lung cancer, metastatic triple-negative breast cancer, and pancreatic cancer. In the Phase II IMMU-132-01 clinical trial, IMMU-132 demonstrated an overall response rate of 33.3% in patients with metastatic triple-negative breast cancer who had failed prior lines of therapy. The median duration of response was 7.7 months. Based on these results, IMMU-132 became the first anti-Trop-2 ADC approved by the FDA. Although this anti-Trop-2 ADC has shown favorable clinical efficacy, improvements in the use of anti-Trop-2 antibodies for treatment of cancers and other diseases associated with Trop-2 activity are still needed.

[0122] As used herein, the term "Trop-2" includes human Trop-2 (hTrop-2), variants, isoforms, and species homologs of hTrop-2, as well as analogs that share at least one common epitope with hTrop-2. In various embodiments, the hTrop-2 polypeptide as used herein may include the amino acid sequence set forth in NCBI Reference Sequence: NP_002344.2 (SEQ ID NO:2): MARGPGLAPPPLRLPLLLLV LAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAV H YEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVV VVVALVAGMAV L VITNRRKS GKYKKVEIKELGELRKEPSL (SEQ ID NO: 2)

[0123] In various embodiments, Trop-2 polypeptides comprise a shared amino acid sequence with the human Trop-2 sequence of SEQ ID NO: 2, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology observed. Polypeptide variants of Trop-2 may be described herein with reference to the addition, deletion, or substitution of an amino acid residue present at a given position in the 223 amino acid sequence of SEQ ID NO: 2. Thus, for example, the term "P21W" indicates that the "P" (proline in the standard single letter code) residue at position 21 of SEQ ID NO: 2 has been replaced with a "W" (tryptophan in the standard single letter code).

[0124] antibody Antibodies can be engineered in a variety of ways. Antibodies can be made as single chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')2 fragments, etc., and can be humanized, chimerized, deimmunized, or fully humanized. Numerous publications describe many types of antibodies and methods for engineering such antibodies. See, for example, U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.

[0125] Methods for generating novel antibodies that bind to human Trop-2 are known to those skilled in the art. The present inventors have generated novel antibodies that bind to human Trop-2. Briefly, a method for generating monoclonal antibodies that specifically bind to Trop-2 may include administering to a mouse an immunogenic composition that includes an effective amount of Trop-2 to stimulate a detectable immune response, obtaining antibody-producing cells from the mouse (e.g., cells from the spleen), fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify hybridomas that produce monoclonal antibodies that specifically bind to Trop-2. Once hybridomas are obtained, they can be grown in cell culture, optionally in culture conditions in which hybridoma-derived cells produce monoclonal antibodies that specifically bind to Trop-2. Monoclonal antibodies can be purified from the cell culture. Chimeric antibodies are produced by recombinant DNA technology. CDR grafting and back mutation methods were used to prepare humanized antibodies.

[0126] Other suitable methods of producing or isolating antibodies with the required specificity can also be used, including, for example, methods based on selecting recombinant antibodies from libraries or immunizing transgenic animals (e.g., mice) capable of producing the entire repertoire of human antibodies. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-2555, 1993; Jakobovits et al., Nature, 362:255-258, 1993; U.S. Patent No. 5,545,806 to Lonberg et al.; U.S. Patent No. 5,545,807 to Surani et al.

[0127] Exemplary Constructs The present application provides a construct comprising a first antigen-binding domain that specifically binds to HER2 (e.g., human HER2) and a second antigen-binding domain that specifically binds to Trop-2 (e.g., human Trop-2).

[0128] In some embodiments, the present application provides a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 A first antigen-binding domain that specifically binds to human HER2, comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 and a second antigen-binding domain that specifically binds to human Trop-2 comprising 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34. H-1 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; L-1and the second antigen-binding domain comprises an scFv comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; H-2 2) an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 31; L-2 and a first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. In some embodiments, the first antigen-binding domain comprises a full-length antibody comprising an Fc fragment comprising: 1) a V H-1 and 2) a V having an amino acid sequence as set forth in SEQ ID NO: 8 or 57. L-1 and the second antigen-binding domain comprises an scFv comprising: 1) a V domain comprising the amino acid sequence set forth in SEQ ID NO:4. H-2 and 2) a V comprising the amino acid sequence set forth in SEQ ID NO:5. L-2 and a full-length antibody comprising an Fc fragment comprising the first antigen-binding domain and the second antigen-binding domain are fused to the N-terminus of both heavy chains of the full-length antibody. In some embodiments, the scFv and the full-length antibody are fused via a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the V H and V Lare fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the construct comprises: a) two heavy chains comprising an amino acid sequence set forth in SEQ ID NO: 14, 60, or 62, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) an amino acid sequence set forth in SEQ ID NO: 15, b) two light chains comprising an amino acid sequence set forth in SEQ ID NO: 15, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) ...

[0129] In some embodiments, the present application provides a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 A first antigen-binding domain that specifically binds to human HER2, comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 and a second antigen-binding domain that specifically binds to human Trop-2 comprising 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34. H-1 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; L-1 and a second antigen-binding domain comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; H-22) an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 31; L-2 and a second antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. In some embodiments, the first antigen-binding domain comprises an scFv comprising: 1) a V H-1 and 2) a V having an amino acid sequence as set forth in SEQ ID NO: 8 or 57. L-1 and a second antigen-binding domain comprising: 1) a V domain comprising the amino acid sequence set forth in SEQ ID NO:4; H-2 and 2) a V comprising the amino acid sequence set forth in SEQ ID NO:5. L-2 and a second antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. In some embodiments, the scFv and the full-length antibody are fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the V H and V L are fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the construct comprises: a) two heavy chains comprising an amino acid sequence set forth in SEQ ID NO: 16, 59, or 61, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%); b) two light chains comprising an amino acid sequence set forth in SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0130] In some embodiments, the present application provides a first heavy chain variable region (V H-1) and the first light chain variable region (V L-1 A first antigen-binding domain that specifically binds to human HER2, comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 and a second antigen-binding domain that specifically binds to human Trop-2 comprising 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34. H-1 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; L-1 and a second antigen-binding domain comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; H-2 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; L-2 and a second antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody. In some embodiments, the first antigen-binding domain comprises an scFv comprising: 1) a V H-1 and 2) a V having an amino acid sequence as set forth in SEQ ID NO: 8 or 57. L-1 and a second antigen-binding domain comprising: 1) a V domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58; H-2 and 2) a V comprising the amino acid sequence set forth in SEQ ID NO:11. L-2and a second antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody. In some embodiments, the scFv and the full-length antibody are fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the V H and V L are fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the construct comprises a) two heavy chains comprising an amino acid sequence as set forth in SEQ ID NO: 18, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) an amino acid sequence as set forth in SEQ ID NO: 19, and b) two light chains comprising an amino acid sequence as set forth in SEQ ID NO: 19, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) an amino acid sequence as set forth in SEQ ID NO: 19.

[0131] In some embodiments, the present application provides a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 A first antigen-binding domain that specifically binds to human HER2, comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 and a second antigen-binding domain that specifically binds to human Trop-2 comprising 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34. H-1 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; L-1and the second antigen-binding domain comprises an scFv comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; H-2 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; L-2 and a first antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody. In some embodiments, the first antigen-binding domain comprises a full-length antibody comprising an Fc fragment comprising: 1) a V H-1 and 2) a V having an amino acid sequence as set forth in SEQ ID NO: 8 or 57. L-1 and the second antigen-binding domain comprises an scFv comprising: 1) a V domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58. H-2 and 2) a V comprising the amino acid sequence set forth in SEQ ID NO:11. L-2 and a full-length antibody comprising an Fc fragment comprising the first antigen-binding domain and the second antigen-binding domain are fused to the C-terminus of both heavy chains of the full-length antibody. In some embodiments, the scFv and the full-length antibody are fused via a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the V H and V Lare fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the construct comprises a) two heavy chains comprising an amino acid sequence as set forth in SEQ ID NO: 20, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) an amino acid sequence as set forth in SEQ ID NO: 21, and b) two light chains comprising an amino acid sequence as set forth in SEQ ID NO: 21, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) an amino acid sequence as set forth in SEQ ID NO: 22.

[0132] In some embodiments, the present application provides a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 A first antigen-binding domain that specifically binds to human HER2, comprising a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 and a second antigen-binding domain that specifically binds to human Trop-2 comprising 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34. H-1 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; L-1 and the second antigen-binding domain comprises an scFv comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; H-22) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; L-2 and a first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. In some embodiments, the first antigen-binding domain comprises a full-length antibody comprising an Fc fragment comprising: 1) a V H-1 and 2) a V having an amino acid sequence as set forth in SEQ ID NO: 8 or 57. L-1 and the second antigen-binding domain comprises an scFv comprising: 1) a V domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58. H-2 and 2) a V comprising the amino acid sequence set forth in SEQ ID NO:11. L-2 and a full-length antibody comprising an Fc fragment comprising the first antigen-binding domain and the second antigen-binding domain are fused to the N-terminus of both heavy chains of the full-length antibody. In some embodiments, the scFv and the full-length antibody are fused via a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the V H and V Lare fused by a linker (e.g., a peptide linker, e.g., a GS linker, e.g., a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54). In some embodiments, the construct comprises a) two heavy chains comprising an amino acid sequence as set forth in SEQ ID NO: 22, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%), and b) two light chains comprising an amino acid sequence as set forth in SEQ ID NO: 23, or a variant comprising an amino acid sequence having at least (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%). In some embodiments, the Fc fragment comprises a heavy chain constant domain, optionally comprising an IgG constant domain (e.g., comprising SEQ ID NO: 12), and a light chain constant domain, optionally comprising a kappa constant region or a lambda constant region (e.g., comprising SEQ ID NO: 13). In some embodiments, the construct is or comprises a bispecific antibody or binding fragment thereof, optionally wherein the bispecific antibody is a humanized antibody. In some embodiments, the construct comprises an antibody drug conjugate (ADC) having the formula: Ab-(LD)n, where Ab is said bispecific antibody or binding fragment thereof, L is a linker or direct bond, D is a therapeutic agent, and n is an integer from 1 to 10.

[0133] Bispecific anti-HER2 / Trop-2 binding molecule In one aspect, the present application provides a construct comprising a bispecific anti-HER2 / Trop-2 antibody, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof of the present application can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand for a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the present application can in fact be derivatized or linked to multiple other functional molecules to generate a multispecific molecule that binds to more than two different binding sites and / or target molecules, and such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To generate a bispecific molecule of the present application, the antibody of the present application can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other methods) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule is prepared. In various embodiments, the present application includes bispecific molecules capable of binding to both effector cells expressing FcγR or FcαR (e.g., monocytes, macrophages, or polymorphonuclear cells (PMN)) and target cells expressing Trop-2. In such embodiments, the bispecific molecules target Trop-2 expressing cells to effector cells and induce Fc receptor-mediated effector cell activity, such as phagocytosis of Trop-2 expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or production of superoxide anions. Methods for preparing the bispecific molecules of the present application are well known in the art.

[0134] In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain and a second immunoglobulin antigen binding domain, where the first immunoglobulin antigen binding domain specifically binds to a HER2 antibody binding site and the second immunoglobulin antigen binding domain specifically binds to a Trop-2 antibody binding site. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain and a second immunoglobulin antigen binding domain, where the first immunoglobulin antigen binding domain specifically binds to a Trop-2 antibody binding site and the second immunoglobulin antigen binding domain binds to a HER2 antibody binding site.

[0135] In various embodiments, the first or second immunoglobulin antigen-binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises: (a) V H and V L (b) a V that further comprises a heavy chain constant region or a fragment thereof H and a V comprising a light chain constant region (LC) or a fragment thereof L , (c) a single chain Fv ("scFv"), (d) a diabody, (e) a minibody, (f) an F(ab')2, and (g) an F(ab).

[0136] In various embodiments, the first immunoglobulin antigen-binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises an scFv antibody fragment and the second antigen-binding domain comprises a V H and V L In various embodiments, the first antigen-binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises V H and V L and the second immunoglobulin antigen-binding domain comprises an scFv antibody fragment.

[0137] In various embodiments, the first or second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody further comprises a V Hand V further comprising a light chain constant region or a fragment thereof. L In various embodiments, the heavy chain constant region or fragment thereof is an IgG constant region. In various embodiments, the IgG constant region or fragment thereof is an IgG1 constant region. In various embodiments, the LC constant region is a kappa constant region. In various embodiments, the light chain constant region is a lambda constant region. In various embodiments, the first or second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody comprises a heavy chain that can include an Fc domain comprising a CH2 and a CH3 region. In various embodiments, the Fc domain is an IgG1 Fc domain. In various embodiments, the IgG1 Fc domain is a native IgG1 Fc domain.

[0138] In various embodiments, the first or second immunoglobulin antigen binding domain is a monoclonal antibody. In various embodiments, the first or second immunoglobulin antigen binding domain is a humanized antibody. In various embodiments, the first or second immunoglobulin antigen binding domain is a human antibody. In various embodiments, the first or second immunoglobulin antigen binding domain is a chimeric antibody. In various embodiments, the first or second immunoglobulin antigen binding domain is an affinity-optimized antibody. In various embodiments, the human antibody is expressed in a transgenic mouse (see, e.g., Bruggemann, "Human antibody expression in transgenic mice," Arch. Immunol. Therap. Exper. 49:203-208, 2001, which is incorporated herein by reference in its entirety).

[0139] In various embodiments, the second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody is covalently linked to the carboxy terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a linker interposed between the second immunoglobulin antigen binding domain and the carboxy terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the peptide linker II comprises the amino acid sequence of SEQ ID NO: 25. In various embodiments, the second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody is covalently linked to the amino terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises a linker interposed between the second immunoglobulin antigen binding domain and the amino terminus of the heavy chain of the first immunoglobulin antigen binding domain. In various embodiments, the peptide linker II comprises the amino acid sequence of SEQ ID NO: 25.

[0140] The scFvs disclosed herein may be available from or produced by any suitable source, whether natural or not, or may be recombinant, synthetic, semi-synthetic, derivatized, fermentation-optimized, fusion proteins or equivalents, variants and derivatives thereof, so long as they retain the required binding specificity of the scFvs disclosed herein. These include scFvs with binding specificity that have amino acid substitutions or have sugars or other molecules attached to amino acid functional groups. The term "derivative" or "derivatized" as used herein with respect to scFvs includes chemical modifications of scFvs. Exemplary of such modifications may be replacement of hydrogen with alkyl, acyl, or amino groups.

[0141] Linker In some embodiments, the constructs described herein provide a separation between two moieties or domains (e.g., between an scFv that specifically binds Trop2 and a full-length antibody of Herceptin, e.g., between a Herceptin scFv and a full-length antibody that binds Trop2, e.g., between the VH or V L The linker may include one or more linkers between the linker(s) (between the linker(s)). The length, degree of flexibility, and / or other properties of the linker(s) used in the construct may affect some properties, including, but not limited to, affinity, specificity, or avidity for one or more particular antigens or epitopes. For example, a longer linker may be selected so that two adjacent domains do not sterically interfere with each other. In some embodiments, the linker (such as a peptide linker) includes flexible residues (e.g., glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0142] Other considerations for the linker include its effect on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (mostly stable but with some planned degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, etc.

[0143] Peptide Linker The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO1996 / 34103.

[0144] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids in length. In some embodiments, the peptide linker is about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids in length. In some embodiments, the length of the peptide linker is from about 1 amino acid to about 10 amino acids, from about 1 amino acid to about 20 amino acids, from about 1 amino acid to about 30 amino acids, from about 5 amino acids to about 15 amino acids, from about 10 amino acids to about 25 amino acids, from about 5 amino acids to about 30 amino acids, from about 10 amino acids to about 30 amino acids in length, from about 30 amino acids to about 50 amino acids, from about 50 amino acids to about 100 amino acids, or from about 1 amino acid to about 100 amino acids.

[0145] A crucial technical feature of such peptide linkers is that they do not have any polymerization activity. The characteristics of peptide linkers with no promotion of secondary structures are known in the art and described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). A particularly preferred amino acid in the context of "peptide linkers" is Gly. Furthermore, peptide linkers that do not promote any secondary structures are also preferred. The link between the domains can be provided, for example, by genetic engineering. Methods for preparing fused, operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0146] The peptide linker may be a stable linker that is not cleaved by proteases, particularly matrix metalloproteases (MMPs).

[0147] The linker may be a flexible linker. Exemplary flexible linkers include glycine polymers (G) n (SEQ ID NO: 48), glycine-serine polymers (e.g., (GS) n (SEQ ID NO: 49), (GSGGS) n (SEQ ID NO:50), (GGGGS) n (SEQ ID NO:51), and (GGGS)n (SEQ ID NO:52), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to function as neutral tethers between components. Glycine has much more access to Φ-Ψ space than alanine and is much less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those skilled in the art will recognize that the design of antibody fusion proteins can include fully or partially flexible linkers, such that the linker can include not only a flexible linker portion, but one or more portions that confer a less flexible structure to provide the desired antibody fusion protein structure.

[0148] Further exemplary linkers also include (GGGGS) n (SEQ ID NO:51), where n is an integer from 1 to 8, e.g., (GGGGS)3 (SEQ ID NO:46), (GGGGS)4 (SEQ ID NO:47), or (GGGGS)6 (SEQ ID NO:53). In some embodiments, the peptide linker is (GSTSGSGKPGSGEGS) n (SEQ ID NO: 54), where n is an integer from 1 to 3.

[0149] Non-peptide linkers The coupling of the two moieties, for example, in the constructs described herein, to HER2 and Trop-2, respectively, can be achieved by any chemical reaction that binds the two molecules, so long as both components retain their respective activities. This linkage can include many chemical mechanisms, such as covalent bonds, affinity bonds, intercalation, coordinate bonds, complexation, etc. In some embodiments, the linkage is a covalent bond. Covalent bonds can be achieved either by direct condensation of existing side chains or by incorporation of external cross-linking molecules. Many bivalent or polyvalent linking agents can be useful for coupling protein molecules in this context. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but rather is illustrative of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0150] Linkers that can be applied in this application have been described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). In some embodiments, non-peptide linkers used herein include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co. catalog number 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co. catalog number 24510) conjugated to EDC.

[0151] The linkers described above contain components with different attributes, which can result in bispecific antibodies with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT can contain a sterically hindered disulfide bond to form antibody fusion proteins with increased stability. Disulfide linkages are generally less stable than other linkages because they are cleaved in vitro, resulting in fewer available antibody fusion proteins. Sulfo-NHS can particularly increase the stability of carbodiimide coupling. Carbodiimide coupling (such as EDC) when used with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0152] Exemplary embodiments In various embodiments of the present application, the isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen-binding domain that specifically binds to HER2 and a second immunoglobulin antigen-binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen-binding domain is an scFv comprising the amino acid sequence set forth in SEQ ID NO:3: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 3) and the second immunoglobulin antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:4. H : QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRLTISKDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS (SEQ ID NO: 4) and V comprising the amino acid sequence set forth in SEQ ID NO:5 L : DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIK (SEQ ID NO:5).

[0153] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:3 and the second immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:10. H : QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 10) and V comprising the amino acid sequence set forth in SEQ ID NO:11 L : DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVDIK (sequence number 11).

[0154] In various embodiments of the present application, the isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen-binding domain that specifically binds Trop-2 and a second immunoglobulin antigen-binding domain that specifically binds HER2, wherein the first immunoglobulin antigen-binding domain is an scFv comprising the amino acid sequence set forth in SEQ ID NO:6: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRLTISKDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS (SEQ ID NO: 6) and the second immunoglobulin antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:7. H : EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 7) and V comprising the amino acid sequence set forth in SEQ ID NO:8 L : DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (sequence number 8).

[0155] In various embodiments of the present application, the isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen-binding domain that specifically binds Trop-2 and a second immunoglobulin antigen-binding domain that specifically binds HER2, wherein the first immunoglobulin antigen-binding domain is an scFv comprising the amino acid sequence set forth in SEQ ID NO:9: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVDIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 9) and the second immunoglobulin antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:7. H and V comprising the amino acid sequence set forth in SEQ ID NO:8 L Includes.

[0156] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen-binding domain that specifically binds to HER2 and a second immunoglobulin antigen-binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen-binding domain comprises the amino acid sequence of SEQ ID NO:7. H and V comprising the amino acid sequence set forth in SEQ ID NO:8 L and the second comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:9.

[0157] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:3 and the second immunoglobulin antigen binding domain comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO:14: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KGGGGSGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED TAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGSQVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSA LMSRLTISKDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV H TFPAV L QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV H NAKTKPREEQYNSTYRVVSV L TV L HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 15: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 15).

[0158] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:3 and the second immunoglobulin antigen binding domain comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO:22: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KGGGGSGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCMSGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLWMGWINTYTGEPTYTDDF KGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV H TFPAV L QSSGLYSLSSVVTVPSSSLGTQTYICWNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV H NAKTKPREEQYNSTYRVVSV L TV L HQDWLNGKEYKCKVSNKALPAPTEKTTSKAKGQPREPQVYTLPPSREEMTKNQVSLTCI.VKGFYPSDIAVEWESNGQPENNYKTTPPV L DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALRNHYTQKSLSLSPG (SEQ ID NO: 22) and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 23:

[0043] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTIIQGLSSPVTKSFNRGEC (sequence number 23).

[0159] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds Trop-2 and a second immunoglobulin antigen binding domain that specifically binds HER2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:6 and the second immunoglobulin antigen binding domain comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO:16: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGT KVEIKGGGGSGGGGSGGGSQVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRLTISKDTSKNQFSLKLSSVTAA DTAVYYCARDENWDGAWFAYWGQGTLVTVSSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV H TFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV H NAKTKPREEQYNSTYRVVSV L TV L HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:16) and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 17:

[0043] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17).

[0160] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds Trop-2 and a second immunoglobulin antigen binding domain that specifically binds HER2, wherein the first immunoglobulin antigen binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:9 and the second immunoglobulin antigen binding domain comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO:20: DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTK VDIKGGGGSGGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQIS SLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV H TFPAV L QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV H NAKTKPREEQYNSTYRVVSV L TV L HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20) and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 21:

[0043] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 21).

[0161] In various embodiments of the present application, an isolated bispecific anti-HER2 / Trop-2 antibody comprises a first immunoglobulin antigen binding domain that specifically binds to HER2 and a second immunoglobulin antigen binding domain that specifically binds to Trop-2, wherein the first immunoglobulin antigen binding domain comprises a heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 18: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV H TFPAV L QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV H NAKTKPREEQYNSTYRVVSV L TV L HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSDIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVDIKGGGGSGGGGSGGGGSQVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 18) and a light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 19: DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) and the second immunoglobulin antigen-binding domain comprises an scFv comprising the amino acid sequence set forth in SEQ ID NO:9.

[0162] In various embodiments, the bispecific antibody is an antibody comprising an scFv domain, a heavy chain and a light chain, wherein the scFv domain comprises a sequence having at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequence set forth in SEQ ID NOs: 3, 6, 9, and the heavy chain comprises a sequence having at least about 80% identity to the amino acid sequence set forth in SEQ ID NOs: 14, 16, 18, 20, 22. , and the light chain comprises a sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the amino acid sequence set forth in SEQ ID NOs: 15, 17, 19, 21, 23.

[0163] In various embodiments, the second immunoglobulin antigen binding domain of the bispecific anti-HER2 / Trop-2 antibody is covalently linked to the carboxy terminus of the HC of the first immunoglobulin antigen binding domain. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises at least one linker interposed between the second immunoglobulin antigen binding domain and the carboxy terminus of the HC of the first immunoglobulin antigen binding domain. In various embodiments, a linker is interposed between the second immunoglobulin antigen binding domain and the carboxy terminus of the HC of the first immunoglobulin antigen binding domain.

[0164] In various embodiments, the second immunoglobulin antigen binding domain is covalently linked to the amino terminus of the HC of the first immunoglobulin antigen binding domain. In various embodiments, the bispecific anti-HER2 / Trop-2 antibody comprises at least one linker interposed between the second immunoglobulin antigen binding domain and the amino terminus of the HC of the first immunoglobulin antigen binding domain. In various embodiments, a linker is interposed between the second immunoglobulin antigen binding domain and the amino terminus of the HC of the first immunoglobulin antigen binding domain.

[0165] In various embodiments of the disclosure, the bispecific antibody can be an anti-HER2 / Trop-2 antibody that has the same or higher antigen binding affinity as an antibody comprising a heavy chain sequence set forth in any of SEQ ID NOs: 14, 16, 18, 20, and 22. In various embodiments, the antibody can be an HER2 / Trop-2 antibody that binds to the same epitope as an antibody comprising a heavy chain sequence set forth in any of SEQ ID NOs: 14, 16, 18, 20, and 22. In various embodiments, the antibody is an anti-HER2 / Trop-2 antibody that competes with an antibody comprising a heavy chain sequence set forth in any of SEQ ID NOs: 14, 16, 18, 20, and 22. In various embodiments, the antibody can be an anti-HER2 / Trop-2 antibody that comprises at least one (e.g., two or three) CDRs of a light chain sequence set forth in any of SEQ ID NOs: 14, 16, 18, 20, and 22.

[0166] In various embodiments of the disclosure, the bispecific antibody can be an anti-HER2 / Trop-2 antibody that has the same or higher antigen binding affinity as an antibody comprising a light chain sequence set forth in any of SEQ ID NOs: 15, 17, 19, 21, and 23. In various embodiments, the antibody can be an HER2 / Trop-2 antibody that binds to the same epitope as an antibody comprising a light chain sequence set forth in any of SEQ ID NOs: 15, 17, 19, 21, and 23. In various embodiments, the antibody is an anti-HER2 / Trop-2 antibody that competes with an antibody comprising a light chain sequence set forth in any of SEQ ID NOs: 15, 17, 19, 21, and 23. In various embodiments, the antibody can be an anti-HER2 / Trop-2 antibody that comprises at least one (e.g., two or three) CDRs of the light chain sequence set forth in any of SEQ ID NOs: 15, 17, 19, 21, and 23.

[0167] The bispecific antibody or antigen-binding fragment thereof of the present application may comprise any constant region known in the art. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, for example, a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, for example, an IgA, IgD, IgE, IgG, and IgM type heavy chain constant region. In various embodiments, the light or heavy chain constant region is a fragment, derivative, variant, or mutant of a naturally occurring constant region.

[0168] Techniques for obtaining antibodies of a different subclass or isotype from an antibody of interest, i.e., subclass switching, are known. Thus, for example, an IgG antibody can be obtained from an IgM antibody, and vice versa. Such techniques allow the preparation of new antibodies that retain the antigen-binding properties of a given antibody (parent antibody) while exhibiting biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques can be employed. In such procedures, cloned DNA encoding a particular antibody polypeptide can be employed, for example, DNA encoding the constant domain of an antibody of a desired isotype. See also Lanitto et al., Methods Mol. Biol. 178:303-16, 2002.

[0169] The antibodies of the present application may also be described or specified in terms of cross-reactivity. Also included in the present application are antibodies that bind to HER2 and Trop-2 having at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity to human HER2 and Trop-2 (as calculated using methods known in the art and described herein).

[0170] In various embodiments, the antibodies of the present application comprise one or both variable regions (i.e., V H and / or V L) or modifying residues in the constant region(s), e.g., to alter the effector function(s) of the antibody. In various embodiments, the variable region of the antibody is modified by performing CDR grafting using framework sequences that can be obtained from public DNA databases or published literature that contain germline antibody gene sequences (e.g., Tomlinson, I. M., et al., J. Mol. Biol. 227:776-798, 1992; and Cox, J. P. Let al., Eur. J. Immunol. 24:827-836, 1994; the contents of each of which are expressly incorporated herein by reference). In various embodiments, the antibody comprises a V mutation(s) that results in improved binding affinity and / or reduced immunogenicity. H and / or V L In various embodiments, antibodies may be modified using site-directed mutagenesis or PCR-mediated mutagenesis to introduce modifications into the Fc region to alter the antibody's serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In various embodiments, antibodies may be modified to modify the glycosylation of the antibody. Methods for making each of the modifications described herein and others are well known to those of skill in the art.

[0171] Characterization of antigen binding of bispecific antibodies The bispecific antibodies of the present application can be tested for binding to human HER2 and Trop-2 expressed on the cell surface, for example, by flow cytometry. Briefly, the antibodies can be added to cells in FACS buffer and incubated at 4°C for 30 minutes. After incubation, the cells are washed to remove unbound antibodies. The cells are then dissociated and stained with fluorescently labeled secondary antibodies for 30 minutes on ice, and then analyzed using a Beckman flow cytometry system. Fluorescence intensity and cell binding percentage can be analyzed by Beckman flow cytometry.

[0172] Pharmaceutical Compositions In another aspect, the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described above. Thus, the pharmaceutical compositions, methods and uses of the present application also encompass embodiments of combination (co-administration) with other active agents, as detailed below.

[0173] In general, the antibody of the present application, or an antigen-binding fragment thereof, is suitable for administration as a formulation with one or more pharma- ceutically acceptable excipient(s). The term "excipient" is used herein to describe any ingredient other than the compound(s) of the present application. The choice of excipient(s) will depend largely on factors such as the particular mode of administration, the excipient's effect on solubility and stability, and the nature of the dosage form. As used herein, "pharma- ceutically acceptable excipient" includes any solvent, dispersion medium, coating agent, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like, that are physiologically compatible. Some examples of pharma- ceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonicity agent, for example, sugar, polyalcohol (such as mannitol, sorbitol, or sodium chloride), in the composition. Further examples of pharma- ceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers which enhance the shelf life or effectiveness of the antibody. The pharmaceutical compositions of the present application and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably manufactured under GMP conditions.

[0174] The pharmaceutical compositions of the present application may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a specified amount of active ingredient.The amount of active ingredient generally corresponds to the dosage of active ingredient administered to a subject or a convenient fraction of the dosage, such as 1 / 2 or 1 / 3 of the dosage.

[0175] Any method accepted in the art for administering peptides, proteins or antibodies may be suitably employed for the antibodies and portions of the present application.

[0176] The pharmaceutical compositions of the present application are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically breaking a tissue of a subject and administering the pharmaceutical composition through the tissue break, thus generally resulting in direct administration into the bloodstream, into a muscle, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial injection or infusion, and kidney dialysis infusion techniques. Various embodiments include intravenous and subcutaneous routes.

[0177] A formulation of a pharmaceutical composition suitable for parenteral administration typically includes the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further include one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form that is reconstituted with a suitable vehicle (e.g., sterile pyrogen-free water) and then the reconstituted composition is administered parenterally. Parenteral formulations may include aqueous solutions (preferably pH 3-9) that may have excipients such as salts, carbohydrates, and buffers, although for some applications they may be more suitably formulated as sterile non-aqueous solutions or as dry forms used with a suitable vehicle such as sterile pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose. Such dosage forms may be suitably buffered, if desired. Other useful parenteral formulations include those that contain the active ingredient in microcrystalline form, or liposomal preparations. Parenteral formulations may be formulated for immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

[0178] For example, in one embodiment, a sterile injectable solution can be prepared by incorporating the bispecific antibody in the required amount in a suitable solvent with one or a combination of the above-mentioned components as required, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other necessary components from those mentioned above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which results in a powder of the active ingredient and any additional desired ingredients from the previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, for example, monostearate salts and gelatin, in the composition.

[0179] The antibodies of the present application may also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, in a mixture, or as mixed component particles, e.g., mixed with suitable pharma- ceutically acceptable excipients) from a dry powder inhaler, as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably a atomizer that uses electrohydrodynamics to produce a fine mist) or nebulizer, with or without the use of a suitable propellant, or as nasal drops.

[0180] The pressurized container, pump, spray, atomizer, or nebulizer will generally contain a solution or suspension of the antibody of the present application, e.g., with a suitable agent for dispersing, solubilizing, or prolonging the release of the active, a propellant(s) as a solvent.

[0181] Prior to use in a dry powder or suspension formulation, the drug is generally micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This can be accomplished by any suitable comminuting method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenizers, or spray drying.

[0182] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound of the present application, a suitable powder base and a performance modifier.

[0183] Suitable flavours, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the present application intended for inhaled / intranasal administration.

[0184] Formulations for inhaled / intranasal administration may be formulated to be immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0185] In the case of dry powder inhalers and aerosols, the dosage unit is determined by using a valve to deliver a metered amount. Units according to the present application are typically arranged to administer a metered amount or "puff" of the antibody of the present application. The total daily dose may typically be administered in a single dose, or more usually as divided doses throughout the day.

[0186] The antibodies and antibody portions of the application can be formulated for oral administration, which may involve swallowing, so that the compound enters the gastrointestinal tract, and / or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.

[0187] Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multiparticulates or nanoparticles, liquids, or powders; lozenges (including liquid-filled); chews; gels; fast-dispersing dosage forms; films; ovules; sprays; and buccal / mucoadhesive patches.

[0188] Pharmaceutical compositions intended for oral use may be prepared according to any method known in the art of pharmaceutical composition manufacture, and such compositions may contain one or more agents selected from the group consisting of sweeteners to provide a pharma-ceutical elegant and palatable preparation. For example, to prepare an orally deliverable tablet, the antibody or antigen-binding fragment thereof is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed according to known methods to form a tablet for delivery to the gastrointestinal tract. Tablet compositions are typically formulated with additives, such as sugar or cellulose carriers, binders such as starch paste or methylcellulose, fillers, disintegrants, or other additives typically used in the manufacture of medical preparations. To prepare an orally deliverable capsule, DHEA is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsule container suitable for delivery to the gastrointestinal tract. Compositions containing antibodies or antigen-binding fragments thereof can generally be prepared as described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) (Chapter 89), incorporated herein by reference.

[0189] In various embodiments, the pharmaceutical composition is formulated as an orally deliverable tablet containing the antibody or antigen-binding fragment thereof mixed with non-toxic pharma- ceutically acceptable excipients suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as corn starch, gelatin or gum arabic, and lubricating agents such as magnesium stearate, stearic acid, or talc. The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be employed alone or with a wax.

[0190] In various embodiments, the pharmaceutical compositions are formulated as hard gelatin capsules in which the antibody or antigen-binding fragment thereof is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the antibody or antigen-binding fragment thereof is mixed with a water or oil medium, such as peanut oil, peanut oil, liquid paraffin, or olive oil.

[0191] Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations can be used as fillers in soft or hard capsules (e.g., made from gelatin or hydroxypropylmethylcellulose), typically containing carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or suitable oils, and one or more emulsifying agents and / or suspending agents. Liquid formulations can also be prepared by the reconstitution of a solid, for example, from a sachet.

[0192] therapeutic use In another aspect, the application provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount (either as monotherapy or in a combination treatment regimen) of an antibody or antigen-binding fragment thereof of the application. Such disorders include solid tumors, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute phosphoinositide, acute myelogenous leukemia ... The cancers include, but are not limited to, chronic or acute leukemias, including leukemia, chronic lymphoblastic leukemia, solid tumors of childhood, lymphocytic lymphomas, bladder cancer, cancer of the kidney or urethra, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, T-cell lymphomas, and combinations of said cancers. In various embodiments, the subject has previously responded to treatment with an anti-cancer therapy, but relapsed when the treatment was discontinued (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer. In various embodiments, the cancerous cells are immunogenic tumors (e.g., tumors that can be vaccinated with the tumor itself to provide immunity to tumor inoculation). In various embodiments, the cancer is selected from the group consisting of colorectal cancer (CRC), renal cancer, non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, and glioma.

[0193] In some embodiments, the cancer is selected from the group consisting of prostate cancer, breast cancer, colon cancer, pancreatic cancer, and gastric cancer.

[0194] In various embodiments, the antibodies and antigen-binding fragments thereof can be utilized to directly kill or eliminate cancerous cells in vivo. Direct killing involves administering the antibodies (optionally fused to a cytotoxic drug) to a subject in need of such treatment. In various embodiments, the cancer comprises cancer cells that express Trop-2 at higher levels than non-cancerous cells of a comparable tissue. Because the antibody recognizes Trop-2 on the cancer cells, any such cells to which the antibody binds are destroyed. When the antibody is used alone to kill or eliminate cancerous cells, such killing or elimination may be subject to the initiation of endogenous host immune functions such as CDC and / or ADCC. Assays for determining whether an antibody kills cells in this manner are within the skill of one of ordinary skill in the art.

[0195] In some embodiments, the cancer is Trop2 positive and / or HER2 positive.

[0196] In some embodiments, the cancer is Trop2 intermediate or high expressing and / or HER2 intermediate or high expressing.

[0197] In some embodiments, the cancer is Trop2 underexpressing and / or HER2 underexpressing.

[0198] The expression levels of Trop2 or HER2 can be assessed by a suitable method, such as, for example, immunohistochemistry (IHC) staining of tumor sections obtained from the subject's specimen, and classification of Trop2 and / or HER2 as low, moderate and high expression is based on the type of cancer and / or criteria widely accepted in the art, such as those exemplified in Zhang et al., BMC Cancer. 2020 Aug 27; 20(1):815; Ahn et al., J Pathol Transl Med. 2020 Jan; 54(1):34-44; and Inamura et al., Oncotarget. 2017 Apr 25; 8(17):28725-28735.

[0199] In some embodiments, a cancer is Trop2 low if it has a level of Trop2 expression comparable to Colo205 or BxPC-3 cells (e.g., within 25% or 50% of the Trop2 expression level in Colo205 or BxPC3). In some embodiments, a cancer is Trop2 high if it has a level of Trop2 expression comparable to NCI-N87 or SK-BR-3 cells (e.g., within 25% or 50% of the Trop2 expression level in NCI-N87 or SK-BR-3 cells). In some embodiments, a cancer is Trop2 mid if it has more than 25%, 50%, 75%, or 100% of the Trop2 expression level in Colo205 or BxPC3 and less than 100%, 75%, 50%, or 25% of the Trop2 expression level in NCI-N87 or SK-BR-3 cells.

[0200] In some embodiments, a cancer is HER2 low if it has a level of HER2 expression equivalent to Colo205 cells (e.g., within 25% or 50% or less of the HER2 expression level in Colo205). In some embodiments, a cancer is HER2 medium if it has a level of HER2 expression equivalent to NCI-N87 or MDA-MB-468 cells (e.g., within 25% or 50% or less of the HER2 expression level in NCI-N87 or MDA-MB-468 cells). In some embodiments, a cancer is HER2 high if it has a level of HER2 expression equivalent to BxPC-3 cells (e.g., within 25% or 50% or less of the HER2 expression level in BxPC-3).

[0201] In various embodiments, the antibodies and antigen-binding fragments thereof can be used to inhibit and / or promote the growth of cancerous tumor cells. These methods can inhibit or prevent the growth of cancer cells in the subject by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. As a result, when the cancer is a solid tumor, the modulation can reduce the size of the solid tumor by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0202] Inhibition of cancer cell proliferation is associated with increased bromodeoxyuridine (BRDU) incorporation (Hoshino et al., Int. J. Cancer 38, 369, 1986; Campana et al., J. Immunol. Meth. 107:79, 1988; 3H]-thymidine incorporation (Chen, J., Oncogene 13:1395-403, 1996; Jeoung, J., J. Biol. Chem. 270:18367-73, 1995; Alamar Blue dye (available from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998). Anchorage-independent growth of cancer cells can be measured by cell-based assays such as soft agar colony formation assays, e.g., by counting the number of cancer cell colonies formed on soft agar (see Examples and Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989).

[0203] The growth inhibition of cancer cells in a subject can be evaluated by monitoring the cancer growth in a subject, for example, an animal model or a human subject. One exemplary monitoring method is a tumorigenicity assay. In one example, the xenograft comprises human cells derived from an existing tumor or tumor cell line. Tumor xenograft assays are known in the art and described herein (see, for example, Ogawa et al., Oncogene 19:6043-6052, 2000). In another embodiment, tumorigenicity is monitored using a hollow fiber assay described in U.S. Patent No. 5,698,413, which is incorporated herein by reference in its entirety.

[0204] The percentage of inhibition is calculated by comparing cancer cell proliferation, anchorage-independent growth, or cancer cell growth under treatment with a modulator to that under negative control conditions (typically without treatment with a modulator). For example, the number of cancer cells or cancer cell colonies (colony formation assay) or PRDU or [ 3 If the incorporation of [H]-thymidine is A (under treatment with modulator) and C (under negative control conditions), then the percentage of inhibition is (CA) / C×100%.

[0205] Examples of tumor cell lines derived from human tumors available for use in in vitro and in vivo testing include leukemia cell lines (e.g., CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPM1-8226, SR, P388 and P388 / ADR, H292, MV-411); non-small cell lung cancer cell lines (e.g., A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H470, NCI-H510, NCI-H610, NCI-H710, NCI-H810, NCI-H910, NCI-H10 ... 522 and LXFL529); small cell lung cancer cell lines (e.g., DMS114 and SHP-77); colon cancer cell lines (e.g., COLO205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620, DLD-1 and KM20L2); central nervous system (CNS) cancer cell lines (e.g., SF-268, SF-295, SF-539, SNB-19, SNB-75, U251, SNB-78 and XF498); melanoma cell lines (e.g., LOX IMVI, MALME-3M, M14, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, RPMI-7951, and M19-MEL; ovarian cancer cell lines (e.g., IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3); kidney cancer cell lines (e.g., 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, RXF-631, and SN12K1); prostate cancer cell lines (e.g., PC-3 and DU-145); breast cancer cell lines (e.g., MCF7, NCI / ADR-RES, MDA-MB-231 / ATCC, HS 578T, MDA-MB-435, BT-549, T-47D and MDA-MB-468); and thyroid cancer cell lines (e.g., SK-N-SH).

[0206] In another aspect, the present application provides a method for treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of an isolated antibody or antigen-binding fragment of the present application (either as monotherapy or in a combination therapy regimen). In various embodiments, the subject is suffering from an infectious disease caused by a pathogenic virus. In various embodiments, the subject is suffering from an infectious disease caused by a pathogenic bacterium. In various embodiments, the subject is suffering from an infectious disease caused by a pathogenic fungus. In various embodiments, the subject is suffering from an infectious disease caused by a pathogenic parasite. In various embodiments, the subject is suffering from an infectious disease that is resistant to or not effectively treated by treatment using conventional vaccines.

[0207] A "therapeutically effective amount" or "therapeutically effective dose" refers to a sufficient quantity of the therapeutic agent being administered to relieve to some extent one or more of the symptoms of the disorder being treated.

[0208] Therapeutically effective doses were first determined using IC 50 The IC determined in cell culture can then be estimated. 50 Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes. Such information can be used to more accurately determine useful doses in humans. Plasma concentrations can be measured, for example, by HPLC. The exact composition, route of administration and dosage can be selected by the individual physician, taking into account the subject's condition.

[0209] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus may be administered, or multiple divided doses (multiple or repeated or maintenance) may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form as used herein refers to physically discrete units suitable for unitary administration to the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to provide the desired therapeutic effect together with the required pharmaceutical carrier. The specifications of the dosage units of the present disclosure are determined by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0210] Therefore, based on the disclosure provided herein, those skilled in the art will understand that dosage and administration regimen are adjusted according to the method well known in the therapeutic field.That is, the maximum tolerated dose can be easily determined, and the effective amount that brings about detectable therapeutic benefit to the subject can also be determined, and the time administration requirement of each agent to bring about detectable therapeutic benefit to the subject can also be determined.Therefore, although certain dosage and administration regimen are exemplified herein, these examples do not limit the dosage and administration regimen that can be provided to the subject when implementing the present disclosure in any way.

[0211] It should be noted that dosage values ​​vary depending on the type and severity of the condition to be alleviated, and include single or multiple administrations. For any particular subject, it should be further understood that the specific dosage regimen should be adjusted over time according to the individual need and the professional judgment of the person administering or directing the administration of the composition, and that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions. Furthermore, the dosage regimen with the compositions of the present disclosure may be based on a variety of factors, such as the type of disease, the subject's age, weight, sex, medical condition, the severity of the condition, the route of administration, and the particular antibody employed. Thus, dosage regimens may vary widely, but can be routinely determined using standard methods. For example, doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or clinical laboratory values. Thus, the present disclosure encompasses intra-subject dose escalation as determined by one of skill in the art. Determination of appropriate dosages and regimens is well known in the relevant art and will be appreciated by those of ordinary skill in the art given the teachings disclosed herein.

[0212] When administered to a human subject, the total monthly dose of the antibody or antigen-binding fragment thereof of the present disclosure will of course depend on the mode of administration, but may be 0.5 to 1200 mg per subject, 0.5 to 1100 mg per subject, 0.5 to 1000 mg per subject, 0.5 to 900 mg per subject, 0.5 to 800 mg per subject, 0.5 to 700 mg per subject, 0.5 to 600 mg per subject, 0.5 to 500 mg per subject, 0.5 to 400 mg per subject, 0.5 to 300 mg per subject, 0.5 to 200 mg per subject, or 0.5 to 100 mg per subject. The dose may range from 0.00 mg per subject, 0.5-100 mg per subject, 0.5-50 mg per subject, 1-1200 mg per subject, 1-1100 mg per subject, 1-1000 mg per subject, 1-900 mg per subject, 1-800 mg per subject, 1-700 mg per subject, 1-600 mg per subject, 1-500 mg per subject, 1-400 mg per subject, 1-300 mg per subject, 1-200 mg per subject, 1-100 mg per subject, or 1-50 mg per subject. For example, a monthly intravenous dose of about 1-1000 mg / subject may be required. In various embodiments, the antibody or antigen-binding fragment thereof of the present disclosure may be administered at about 1-200 mg per subject, 1-150 mg per subject, or 1-100 mg / subject. The total monthly dose may be administered in single or divided doses and, at the physician's discretion, may fall outside of the typical range given herein.

[0213] In some embodiments, the construct is administered to a subject at a dose of at least about 5 mg / kg. In some embodiments, the construct is administered to a human at a dose equivalent to a dose of at least 5 mg / kg in a mouse. See Nair, AB, & Jacob, S. (2016). A simple practice guide for dose conversion between animals and human. Journal of basic and clinical pharmacy, 7(2), 27.

[0214] In the case of repeated administration over several days or more, treatment is continued until desired suppression of symptoms occurs or sufficient therapeutic levels are achieved, e.g., pain is reduced, depending on the condition. An exemplary dosing regimen includes administration of an initial dose of about 2 mg / kg of bispecific anti-HER2 / Trop-2 antibody, followed by a weekly maintenance dose of about 1 mg / kg, or a biweekly maintenance dose of about 1 mg / kg. However, other dosing regimens may be useful, depending on the pharmacokinetic decay pattern the physician wishes to achieve. For example, in some embodiments, administration 1-4 times per week is contemplated. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the HER2 / Trop-2 antagonist(s) used) may vary over time. In various embodiments, the appropriate dosage of the HER2 / Trop-2 antagonist antibody will vary depending on the bispecific anti-HER2 / Trop-2 antibody (or composition thereof) employed, the type and severity of the headache (e.g., migraine) being treated, whether the agent is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the judgment of the attending physician. Typically, the clinician will administer the bispecific anti-HER2 / Trop-2 antibody until a dosage is reached that produces the desired result. Dosage and / or frequency may vary over the course of treatment.

[0215] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or directing the administration of the composition, and it should be further understood that dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0216] In various embodiments, the total dose administered achieves a plasma antibody concentration in the range of, for example, about 1 to 1000 μg / ml, about 1 to 750 μg / ml, about 1 to 500 μg / ml, about 1 to 250 μg / ml, about 10 to 1000 μg / ml, about 10 to 750 μg / ml, about 10 to 500 μg / ml, about 10 to 250 μg / ml, about 20 to 1000 μg / ml, about 20 to 750 μg / ml, about 20 to 500 μg / ml, about 20 to 250 μg / ml, about 30 to 1000 μg / ml, about 30 to 750 μg / ml, about 30 to 500 μg / ml, or about 30 to 250 μg / ml.

[0217] The toxicity and therapeutic index of the pharmaceutical compositions of the present application can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (the dose that is lethal to 50% of the population) and ED 50 The LD (the dose that is therapeutically effective in 50% of the population) can be determined by procedures for determining the dose ratio between the toxic and therapeutically effective doses, which is the therapeutic index, and is 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0218] In various embodiments, single or multiple administrations of the pharmaceutical composition are performed according to the dosage and frequency required and tolerated by the subject.In any case, the composition should provide at least one of the antibodies or antigen-binding fragments thereof disclosed herein in an amount sufficient to effectively treat the subject.Dosage may be a one-time administration, but may also be performed periodically until therapeutic results are achieved or side effects make it difficult to continue the treatment.

[0219] The frequency of administration of the antibody or antigen-binding fragment thereof pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The subject can be treated at regular intervals, such as weekly or monthly, until the desired therapeutic result is achieved. Exemplary administration frequencies include, but are not limited to, once a week without a break; once a week, every other week; once every two weeks; once every three weeks; just under two weeks without a break, then once a month; just under three weeks without a break, then once a month; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months, or once a year.

[0220] Combination therapy As used herein, the terms "co-administration," "co-administered," and "in combination" with respect to an antibody or antigen-binding fragment thereof of the present disclosure and one or more other therapeutic agents are intended to mean, refer to, and include: simultaneous administration of a combination of an antibody or antigen-binding fragment thereof and a therapeutic agent(s) to a subject in need of treatment, where such components are formulated together in a single dosage form and such components are released to the subject substantially simultaneously; substantially simultaneous administration of a combination of an antibody or antigen-binding fragment thereof and a therapeutic agent(s) to a subject in need of treatment, where such components are formulated separately from one another in separate dosage forms and taken by the subject substantially simultaneously and such components are released to the subject substantially simultaneously. sequential administration to a subject in need of treatment of a combination of an antibody or antigen-binding fragment thereof and a therapeutic agent(s) wherein such components are formulated together in separate dosage forms and taken by the subject sequentially with a significant time interval between each administration, and wherein the components are released to the subject at substantially different times; and sequential administration to a subject in need of treatment of a combination of an antibody or antigen-binding fragment thereof and a therapeutic agent(s) wherein such components are formulated together in a single dosage form and wherein the components are released in a controlled manner and released to the subject simultaneously, sequentially, and / or overlappingly at the same and / or different times, and wherein each portion may be administered by either the same route or by different routes.

[0221] In another aspect, the present application relates to a combination therapy designed to treat cancer or an infectious disease in a subject, comprising a) administering to the subject a therapeutically effective amount of an isolated antibody or antigen-binding fragment of the present application; and b) administering one or more additional therapies selected from the group consisting of immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, vaccination protocols, and stem cell transplantation, wherein the combination therapy increases cell killing of tumor cells, i.e., there is a synergistic effect between the isolated antibody or antigen-binding fragment and the additional therapy when administered together.

[0222] In various embodiments, immunotherapy includes treatment with agonist, antagonist, or blocking antibodies against costimulatory or co-inhibitory molecules (immune checkpoints), such as PD-1, PD-L1, OX-40, CD137, GITR, LAG3, TIM-3, and VISTA; treatment with bispecific T cell engaging antibodies (BiTEs®), such as blinatumomab; treatment with administration of biological response modifiers, such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines, such as sipuleucel-T; treatment with dendritic cell vaccines, or tumor antigen peptide vaccines; treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TIL); adoptively transferred anti-tumor T cells (ex and treatment with immune activators such as Toll-like receptor (TLR) agonists CpG and imiquimod.

[0223] A wide variety of conventional compounds have been shown to have antineoplastic activity. These compounds are used as medicines in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or myeloid malignancies. Chemotherapy is effective in treating various types of malignancies, but many antineoplastic compounds induce undesirable side effects. It has been shown that combining two or more different therapies can reduce the dosage of each treatment because the treatments act synergistically, thereby reducing the harmful side effects caused by higher dosages of each compound. In other cases, malignancies that are refractory to one treatment may respond to a combination of two or more different therapies.

[0224] When the bispecific antibodies disclosed herein are administered in combination with another conventional anti-neoplastic agent, either simultaneously or sequentially, such antibodies or antigen-binding fragments may improve the therapeutic efficacy of the anti-neoplastic agent or overcome cellular resistance to such an anti-neoplastic agent, thereby allowing the dosage of the antineoplastic agent to be reduced, reducing undesirable side effects, or restoring the efficacy of the antineoplastic agent in resistant T cells.

[0225] Pharmaceutical compounds that may be used in combination antitumor therapy include, by way of example only, aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, ribavirin ... A, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin These include, but are not limited to, cyclosporine, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0226] These chemotherapeutic antitumor compounds can be classified according to their mechanism of action, for example, into the following groups: antimetabolites / anticancer drugs, for example pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogues, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, for example natural products such as the vinca alkaloids (vinblastine, vincristine and vinorelbine); Microtubule disrupting agents such as vincristine, vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melpha orchids, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (which metabolize L-asparagine systemically and synthesize asparagine themselves) L-asparaginase to reduce incompetent cells; antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl-busulfans, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazene-dacarbazinin (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate);Platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (bleverdin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP-470, genistein) and proliferation factors. inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0227] In various embodiments, chemotherapy is selected from the group consisting of daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin. , carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fluradabin, ifosfamide, hydroxyureataxanes (such as paclitaxel and doxitaxel) and / or anthracycline antibiotics, and combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX.

[0228] In various embodiments, the small molecule kinase inhibitor targeted therapy comprises a small molecule kinase inhibitor selected from the group consisting of a Bruton's tyrosine kinase (BTK) inhibitor, a phosphatidylinositol-3-kinase (PI3K) inhibitor, a SYK inhibitor (e.g., entospletinib), an AKT inhibitor, an mTOR inhibitor, a Src inhibitor, a JAK / STAT inhibitor, a Ras / Raf / MEK / ERK inhibitor, and an Aurora inhibitor (see D'Cruz et al, Expert Opin Pharmacother, 14(6):707-21, 2013).

[0229] In various embodiments, the combination therapy comprises administration of an antibody or antigen-binding fragment thereof simultaneously with one or more additional therapies, hi various embodiments, the antibody or antigen-binding fragment thereof composition and the one or more additional therapies are administered sequentially, i.e., the antibody or antigen-binding fragment thereof composition is administered either before or after administration of the one or more additional therapies.

[0230] In various embodiments, administration of the antibody or antigen-binding fragment thereof composition and the one or more additional therapies is concurrent, i.e., the administration period of the antibody or antigen-binding fragment thereof composition and the administration of the one or more additional therapies overlap.

[0231] In various embodiments, the administration of the antibody or antigen-binding fragment thereof composition and the administration of one or more additional therapies are not simultaneous. For example, in various embodiments, the administration of the antibody or antigen-binding fragment thereof composition is terminated before the administration of one or more additional therapies. In various embodiments, the administration of one or more additional therapies is terminated before the administration of the antibody or antigen-binding fragment thereof composition.

[0232] When the bispecific antibodies disclosed herein are administered in combination with one or more additional therapies, either simultaneously or sequentially, such antibodies or antigen-binding fragments thereof may improve the therapeutic efficacy of the one or more additional therapies or overcome cellular resistance to the one or more additional therapies, thereby allowing the dosage or duration of the one or more additional therapies to be reduced, reducing undesirable side effects, or restoring the efficacy of the one or more additional therapies.

[0233] Diagnostic Uses In another aspect, the application provides methods for detecting the presence of human HER2 and Trop-2 peptides in a sample in vitro or in vivo, for example to diagnose human HER2 and Trop-2 associated disorders. In some methods, this is accomplished by contacting the bispecific antibody with a sample to be tested, in addition to a control sample, under conditions that allow for the formation of a complex between the antibody and human HER2 and Trop-2. The formation of the complex in both samples is then detected (e.g., using ELISA), and a statistically significant difference in complex formation between the samples indicates the presence of human HER2 and Trop-2 antigens in the test sample.

[0234] In various embodiments, a method for detecting cancer or confirming a diagnosis of cancer in a subject is provided. The method includes contacting a biological sample obtained from the subject with an isolated antibody or antigen-binding fragment thereof of the present application and detecting binding of the isolated human monoclonal antibody or antigen-binding fragment thereof to the sample. Increased binding of the isolated human monoclonal antibody or antigen-binding fragment thereof to the sample compared to binding of the isolated human monoclonal antibody or antigen-binding fragment thereof to a control sample detects cancer in the subject or confirms the diagnosis of cancer in the control. The control can be a sample from a subject known to be cancer-free or a standard value. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies, and pathology specimens. Biological samples also include tissue sections, e.g., frozen sections taken for histological purposes. Biological samples further include bodily fluids such as blood, serum, plasma, sputum, and cerebrospinal fluid.

[0235] In one embodiment, a kit is provided for detecting HER2 and Trop-2 in a biological sample, such as a blood sample. The kit for detecting a polypeptide typically includes a human antibody that specifically binds to HER2 and Trop-2, such as any of the antibodies disclosed herein. In some embodiments, an antibody fragment, such as an Fv fragment, is included in the kit. For in vivo use, the antibody may be an scFv fragment. In a further embodiment, the antibody is labeled (e.g., fluorescently, radioactively, or enzymatically labeled).

[0236] In one embodiment, the kit includes instructional materials disclosing means of use of the antibodies that specifically bind to HER2 and Trop-2. The instructional materials may be written, electronic (such as a computer disk or compact disk) or visual (such as a video file). The kit may also include additional components that facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means for detecting the label (such as an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an appropriate secondary label such as a secondary antibody, etc.). The kit may additionally include buffers and other reagents routinely used in the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

[0237] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary depending on the particular format employed, a method for detecting HER2 and Trop-2 in a biological sample generally involves contacting the biological sample with an antibody that specifically reacts with HER2 and Trop-2 under immunologically reactive conditions. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (binding of the antibody) is detected directly or indirectly.

[0238] In various embodiments, the antibody or antigen-binding fragment may be labeled or unlabeled for diagnostic purposes. Typically, diagnostic assays involve detecting the formation of a complex resulting from the binding of the antibody to HER2 or Trop-2. The antibody may be directly labeled. A variety of labels may be employed, including but not limited to radionuclides, fluorescent materials, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens). Many suitable immunoassays are known to those skilled in the art (see, for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and 4,098,876). When unlabeled, the antibody may be used in assays such as agglutination assays. An unlabeled antibody can also be used in combination with another suitable reagent (one or more) that can be used to detect the antibody, e.g., a labeled antibody (e.g., a second antibody) reactive with the first antibody (e.g., an anti-idiotypic antibody or other antibody specific for the unlabeled immunoglobulin) or other suitable reagent (e.g., labeled Protein A).

[0239] The antibodies or antigen-binding fragments provided herein may also be used in a method for detecting susceptibility of a mammal to a particular disease. Illustratively, the method can be used to detect susceptibility of a mammal to a disease that develops based on the amount of HER2 and Trop-2 present on the cells of the mammal and / or the number of HER2 and Trop-2 positive cells. In one embodiment, the application provides a method for detecting susceptibility of a mammal to a tumor. In this embodiment, the sample to be tested is contacted with an antibody or portion thereof that binds to HER2 and Trop-2 under conditions suitable for binding to the antibody. The sample comprises cells that express HER2 and Trop-2 in a normal individual. The binding and / or amount of binding of the antibody is detected, which is indicative of susceptibility of the individual to the tumor, where higher receptor levels correlate with higher susceptibility of the individual to the tumor.

[0240] In various embodiments, the antibody or antigen-binding fragment is conjugated to a detectable label (e.g., the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor). The active moiety can be a radioactive agent, e.g., a radioactive heavy metal such as an iron chelate, a radioactive chelate of gadolinium or manganese, a positron emitter of oxygen, nitrogen, iron, carbon, or gallium, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 123 I, 125 I, 131 I, 132 I, or 99 The binding agent bound to such a moiety can be used as an imaging agent and is administered in an amount effective for diagnostic use in a mammal, such as a human, and then the localization and accumulation of the imaging agent is detected. The localization and accumulation of the imaging agent can be detected by radioscintigraphy, nuclear magnetic resonance imaging, computed tomography, or positron emission tomography.

[0241] Immunoscintigraphy using antibodies or antigen-binding fragments directed against HER2 and Trop-2 can be used to detect and / or diagnose cancer and the vasculature. For example, 99 technetium, 111 Indium, or 125Monoclonal antibodies against HER2 and Trop-2 markers labeled with iodine can be effectively used in such imaging methods. As will be appreciated by those skilled in the art, the amount of radioisotope administered will depend on the radioisotope. Those skilled in the art can readily determine the amount of imaging agent administered based on the specific activity and energy of a given radionuclide used as the active moiety. Typically, 0.1-100 millicuries or 1-10 millicuries, or 2-5 millicuries are administered per dose of imaging agent. Thus, the compositions of the present disclosure are useful as imaging agents comprising a targeting moiety conjugated to a radioactive moiety, comprising 0.1-100 millicuries, in some embodiments 1-10 millicuries, in some embodiments 2-5 millicuries, and in some embodiments 1-5 millicuries.

[0242] Immunoconjugates The present application further provides an immunoconjugate comprising at least one of the bispecific anti-HER2 / Trop-2 antibodies or antigen-binding fragments of the present application directly or indirectly conjugated (or linked) to an effector molecule. In this regard, the term "conjugated" or "linked" refers to making two polypeptides into one continuous polypeptide molecule. This linking can be by either chemical or recombinant means. In one embodiment, the linking is chemical, where a reaction between the antibody moiety and the effector molecule forms a covalent bond between the two molecules to form one molecule. Optionally, a peptide linker (a short peptide sequence) can be included between the antibody and the effector molecule. In various embodiments, the antibody or antigen-binding fragment is connected to the effector molecule. In other embodiments, the antibody or antigen-binding fragment connected to the effector molecule is further connected to a lipid, protein, or peptide to increase half-life in the body. Thus, in various embodiments, the antibodies of the present disclosure can be used to deliver a variety of effector molecules.

[0243] The effector molecule can be a radioisotope, a radioisotope, an immunomodulatory agent, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide lipid, a carbohydrate, a chelator, a therapeutic agent, or a chemotherapeutic agent.

[0244] Specific non-limiting examples of immunotoxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxins (PEs such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, Vibrio cholerae toxin or modified toxins thereof, or other toxic substances that directly or indirectly inhibit cell growth or kill cells.

[0245] A "cytokine" is a protein or peptide released by one cell population that acts on another cell as an intercellular messenger. Cytokines may also act as immunomodulators. Examples of cytokines include lymphokines, monokines, growth factors, and traditional polypeptide hormones. Thus, embodiments include interferons (e.g., IFN-α, IFN-β, and IFN-γ); tumor necrosis factor superfamily (TNFSF) members; human growth hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; follicle stimulating hormone (FSH); thyroid stimulating hormone (TSH); luteinizing hormone (LH); hepatic growth factors; prostaglandins, fibroblast growth factors; prolactin; placental lactogen, OB protein; TNF-α; TNF-β; integrins; thrombopoiesis hormone (THM); endothelial growth factor (EGF ... Cytokines that may be utilized include thrombopoietin (TPO), nerve growth factors such as NGF-β, platelet growth factors, transforming growth factor (TGF-α), transforming growth factor (TGF-β), insulin-like growth factors-I and -II, erythropoietin (EPO), colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL-1 through IL-21), kit-ligand or FLT-3, angiostatin, thrombospondin, or endostatin. These cytokines include proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0246] Chemokines may also be conjugated to the antibodies disclosed herein. Chemokines are a superfamily of small (approximately about 4 to about 14 kDa), inducible and secreted proinflammatory cytokines that act primarily as chemoattractants for certain leukocyte subtypes. Chemokine production is induced by inflammatory cytokines, growth factors, and pathogenic stimuli. Chemokine proteins are divided into subfamilies (alpha, beta, and delta) based on conserved amino acid sequence motifs and into four highly conserved groups, CXC, CC, C, and CX3C, based on the position of the first two cysteines adjacent to the amino terminus. To date, over 50 chemokines have been discovered and there are at least 18 human seven transmembrane domain (7TM) chemokine receptors. Chemokines used include, but are not limited to, RANTES, MCAF, MCP-1, and fractalkine.

[0247] The therapeutic agent may be a chemotherapeutic agent. Those skilled in the art can readily identify the chemotherapeutic agent to be used (see, for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 nded. (registered trademark). 2000 Churchill Livingstone, Inc.; Baltzer L., Berkery R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeutic agents useful in the preparation of immunoconjugates include auristatins, dolastatins, MMAE, MMAF, tubulysin, AFP, DM1, DM4, calicheamicins, duocarmycins, pyrrolobenzodiazepines, AEB, doxorubicin, daunorubicin, methotrexate, melphalan, chlorambucil, vinca alkaloids, 5-fluorouridine, mitomycin-C, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine ... The chemotherapeutic agents include clophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, etoposide, BCNU, irinotecan, camptothecin, bleomycin, idarubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, as well as their salts, solvents, and derivatives. In various embodiments, the chemotherapeutic agent is auristatin E (also known in the art as dolastatin-10) or its derivatives and pharmaceutical salts or solvates thereof. Exemplary auristatin derivatives include DM1, AEB, AEVB, AFP, MMAF, and MMAE. The synthesis and structure of auristatin E and its derivatives, as well as linkers, are described, for example, in U.S. Patent Application Publication No. 20030083263; U.S. Patent Application Publication No. 20050238629; and U.S. Patent No. 6,884,869, each of which is incorporated by reference in its entirety.In various embodiments, the therapeutic agent is an auristatin or an auristatin derivative. In various embodiments, the auristatin derivative is dovaline-valine-dolaisolonine-dolaproine-phenylalanine (MMAF) or monomethylauristatin E (MMAE). In various embodiments, the therapeutic agent is a maytansinoid or a maytansinol analog. In various embodiments, the maytansinoid is a DM1 or DM4 derivative. In various embodiments, the therapeutic agent is a calicheamicin, variant, or derivative. In various embodiments, the therapeutic agent is a duocarmycin, variant, or derivative.

[0248] Effector molecules can be linked to the antibodies or antigen-binding fragments of the present application using any number of means known to those of skill in the art. Both covalent and non-covalent attachment means can be used. The procedure for attaching an effector molecule to an antibody varies depending on the chemical structure of the effector molecule. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (--NH2) or sulfhydryl (--SH) groups, available for reaction with suitable functional groups on the antibody, resulting in attachment of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attachment of any of a number of linker molecules, such as those available from Pierce Chemical Company, Rockford, Ill. The linker can be any molecule used to connect the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight or branched chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibodies and effector molecules are polypeptides, the linkers may be attached to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

[0249] In some situations, it is desirable for the effector molecule to be released from the antibody when the immunoconjugate reaches its target site. Thus, in these situations, the immunoconjugate includes a linkage that is cleavable near the target site. Cleavage of the linker to release the effector molecule from the antibody can be facilitated by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or near the target site.

[0250] Procedures for conjugating antibodies with effector molecules have been previously described and are within the skill of the art. For example, procedures for preparing enzymatically active polypeptides of immunotoxins are described in WO84 / 03508 and WO85 / 03508, which are incorporated herein by reference for their specific teachings. Other techniques are described in Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); Shih et al., U.S. Patent No. 5,057,313; Shih Cancer Res. 51:4192, International Publication WO02 / 088172; U.S. Patent No. 6,884,869; International Patent Publication WO2005 / 081711; U.S. Patent Application 2003-0130189A; and U.S. Patent Application 20080305044, each of which is incorporated by reference herein for the purposes of teaching such techniques.

[0251] The immunoconjugates of the present application retain the immunoreactivity of the antibody or antigen-binding fragment, e.g., the antigen-binding ability of the antibody or antigen-binding fragment after conjugation is approximately the same as or only slightly reduced compared to before conjugation.

[0252] Antibody Drug Conjugates (ADCs) As used herein, an immunoconjugate is also referred to as an antibody drug conjugate (ADC). An antibody drug conjugate (ADC) according to one embodiment of the present application has the formula: Ab-(LD)n, where Ab is a bispecific anti-HER2 / Trop-2 binding antibody, L is a linker, D is an effector molecule that is an immunotoxin, a drug moiety, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent, and n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0253] In various embodiments, the ADC comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 therapeutic moieties. In various embodiments, the ADC comprises 2, 3, or 4 therapeutic moieties. In various embodiments, all of the therapeutic moieties are the same. In various embodiments, at least one therapeutic moiety is different from the rest. In various embodiments, all of the therapeutic moieties are different.

[0254] Expression of Polynucleotides and Antibodies The present application further provides polynucleotides comprising nucleotide sequences encoding anti-HER2 / Trop-2 antibodies or antigen-binding fragments thereof. Due to the degeneracy of the genetic code, a variety of nucleic acid sequences encode the amino acid sequence of each antibody. The present application further provides polynucleotides that hybridize to polynucleotides encoding antibodies that bind to human HER2 and Trop-2, e.g., under stringent or less stringent hybridization conditions as defined herein.

[0255] Stringent hybridization conditions include, but are not limited to, highly stringent conditions such as hybridization to filter-bound DNA in 6×SSC at about 45° C. followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C., hybridization to filter-bound DNA in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC / 0.2% SDS at about 60° C., or any other stringent hybridization conditions known to those of skill in the art (see, for example, Ausubel, FM et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

[0256] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which briefly involves synthesizing overlapping oligonucleotides containing a portion of the antibody-encoding sequence, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR. In one embodiment, the codons used are typical of human or mouse (see, e.g., Nakamura, Y., Nucleic Acids Res. 28:292 (2000)).

[0257] Polynucleotides encoding antibodies can also be generated from nucleic acid of a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding an immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library or isolated nucleic acid generated from any tissue or cell that expresses the antibody, e.g., hybridoma cells selected to express the antibody, preferably polyA+RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence, for example, by identifying a cDNA clone from a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0258] The present application is also directed to host cells expressing the anti-HER2 / Trop-2 antibodies of the present application. A wide variety of host expression systems known in the art can be used to express the antibodies of the present application, including, for example, prokaryotic (bacterial) and eukaryotic expression systems (such as yeast, baculovirus, plants, mammalian and other animal cells, transgenic animals, and hybridoma cells), as well as phage display expression systems.

[0259] The bispecific antibodies of the present application can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To recombinantly express an antibody, a host cell is transformed, transduced, infected, etc. with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and / or heavy chains of the antibody, such that the light and / or heavy chains are expressed in the host cell. The heavy and light chains may be expressed independently from different promoters operably linked in one vector, or the heavy and light chains may be expressed independently from different promoters operably linked in two vectors, one expressing the heavy chain and the other expressing the light chain. Optionally, the heavy and light chains may be expressed in different host cells.

[0260] Additionally, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody light and / or heavy chain from a host cell. The antibody light and / or heavy chain gene can be cloned into the vector such that the signal peptide is operably linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Preferably, the recombinant antibody is secreted into the medium in which the host cells are cultured, from which the antibody can be recovered or purified.

[0261] The isolated DNA encoding the HCVR can be converted into a full-length heavy chain gene by operably linking the DNA encoding the HCVR to another DNA molecule encoding a heavy chain constant region. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained, for example, by standard PCR amplification. The heavy chain constant region can be of any type (e.g., IgG, IgA, IgE, IgM or IgD), class (e.g., IgG1, IgG2, IgG3 and IgG4) or subclass of constant region and any allotypic variants thereof, as described in Kabat (supra).

[0262] The isolated DNA encoding the LCVR region can be converted to a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding the LCVR to another DNA molecule encoding a light chain constant region. The sequences of human and other mammalian light chain constant region genes are known in the art. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0263] In addition to the antibody heavy and / or light chain gene(s), the recombinant expression vector of the application carries regulatory sequences that control the expression of the antibody chain gene(s) in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene(s), as appropriate. The design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for expression in mammalian host cells include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and / or polyoma virus.

[0264] Furthermore, the recombinant expression vector of the present application may have additional sequences, such as sequences regulating replication of the vector in a host cell (e.g., origin of replication) and one or more selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced. For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (dhfr) gene (used for methotrexate selection / amplification in dhfr minus host cells), the neo gene (G418 selection), and glutamine synthetase (GS) for selection / amplification in GS negative cell lines (such as NSO).

[0265] For expression of the light and / or heavy chains, the expression vector(s) encoding the heavy and / or light chains are introduced into a host cell by standard techniques, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, transduction, infection, etc. Although the antibodies of the present application can theoretically be expressed in either prokaryotic or eukaryotic host cells, eukaryotic cells are preferred, and most preferably mammalian host cells, since such cells are more likely to assemble and secrete properly folded, immunologically active antibodies. Preferred mammalian host cells for expressing the recombinant antibodies of the present application include Chinese hamster ovary (CHO cells) [including dhfr minus CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-20, 1980, e.g., used with a DHFR selection marker as described in Kaufman and Sharp, J. Mol. Biol. 159:601-21, 1982], NSO myeloma cells, COS cells, and SP2 / 0 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell under appropriate conditions known in the art for a period of time sufficient for the antibody to be expressed in the host cell or, more preferably, secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the host cell and / or culture medium using standard purification methods.

[0266] The present application provides a host cell comprising the nucleic acid molecule of the present application. Preferably, the host cell of the present application comprises one or more vectors or constructs comprising the nucleic acid molecule of the present application. For example, the host cell of the present application is a cell into which a vector of the present application is introduced, the vector comprising a polynucleotide encoding the LCVR of the antibody of the present application and / or a polynucleotide encoding the HCVR of the present application. The present application also provides a host cell into which two vectors of the present application are introduced, one comprising a polynucleotide encoding the LCVR of the antibody of the present application and one comprising a polynucleotide encoding the HCVR present in the antibody of the present application, each operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, adenovirus, etc., e.g., a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high gene transcription levels.

[0267] Once expressed, the intact antibodies, individual light and heavy chains, or other immunoglobulin forms of the present application can be purified according to standard procedures in the art, including ammonium sulfate precipitation, ion exchange, affinity (e.g., protein A), reverse phase, hydrophobic interaction column chromatography, hydroxyapatite chromatography, gel electrophoresis, etc. Standard procedures for the purification of therapeutic antibodies are described, for example, in Feng Ll, Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee, in an article entitled "Current Therapeutic Antibody Production and Process Optimization," BioProcessing Journal, September / October 2005, which is incorporated by reference in its entirety for its teaching of the purification of therapeutic antibodies. Furthermore, standard techniques for removing viruses from recombinantly expressed antibody preparations are also known in the art (see, e.g., Gerd Kern and Mani Krishnan, “Viral Removal by Filtration: Points to Consider” (Biopharm International, October 2006)). It is known that the effectiveness of filtration to remove viruses from therapeutic antibody preparations depends at least in part on the protein and / or antibody concentration in the filtered solution. The purification process for the antibody of the present application may include a step of filtering to remove viruses from the mainstream of one or more chromatography operations. Preferably, the chromatographic mainstream containing the antibody of the present application is diluted or concentrated to obtain a total protein and / or antibody concentration of about 1 g / L to about 3 g / L before filtering through a pharmaceutical grade nanofilter to remove viruses. Even more preferably, the nanofilter is a DV20 nanofilter (e.g., Pall Corporation; East Hills, NY). For therapeutic uses, substantially pure immunoglobulins of at least about 90%, about 92%, about 94% or about 96% homogeneity are preferred, and about 98 to about 99% or more homogeneity are most preferred.Once purified, partially or to homogeneity as desired, the sterile antibodies can be used therapeutically as described herein.

[0268] In view of the above considerations, the present application is further directed to antibodies obtainable by a process comprising culturing host cells, such as but not limited to mammalian, plant, bacterial, transgenic animal, or transgenic plant cells, transformed with a polynucleotide or vector comprising a nucleic acid molecule encoding an antibody of the present application, such that the nucleic acid is expressed, and optionally recovering the antibody from the culture medium of the host cells.

[0269] In certain aspects, the present application provides hybridoma cell lines and monoclonal antibodies produced by the hybridoma cell lines. The disclosed cell lines have uses other than the production of monoclonal antibodies. For example, the cell lines can be fused with other cells (such as suitably drug-labeled human myeloma, mouse myeloma, human-mouse heteromyeloma or human lymphoblastoid cells) to generate additional hybridomas, thereby providing for the introduction of genes encoding monoclonal antibodies. In addition, the cell lines can be used as a source of nucleic acid encoding anti-HER2 immunoglobulin chains, allowing the isolation and expression of the nucleic acid (e.g., by introduction into other cells using any suitable technique (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter, U.S. Pat. No. 5,225,539)). For example, clones containing rearranged anti-HER2 light or heavy chains can be isolated (e.g., by PCR), or a cDNA library can be prepared from mRNA isolated from cell lines and cDNA clones encoding anti-HER2 immunoglobulin chains can be isolated. Thus, nucleic acids encoding antibody heavy and / or light chains or portions thereof can be obtained and used according to recombinant DNA techniques to produce specific immunoglobulins, immunoglobulin chains, or variants thereof (e.g., humanized immunoglobulins) in various host T cells or in vitro translation systems. For example, nucleic acids containing cDNAs encoding variants such as humanized immunoglobulins or immunoglobulin chains, or derivatives thereof, can be placed in a suitable prokaryotic or eukaryotic vector (e.g., expression vector) and introduced into a suitable host T cell by a suitable method (e.g., transformation, transfection, electroporation, infection). The nucleic acid is thereby operably linked to one or more expression control elements (e.g., in a vector or integrated into the host T cell genome). For production, the host T cells can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide.If desired, the encoded protein can be recovered and / or isolated (e.g., from the host T cells or culture medium). It will be understood that production methods include expression in host T cells of a transgenic animal (see, e.g., WO 92 / 03918, GenPharm International, published March 19, 1992) (incorporated by reference in its entirety).

[0270] Host cells can also be used to produce portions or fragments of intact antibodies, such as Fab fragments or scFv molecules, by conventional techniques. For example, it may be desirable to transfect host cells with DNA encoding either the light or heavy chains of the antibodies of the present application. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to human HER2 and Trop-2. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present application.

[0271] Methods for expressing and / or refolding single chain antibodies into a suitable active form, including single chain antibodies derived from bacteria such as E. coli, have been described and are well known and are applicable to the antibodies disclosed herein (see, e.g., Buchner et al., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275, 1989, and Ward et al., Nature 341:544, 1989, all of which are incorporated herein by reference).

[0272] In many cases, functional heterologous proteins from E. coli or other bacteria need to be isolated from inclusion bodies, solubilized using strong denaturants, and then refolded. In the solubilization step, a reducing agent must be present to break disulfide bonds, as is well known in the art. An exemplary buffer containing a reducing agent is 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of disulfide bonds can occur in the presence of reduced and oxidized low molecular weight thiol reagents, as described in Saxena et al., Biochemistry 9:5015-5021, 1970 (incorporated herein by reference), and in particular in Buchner et al. (supra).

[0273] Renaturation is typically accomplished by diluting (e.g., 100-fold) the denatured and reduced protein into a refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

[0274] As an improvement to the purification protocol of two-chain antibodies, the heavy and light chain regions are solubilized and reduced separately, and then combined in the refolding solution. Exemplary yields are obtained when these two proteins are mixed in a molar ratio such that one protein is not more than 5-fold molar excess over the other. Excess oxidized glutathione or other oxidized low molecular weight compounds can be added to the refolding solution after redox shuffling is completed.

[0275] In addition to recombinant methods, the antibodies, labeled antibodies and antigen-binding fragments thereof disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of polypeptides less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support, followed by sequential addition of the remaining amino acids of the sequence. Techniques for solid phase synthesis are described in Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85: 2149-2156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Longer proteins can be synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods for forming peptide bonds by activation of the carboxyl terminus (such as the use of the coupling reagent N,N'-dicyclohexylcarbodiimide) are well known in the art.

[0276] The following examples are provided to more fully illustrate the present application, but are not to be construed as limiting its scope. As detailed in the specific examples provided below, highly potent bispecific antibodies were generated by combining Herceptin antibody or Herceptin scFv with anti-Trop-2 antibody or anti-Trop-2 scFv. Many different bispecific antibody structures were generated and tested. The unique bispecific antibodies provided exhibit superior biological activity to both monospecific anti-HER2 and monospecific anti-Trop-2 antibodies tested. In many assays, bispecific antibodies also exhibit synergistic activity over monospecific anti-HER2 antibodies. Bispecific anti-HER2 / Trop-2 antibodies have shown superior anti-tumor activity in vitro and in vivo when conjugated with DM1 derivative cytotoxic agents. These data suggest that bispecific antibodies may have therapeutic use, particularly as ADC or ADCC enhancing antibodies, in the treatment of cancers expressing HER2 and / or Trop-2 levels, including patients who are currently unsuitable for treatment with trastuzumab, pertuzumab or T-DM1.In addition, bispecific antibodies may have therapeutic use for the treatment of cancer patients who have failed existing anti-HER2 and / or anti-Trop-2 therapies.

[0277] Further exemplary embodiments 1. A construct comprising a first antigen-binding domain that specifically binds to human HER2 and a second antigen-binding domain that specifically binds to human Trop-2.

[0278] 2. The first antigen-binding domain that specifically binds to human HER2 is a first heavy chain variable region (V H-1 ) and the first light chain variable region (V L-1 ), and / or the second antigen-binding domain that specifically binds to human Trop-2 comprises a second heavy chain variable region (V H-2 ) and a second light chain variable region (V L-2 2. The construct of embodiment 1, comprising:

[0279] 3.a) The above V H-2comprises an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; The above V L-2 comprises an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31; b) The above V H-2 comprises an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; The above V L-2 comprising an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; 3. The construct of embodiment 2.

[0280] 4. The above V H-1 comprises an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The above V L-1 comprising an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; A construct according to embodiment 2 or embodiment 3.

[0281] 5.a) the V, wherein HC-CDR1, HC-CDR2, and HC-CDR3 have the sequences set forth in SEQ ID NO:7; H-1 and LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid sequences set forth in SEQ ID NO: 8, respectively. L-1and / or b1) the V, in which HC-CDR1, HC-CDR2, and HC-CDR3 have the sequences set forth in SEQ ID NO: 4; H-2 and wherein LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid sequences set forth in SEQ ID NO:5. L-2 or b2) the V, in which HC-CDR1, HC-CDR2, and HC-CDR3 have the sequences set forth in SEQ ID NO: 10; H-2 and LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid sequences set forth in SEQ ID NO: 11, respectively. L-2 Each of the amino acid sequences of CDR1, CDR2, and CDR3 is A construct according to any one of embodiments 2 to 4.

[0282] 6. The construct of any one of embodiments 1 to 5, wherein the first antigen-binding domain and / or the second antigen-binding domain is selected from the group consisting of a full-length antibody, Fab, Fab', (Fab')2, Fv, single-chain Fv (scFv) fragment, scFv-scFv, minibody, diabody, or sdAb.

[0283] 7. The construct of embodiment 6, wherein the first antigen-binding domain comprises a full-length antibody comprising an Fc fragment.

[0284] 8. The second antigen-binding domain comprises the V H-2 and V L-2 8. The construct of embodiment 7, comprising an scFv comprising:

[0285] 9. The construct of embodiment 8, wherein the second antigen-binding domain is fused to one or both of the heavy chains of the full-length antibody.

[0286] 10. The construct of embodiment 8, wherein the second antigen-binding domain is fused to one or both of the light chains of the full-length antibody.

[0287] 11. The construct of embodiment 9 or 10, wherein the second antigen-binding domain is fused to the N-terminus of one or both of the heavy or light chains of the full-length antibody.

[0288] 12. The construct of any one of embodiments 9 to 11, wherein the second antigen-binding domain is fused to the C-terminus of one or both of the heavy or light chains of the full-length antibody.

[0289] 13. The construct of any one of embodiments 9 to 12, wherein the second antigen-binding domain is fused to the full-length antibody via a first linker.

[0290] 14. The first antigen-binding domain is H-1 and V L-1 7. The construct of embodiment 6, comprising an scFv comprising:

[0291] 15. The construct of embodiment 14, wherein the second antigen-binding domain comprises a full-length antibody comprising an Fc fragment.

[0292] 16. The construct of embodiment 15, wherein the first antigen-binding domain is fused to one or both of the heavy chains of the full-length antibody.

[0293] 17. The construct of embodiment 15, wherein the first antigen-binding domain is fused to one or both of the light chains of the full-length antibody.

[0294] 18. The construct of embodiment 16 or 17, wherein the first antigen-binding domain is fused to the N-terminus of one or both of the heavy or light chains of the full-length antibody.

[0295] 19. The construct of any one of embodiments 16 to 18, wherein the first antigen-binding domain is fused to the C-terminus of one or both of the heavy or light chains of the full-length antibody.

[0296] 20. The construct of any one of embodiments 16 to 19, wherein the second antigen-binding domain is fused to the full-length antibody via a first linker.

[0297] 21. The construct of embodiment 13 or embodiment 20, wherein the first linker is a GS linker selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54.

[0298] 22. The V of the scFv in the first antigen-binding domain H-1 and V L-1 or the V of the scFv in the second antigen-binding domain H-2 and V L-2 are linked to each other via a second linker.

[0299] 23. The construct of embodiment 22, wherein the second linker is a GS linker selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54.

[0300] 24. a) the first antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; H-1 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. L-1 and the second antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28. H-2and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31. L-2 and wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody, or b) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; H-1 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. L-1 and a full-length antibody comprising an Fc fragment, the second antigen-binding domain comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28. H-2 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31. L-2 and wherein the second antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody; or c) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; H-1 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. L-1and a full-length antibody comprising an Fc fragment, the second antigen-binding domain comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. H-2 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43. L-2 and wherein the second antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or d) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; H-1 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. L-1 and the second antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. H-2 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43. L-2 and wherein the first antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or e) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34;H-1 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37. L-1 and the second antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. H-2 and 2) an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43. L-2 and a full-length antibody comprising an Fc fragment, wherein the first antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody. A construct according to any one of embodiments 2 to 23.

[0301] 25.a) The above V H-1 7, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, L-1 comprises an amino acid sequence of SEQ ID NO:8, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; b) The above V H-2 4, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, L-2comprising an amino acid sequence of SEQ ID NO:5, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; A construct according to any one of embodiments 2 to 24.

[0302] 26.a) The above V H-1 7, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, L-1 comprises an amino acid sequence of SEQ ID NO:8, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; b) The above V H-2 10, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, L-2 comprising an amino acid sequence of SEQ ID NO: 11, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto; A construct according to any one of embodiments 2 to 24.

[0303] 27. a) the first antigen-binding domain comprises 1) the V comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; H-1 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. L-1 and wherein the second antigen-binding domain comprises an scFv comprising: 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO:4. H-2 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:5. L-2and wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody, b) the first antigen-binding domain comprises 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; H-1 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. L-1 and wherein the second antigen-binding domain comprises a full-length antibody comprising an Fc fragment, the second antigen-binding domain comprising: 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO:4. H-2 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:5. L-2 and wherein the second antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody; or c) the first antigen-binding domain comprises 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; H-1 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. L-1 and wherein the second antigen-binding domain comprises a full-length antibody comprising an Fc fragment, the second antigen-binding domain comprising: 1) the V domain comprising an amino acid sequence set forth in SEQ ID NO: 10 or 58. H-2 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:11. L-2 and wherein the second antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or d) the first antigen-binding domain comprises 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; H-1 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. L-1 and wherein the second antigen-binding domain comprises an scFv comprising: 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58. H-2 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:11. L-2 and wherein the first antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or e) the first antigen-binding domain comprises 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; H-1 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. L-1 and wherein the second antigen-binding domain comprises an scFv comprising: 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58. H-2 and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:11. L-2 and a full-length antibody comprising an Fc fragment, wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. A construct according to any one of embodiments 2 to 24.

[0304] 28. The construct according to any one of embodiments 7 to 13 and 15 to 27, wherein the Fc fragment comprises a heavy chain constant domain optionally comprising an IgG constant domain, and a light chain constant domain optionally comprising a kappa constant region or a lambda constant region.

[0305] 29.a) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 14, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO: 15, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; b) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; c) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; d) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO:20, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO:21, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; e) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO:22, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO:23, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; f) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 60, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; g) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 62, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; h) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO:59, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; two light chains comprising the amino acid sequence set forth in SEQ ID NO:17, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; or i) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 61, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 17, or a variant comprising an amino acid sequence having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; A construct according to any one of embodiments 1 to 28.

[0306] 30. A construct according to any one of the preceding embodiments, wherein the construct specifically binds to HER2 and / or Trop-2 expressing cells and induces internalization upon binding to the target(s).

[0307] 31. A construct according to any one of the preceding claims, wherein the construct is or comprises a bispecific antibody or a binding fragment thereof, optionally wherein the bispecific antibody is a humanized antibody.

[0308] 32. The construct of embodiment 31, wherein the construct comprises an antibody drug conjugate (ADC) having the formula: Ab-(LD)n, where Ab is the bispecific antibody or binding fragment thereof, L is a linker or a direct bond, D is a therapeutic agent, and n is an integer from 1 to 10.

[0309] 33. The construct according to embodiment 32, wherein the therapeutic agent is a cytotoxic agent.

[0310] 34. The construct of embodiment 33, wherein the cytotoxic agent is monomethylauristatin E (MMAE) or B1-P203.

[0311] 35. The construct of embodiment 33, wherein the cytotoxic agent is maytansinoid (DM1) or a derivative thereof.

[0312] 36. A construct that specifically binds to human Trop2 and human HER2, which binds to human HER2 and / or Trop2 competitively with a construct according to any one of embodiments 2 to 35.

[0313] 37. A pharmaceutical composition comprising a construct according to any one of embodiments 1 to 36 and a pharma- ceutically acceptable carrier.

[0314] 38. An isolated nucleic acid encoding a construct according to any one of embodiments 1 to 31 and 36.

[0315] 39. A vector comprising the isolated nucleic acid of embodiment 38.

[0316] 40. An isolated host cell comprising the isolated nucleic acid of embodiment 38 or the vector of embodiment 39.

[0317] 41. A method for producing a construct, comprising: a) culturing the isolated host cell of embodiment 40 under conditions effective to express said construct or a portion thereof; b) obtaining said expressed construct or a portion thereof from said host cell; The method comprising:

[0318] 42. A method for treating a disease or condition in a subject, comprising administering to the subject a construct according to any one of embodiments 1 to 36 or a pharmaceutical composition according to embodiment 37.

[0319] 43. The method of embodiment 42, wherein the disease or condition is cancer.

[0320] 44. The method of embodiment 43, wherein the cancer is Trop2 positive and / or HER2 positive.

[0321] 45. The method of embodiment 44, wherein the cancer is Trop2 intermediate or high and / or HER2 intermediate or high.

[0322] 46. ​​The method of embodiment 43, wherein the cancer is Trop2 underexpressing and / or HER2 underexpressing.

[0323] 47. The method of any one of embodiments 43-46, wherein the cancer is selected from the group consisting of prostate cancer, breast cancer, colon cancer, pancreatic cancer, and gastric cancer.

[0324] 48. The method of any one of embodiments 41 to 47, wherein the method further comprises a second therapy, optionally selected from the group consisting of immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, vaccination protocols, and stem cell transplantation. EXAMPLES

[0325] Example 1 Construction of bispecific antibodies Schematic diagrams of the anti-HER2 / Trop-2 bispecific antibody formats described in this application are shown in Figures 1-5. These constructs are designed to simultaneously target the HER2 and Trop-2 antigens. The scFv portion in all antibody formats is, for example, H The domains were constructed by linking them using a linker (e.g., a synthetic flexible linker, e.g., a GS linker, e.g., the linker sequence set forth in SEQ ID NO: 25). The scFv is covalently linked to the N-terminus of the heavy chain and the C-terminus of the CH3 domain, e.g., via a flexible linker, e.g., a GS linker, e.g., the linker sequence set forth in SEQ ID NO: 25.

[0326] To generate the bispecific antibody HT2 (Figure 1), the anti-HER2 antibody Herceptin scFv (SEQ ID NO: 3), the anti-Trop-2 antibody A1,2X4 V H (SEQ ID NO: 4), and a heavy chain (HC) containing an IgG1 constant (CH) region were synthesized. H were linked via a GS linker. H The -CH fusion was cloned into the pUC57 expression vector. Anti-Trop-2 antibody A1,2X4 V L A light chain (LC) containing the heavy chain (HC) (SEQ ID NO: 5) and CL regions was also synthesized and cloned into a pUC57 expression vector. The HC and LC were then co-transfected into the expression vector. The resulting bispecific antibody was composed of the scFv Herceptin and the Fab anti-Trop2 antibody A1,2X4, in which the Herceptin scFv was linked to the VK-linker-V when fused to the N-terminus of both heavy chains of the anti-Trop-2 antibody. H It is oriented as.

[0327] To generate the bispecific antibody TH2 (Figure 2), the anti-Trop-2 antibody A1,2X4 scFv (SEQ ID NO: 6) and the anti-HER2 antibody Herceptin V were used. H (SEQ ID NO: 7) and HC containing the IgG1 CH region were synthesized. Hwere linked via a GS linker. H The -CH fusion was cloned into the pUC57 expression vector. L A LC containing the HC (SEQ ID NO: 8) and CL (SEQ ID NO: 13) regions was also synthesized and cloned into a pUC57 expression vector. The HC and LC were then co-transfected into the expression vector. The resulting bispecific antibody was composed of the scFv anti-Trop-2 antibody A1,2X4 and the Fab Herceptin, where the anti-Trop-2 scFv was fused to the N-terminus of both heavy chains of Herceptin via the VK-linker-V H It is oriented as.

[0328] To generate the bispecific antibody HB1 (Figure 3), we used the anti-HER2 antibody Herceptin V. H (SEQ ID NO: 7) and HC containing the IgG1 CH region and the anti-Trop-2 antibody hRS7 scFv (SEQ ID NO: 9) were synthesized. H and scFv were linked via a GS linker. H The -CH-scFv fusion was cloned into the pUC57 expression vector. L (SEQ ID NO: 8) and LC containing the CL region were also synthesized and cloned into pUC57 expression vector. HC and LC were then co-transfected into the expression vector. The resulting bispecific antibody is composed of the Fab Herceptin and the scFv anti-Trop-2 antibody hRS7, with the anti-Trop-2 scFv oriented as VK-linker-CH when fused to the C-terminus of both heavy chains of Herceptin.

[0329] To generate the bispecific antibody BH1 (FIG. 4), a HC containing the anti-HER2 antibody Herceptin scFv (SEQ ID NO: 9) and an IgG1 constant region was synthesized. H were linked via a GS linker. H The -CH fusion was cloned into the pUC57 expression vector. LThe LC, containing the HC (SEQ ID NO: 11) and CL regions, was also synthesized and cloned into the pUC57 expression vector. The HC and LC were then co-transfected into the expression vector. The resulting bispecific antibody was composed of the scFv Herceptin and the Fab anti-Trop-2 antibody hRS7, in which the anti-Herceptin scFv was linked to the VK-linker-V when fused to the C-terminus of both heavy chains of the hRS7 antibody. H It is oriented as.

[0330] To generate the bispecific antibody BH2 (Figure 5), the anti-HER2 antibody Herceptin scFv (SEQ ID NO: 3) and the anti-Trop-2 antibody hRS7 V were H (SEQ ID NO: 10) and a heavy chain (HC) containing the IgG1 CH region. H were linked via a GS linker. H The -CH fusion was cloned into the pUC57 expression vector. L The LC, containing the HC (SEQ ID NO: 11) and CL regions, was also synthesized and cloned into a pUC57 expression vector. The HC and LC were then co-transfected into the expression vector. The resulting bispecific antibody was composed of the scFv Herceptin and the Fab anti-Trop2 antibody hRS7, in which the Herceptin scFv was linked to the VK-linker-V when fused to the N-terminus of both heavy chains of the hRS7 antibody. H It is oriented as.

[0331] All antibody constructs were transiently expressed in HEK293F cells, with expression levels of 1.08 mg / mL for HT2 (HT002), 0.55 mg / mL for TH2 (TH002), and 0.695 mg / mL for HB1 (HT001a), with the expression level of HT2 (HT002) being particularly favorable.

[0332] All bispecific antibodies were purified to homogeneity (>90%) by standard Protein A purification and analyzed by reducing and non-reducing SDS-PAGE.

[0333] Example 2 Bispecific antibody binding affinity The binding affinity of the purified anti-HER2 / Trop-2 bispecific antibody was determined by FACS analysis. Prostate cancer cells LNCap, which express HER2 but not Trop-2, breast cancer cells MDA-MB-468, which express Trop-2 but not HER2, and colon cancer cells Colo205, which express both HER2 and Trop-2, were harvested and diluted in FACS buffer at 0.5–1 × 10 6 The cells were then suspended at 1.25, 2.5, 5, 10, 20, and 40 g / ml of anti-HER2 antibody Herceptin, anti-Trop-2 antibody A1, 2X4 bispecific anti-HER2 / Trop-2 antibodies HT2 (HT002) and TH2 (TH001) were added to the cells and incubated at 4°C for 30 minutes. After incubation, the cells were washed to remove unbound antibodies. The cells were then dissociated and stained with fluorescently labeled secondary antibodies at 20 g / ml for 30 minutes on ice in the dark. After incubation, the cells were washed and resuspended in staining buffer before analysis using a Beckman flow cytometry system (BD Accuri C6). Mean fluorescence intensity (MFI) was analyzed by Beckman flow cytometry software. The equilibrium binding constant Kd was obtained using the "one site-specific binding" analysis model in GraphPad Prism7. Kd is the concentration of radioligand required to achieve half-maximal binding at equilibrium.

[0334] Figure 6B shows the results for the bispecific anti-HER2 / Trop-2 antibody HB1. In LNCap cells, which express only Her2 on the cell surface, and in MDA-MB-468 cells, which express only Trop-2, HB1 showed less binding compared to the parental monospecific antibodies Herceptin or hRS7. In Colo205 cells, which express both HER2 and Trop-2, HB1 had comparable binding affinity compared to the parental antibodies.

[0335] FIG. 6C shows results for bispecific anti-HER2 / Trop-2 antibodies BH1 (TH001a) and BH2 (TH001b). In LNCap cells, which express only Her2 on the cell surface, bispecific antibody TH001b had similar binding affinity to the parental monospecific antibody Herceptin, while bispecific antibody TH001a had intermediate binding affinity compared to Herceptin. In MDA-MB-468 cells, which express only Trop-2 on the cell surface, TH001a showed better binding than TH001b and comparable to the parental monospecific antibody A1,2X4. In Colo205 cells, which express both antigens, the bispecific antibodies showed binding equal to or better than the parental monospecific antibodies.

[0336] FIG. 6A shows the results for the bispecific anti-HER2 / Trop-2 antibodies HT2 (HT002) and TH2 (TH002). In LNCap cells, which express only Her2 on the cell surface, HT002 and TH002 bound to the cells with similar binding affinity as the parent monospecific antibody Herceptin. However, to MDA-MB-468 cells, which express only Trop-2, both bispecific antibodies had intermediate binding affinity compared to the monospecific antibody A1,2X4. Impressively, in Colo205 cells, which express both antigens, the amount of HT002 and TH002 bispecific antibodies detected bound on the cell surface was dramatically increased compared to either monospecific antibody alone. Such an effect provides a special advantage for the treatment of patients with both Trop2- and HER2-positive cancer cells.

[0337] Example 3 Bispecific antibody internalization The internalization and degradation of the bispecific anti-HER2 / Trop-2 antibody was measured by flow cytometry, in which a fluorescently labeled secondary antibody was used to detect remaining primary antibody after internalization.

[0338] HER2 low expression / Trop-2 low expression colon cancer cell Colo205, HER2 negative / Trop-2 high expression breast cancer cell MDA-MB-468, and HER2 high expression / Trop-2 intermediate expression ovarian cancer cell SK-BR-3 were cultured at 1 × 10 5 Cells were seeded with 1000 cells / well. Then, either the bispecific antibody or the parental monospecific antibody was added to the cells at a concentration of 400, 1000, 3000, or 10000ng / ml, respectively, in FACS buffer and incubated at 4°C for 30 minutes. After incubation, the cells were washed three times with FACS buffer to remove unbound antibody. The cells were then incubated at 37°C, 5% CO2 for antibody internalization. After 2 hours, the cells were dissociated with trypsin and stained with a fluorescently labeled goat anti-human IgG secondary antibody for 30 minutes on ice in the dark, and then analyzed using a Beckman flow cytometry system. The fluorescence intensity and cell binding percentage at each time point were analyzed by Beckman flow cytometry software. The amount of antibody internalized into the cells at each concentration was calculated using the following formula: (MFI 4℃ -MFI 37℃ ) x 10 3 was determined using

[0339] Figure 7 shows the results of the study. The results show that the bispecific anti-HER2 / Trop-2 antibodies HT2 (HT002), BH1 (TH001a), and BH2 (TH001b) of the present application can be internalized by cells expressing HER2 and / or Trop-2. The amount of internalization of the bispecific antibodies was similar to that of the parental anti-Trop-2 antibodies A1, 2X4 or hRS7 and the anti-HER2 antibody Herceptin in MDA-MB-468 cells, which express Trop-2 but no HER2 levels, and in Colo205 cells, which have low Her2 and low Trop-2 expression. In SK-BR-3 cells, which express both HER2 and Trop-2 at moderate or high levels, the bispecific antibodies HT2 and BH2 induced increased internalization over the parental monospecific antibodies, while BH1 had similar internalization compared to the monospecific antibodies. The amount of internalization of HT2 and BH2 was greater than that of the monospecific antibodies Herceptin and A1,2X4 combined. These results indicate the particular advantage of using HT2 or BH2 ​​for the treatment of patients whose cancer cells express intermediate or high levels of HER2 and / or Trops.

[0340] Example 4 Creation of bispecific antibody drug conjugates (ADCs) The constructs described herein, including anti-HER2 / Trop-2 bispecific antibodies, including HT2 (HT002) and HB1 (HT001a), conjugated to BI-P203, DM1, or MMAE to form ADCs, were evaluated for their ability to inhibit the growth of multiple cancer cell lines expressing different levels of HER2 and Trop-2.

[0341] Some embodiments of the present application relate to BI-P203 or DM1 or MMAE linked via a non-cleavable linker to a bispecific antibody. In this example, bispecific anti-HER2 / Trop-2 antibodies HT2 (HT002) or HB1 (HT001a) were reduced by adding the antibody to TCEP (tris(2-carboxyethyl)phosphine) dissolved in pH-adjusted PBS EDTA buffer. The antibody / TCEP solution was incubated at 37°C for 2-3 hours. The reduced antibody was buffer exchanged into the conjugation reaction buffer. To the solution of reduced anti-Trop-2 monoclonal antibody, BI-P203, DM1, or MMAE payload dissolved in DMSO was added at a payload / antibody ratio of 7-30:1 to achieve different drug-antibody ratios (DAR). The payload / antibody solution was incubated at 20°C for 1-2 hours during which the reduced antibody was conjugated to the BI-P203 payload via the maleimide group. Upon completion of conjugation, the reaction mixture was desalted and concentrated to obtain anti-HER2 / Trop-2-BI-P203 ADC. The biochemical properties of the resulting ADC were characterized using size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to determine purity and amount of aggregation, and hydrophobic interaction chromatography-HPLC (HIC-HPLC) to confirm drug loading (DAR). The conjugation procedure is shown in Figure 8. The final ADC product contains 4 or 6 or 7 BI-P203 and DM1-linker molecules and 2 or 4 MMAE-linker molecules. The cysteine ​​conjugation method used in the conjugation process results in a more homogenous ADC compared to the lysine conjugation method.

[0342] Example 5 In vitro activity of bispecific ADCs The ADCs of the present application containing a cytotoxic payload can be used to kill target cells, such as killing cancer cells. The HT2-BI-P203, HT2-MMAE, and HB1-DM1 ADCs were tested in an in vitro cytotoxicity assay. Naked HT2 antibody was also included in the assay. In the in vitro assay of HT2-BI-P203, BxPC-3 pancreatic cancer cells, N87 gastric cancer cells, MDA-MB-468 breast cancer cells, and A549 lung cancer cells were used. BxPC-3 cells had low HER2 and high Trop-2 expression, NCI-N87 cells had high HER2 and moderate Trop-2 expression, MDA-MB-468 cells did not express HER2 but expressed moderate levels of Trop-2, and A549 cells had undetectable levels of HER2 and Trop-2. All cell lines were cultured in appropriate culture media at 37°C in a humidified incubator atmosphere of 5% CO2. Cells were plated in 96-well flat-bottom plates. Cell seeding numbers ranged from 500 cells / 100ul / well to 6,000 cells / 100μl / well. Cells were allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. HT2-BI-P203 ADC or HT2 antibody were prepared from stock solutions and diluted to appropriate working concentrations 24 hours after cell seeding. Seven 10-fold serial dilutions were performed using culture media. Final concentrations ranged from 1000nM to 0.001nM. Cells were incubated with ADC for 72 hours. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, Lot No. PL701) was added to the wells for 1-4 hours at 37°C, and the absorbance at 450 nm was measured using a Microplate Reader (SpectraMax M5, Molecular Devices) and SoftMax Pro5.4.1 software. Percent inhibition was calculated by the following formula: (mean absorbance of tested samples / mean absorbance of control samples) x 100. For all cell assays, dose-response curves were generated using three-parameter curve fitting in GraphPad Prism 7.

[0343] Table 2 summarizes the expression levels of HER2 and Trop-2 in BxPC-3, NCI-N87, MDA-MB-468, and A549 cells, as well as the IC50 of each test substance. The killing curves are shown in Figure 9A. The results show that the anti-HER2 / Trop-2 bispecific antibody HT2-BI-P203 effectively kills all cancer cells expressing different levels of HER2 and Trop-2. In HER2- and Trop-2-negative A549 cells, IC50 could not be calculated for all HT2-BI-P203, indicating that specific killing activity was not achieved. Naked HT2 antibody had no specific killing activity in all cell lines tested. [Table 2]

[0344] In vitro assays of HT2-MMAE used MDA-MB-468 breast cancer cells, LNCaP prostate cancer cells, SK-BR-3 ovarian cancer cells, and A549 lung cancer cells. HT2(HT002)-MMAE was tested head-to-head with the parental monospecific antibody Herceptin ADC and the A1,2X4-MMAE ADC. In vitro cytotoxicity assays were performed according to the procedures described above.

[0345] Table 3 summarizes the expression levels of HER2 and Trop-2 in MDA-MB-468, SK-BR-3, and A549 cells, as well as the IC50 of each test substance. Killing curves are shown in Figure 9B. In MDA-MB-468 cells expressing only Trop-2, Herceptin-MMAE showed no specific killing, and HT002-MMAE had weak cytotoxicity compared to A1,2X4-MMAE. All three ADCs were potent against SK-BR-3 cells expressing both HER2 and Trop-2.

[0346] The results show that the anti-HER2 / Trop-2 bispecific antibody HT2-BI-P203 effectively kills all cancer cells expressing different levels of HER2 and Trop-2. In HER2 and Trop-2 negative A549 cells, IC50 could not be calculated for all HT2-BI-P203, indicating that specific killing activity was not achieved. Naked HT2 antibody had no specific killing activity in all cell lines tested. In HER2 and Trop-2 negative A549 cells, IC50 was above 200 nM or could not be calculated for all three ADCs, indicating that specific killing activity was not achieved. [Table 3]

[0347] In vitro assays of HB1-DM1 were performed using BxPC-3 pancreatic cancer cells, MDA-MB-468 breast cancer cells, LNCaP prostate cancer cells, and SK-BR-3 ovarian cancer cells. MDA-MB-468 cells express only Trop-2, LnCaP cells express only HER2, and SK-BR-3 cells have both HER2 and Trop-2 expression. In vitro cytotoxicity assays were performed according to the procedure described above.

[0348] Table 4 summarizes the IC50 of HB1-DM1 in each cell line. Killing curves are shown in Figure 9C. The HB1-DM1 ADC was most potent against SK-BR-3 cells, resulting in an IC50 value of less than 1 nM. [Table 4]

[0349] Example 6 In vivo activity of bispecific ADCs The anti-tumor activity of the anti-HER2 / Trop-2 bispecific antibody HT002-BI-P203 ADC was evaluated in mouse subjects using Trop-2 positive / Her2 negative MDA-MB-468 and Trop-2 positive / HER2 positive NCI-N87 xenograft models. Five million BxPC3 or NCI-N87 cells were harvested from culture flasks and implanted subcutaneously into the right flank of 6-7 week old BALB / c nude mice. Tumors were measured twice weekly throughout the experimental period and tumor volumes were calculated according to the following formula: tumor volume (mm 3 )=(height x width 2 ) / 2.

[0350] The ADCs tested in the BxPC3 model were conjugated with different DARs: A1,2X4-BI-P203 had a DAR of 7.2, T-DM1 had a DAR of 3.2, and HT002-BI-P203 had a DAR of 5.2. The ADCs were administered intravenously as a single bolus dose.

[0351] Figure 10A shows the results of an in vivo BxPC-3 xenograft study. As shown, a single dose of 3 mg / kg of the bispecific ADC of the present application induced sustained disease stabilization, while a 3 mg / kg dose of the monospecific antibody Trop-2 ADC or T-DM1 resulted in inhibition of tumor growth. As shown in Figure 10B, there was no significant change in body weight with all ADC treatments compared to the vehicle control and the IgG isotype ADC control.

[0352] To further evaluate the antitumor activity of the bispecific ADC, HT002-BI-P203 was tested at two dose levels, 1.5 mg / kg and 5 mg / kg, in the N87 xenograft model. The ADC was administered intravenously as a single bolus dose.

[0353] Figure 11A shows the results of an in vivo N87 xenograft study. The results show that the bispecific ADC was able to achieve sustained disease stabilization at a single dose of 5 mg / kg. As shown in Figure 11B, there was no significant change in body weight with all ADC treatments compared to the vehicle control and the IgG isotype ADC control.

[0354] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the present application have been described in terms of preferred embodiments, it will be apparent to one skilled in the art that modifications can be made to the articles and methods without departing from the spirit and scope of the present application. All such modifications and equivalents, whether now existing or later developed, apparent to those skilled in the art are deemed to be within the spirit and scope of the present application as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of skill in the art to which this application pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The present application illustratively described herein may be suitably practiced in the absence of any element or elements not specifically disclosed herein. Thus, while the present application has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be utilized by those skilled in the art, and such modifications and variations are deemed to be within the scope of the present application as defined by the appended claims.

[0355] Example 7 HT002-MMAE (DAR3.5) was tested against T-DM1 in the BxPC-3 xenograft model. The unconjugated bispecific antibody HT002 was also included. Test articles were administered intravenously on days 0 and 7.

[0356] Figure 12 shows the results of an in vivo BxPC-3 xenograft study. As shown, administration of the bispecific ADC HT002 of the present application at 5 mg / kg resulted in tumor regression that persisted until the end of the study (day 43), and T-DM1 at a dose of 5 mg / kg induced tumor growth inhibition. Tumor growth inhibition was also observed with administration of the unconjugated bispecific antibody HT002.

[0357] Example 8 The bispecific antibody ADC HT002-MMAE was tested in an in vitro cytotoxicity assay in JIMT-1 breast cancer cells along with the parent antibody ADCs A1,2X4-MMAE and Herceptin-MMAE. Unconjugated HT002 antibody and Herceptin were also included in the assay. JIMT-1 cells were derived from a patient with Herceptin-resistant breast cancer and express moderate to high levels of Trop-2 and HER2. Killing curves are shown in Figure 13. The results show that the bispecific antibody ADC HT002-MMAE effectively inhibited JIMT-1 cell growth, with an IC50 value of 0.4301 nM, similar to the two parent antibody ADCs, which had IC50 values ​​of 0.3874 nM and 0.5043 nM, respectively. The unconjugated bispecific antibody HT002 alone was able to inhibit JIMT-1 cell growth with an IC50 value of 0.1853 nM. As expected, Herceptin had minimal antiproliferative activity in this cell line. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

Table 5-7

Table 5-8

Table 5-9

Claims

1. A construct comprising a first antigen-binding domain that specifically binds to human HER2 and a second antigen-binding domain that specifically binds to human Trop-2.

2. The first antigen-binding domain that specifically binds to human HER2 comprises a first heavy chain variable region (V Ｈ－１ ) and a first light chain variable region (V Ｌ－１ ), and / or the second antigen-binding domain that specifically binds human Trop-2 comprises a second heavy chain variable region (V Ｈ－２ ) and a second light chain variable region (V Ｌ－２ 2. The construct of claim 1 , comprising:

3. a) the V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28; The V Ｌ－２ comprises an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:31; or b) the V Ｈ－２ comprises an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:40; The V Ｌ－２ comprises an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; The construct of claim 2.

4. The V Ｈ－１ comprises an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The V Ｌ－１ comprises an LC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37; A construct according to claim 2 or claim 3.

5. a) the VH-CDR1, HC-CDR2, and HC-CDR3 have the sequences set forth in SEQ ID NO:7; Ｈ－１ and LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid sequences set forth in SEQ ID NO:

8. Ｌ－１ and / or b1) the V, wherein HC-CDR1, HC-CDR2, and HC-CDR3 have the sequence set forth in SEQ ID NO:4; Ｈ－２ and LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid sequences set forth in SEQ ID NO:

5. Ｌ－２ or b2) the V, wherein HC-CDR1, HC-CDR2, and HC-CDR3 have the sequences set forth in SEQ ID NO: 10; Ｈ－２ and LC-CDR1, LC-CDR2, and LC-CDR3 have the amino acid sequences set forth in SEQ ID NO:

11. Ｌ－２ and each of the CDR1, CDR2, and CDR3 amino acid sequences of A construct according to any one of claims 2 to 4.

6. The construct of any one of claims 1 to 5, wherein the first antigen-binding domain and / or the second antigen-binding domain is selected from the group consisting of a full-length antibody, a Fab, a Fab', a (Fab')2, an Fv, a single-chain Fv (scFv) fragment, a scFv-scFv, a minibody, a diabody, or an sdAb.

7. The construct of claim 6 , wherein the first antigen-binding domain comprises a full-length antibody comprising an Fc fragment.

8. The second antigen-binding domain is Ｈ－２ and V Ｌ－２ The construct of claim 7, comprising an scFv comprising:

9. The construct of claim 8 , wherein the second antigen-binding domain is fused to one or both of the heavy chains of the full-length antibody.

10. The construct of claim 8 , wherein the second antigen-binding domain is fused to one or both of the light chains of the full-length antibody.

11. 11. The construct of any one of claims 1 to 10, wherein the second antigen-binding domain is fused to the full-length antibody via a first linker, optionally wherein the first linker is a GS linker selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54.

12. The V of the scFv in the first antigen-binding domain Ｈ－１ and V Ｌ－１ or the V of the scFv in the second antigen-binding domain Ｈ－２ and V Ｌ－２ are linked to each other via a second linker, said second linker being a GS linker selected from the group consisting of SEQ ID NOs: 24, 25, and 44-54.

13. a) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Ｈ－１ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

37. Ｌ－１ and the second antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

28. Ｈ－２ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO:29, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO:30, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

31. Ｌ－２ and wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody, b) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Ｈ－１ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

37. Ｌ－１ and a full-length antibody comprising an Fc fragment, the second antigen-binding domain comprising: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:26, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:27, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

28. Ｈ－２ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO:29, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO:30, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

31. Ｌ－２ and wherein the second antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody; or c) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Ｈ－１ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

37. Ｌ－１ and wherein the second antigen-binding domain comprises: 1) a HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, a HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

40. Ｈ－２ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

43. Ｌ－２ and wherein the second antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or d) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Ｈ－１ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

37. Ｌ－１ and the second antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

40. Ｈ－２ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

43. Ｌ－２ and wherein the first antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or e) the first antigen-binding domain comprises: 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Ｈ－１ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 35, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

37. Ｌ－１ and the second antigen-binding domain comprises 1) an HC-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HC-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and an HC-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

40. Ｈ－２ and 2) the V comprising an LC-CDR1 having the amino acid sequence set forth in SEQ ID NO: 41, an LC-CDR2 having the amino acid sequence set forth in SEQ ID NO: 42, and an LC-CDR3 having the amino acid sequence set forth in SEQ ID NO:

43. Ｌ－２ and a full-length antibody comprising an Fc fragment, wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. A construct according to any one of claims 2 to 12.

14. a) the V Ｈ－１ 7, or a variant thereof having an amino acid sequence with at least about 80% sequence identity, Ｌ－１ comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having an amino acid sequence with at least about 80% sequence identity; b) the V Ｈ－２ comprises the amino acid sequence of SEQ ID NO: 4, or a variant thereof having an amino acid sequence having at least about 80% sequence identity, Ｌ－２ comprises the amino acid sequence of SEQ ID NO:5, or a variant having an amino acid sequence having at least about 80% sequence identity; A construct according to any one of claims 2 to 13.

15. a) the V Ｈ－１ 7, or a variant thereof having an amino acid sequence with at least about 80% sequence identity, Ｌ－１ comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having an amino acid sequence with at least about 80% sequence identity; b) the V Ｈ－２ comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having an amino acid sequence with at least about 80% sequence identity, Ｌ－２ comprises the amino acid sequence of SEQ ID NO: 11, or a variant having an amino acid sequence having at least about 80% sequence identity; A construct according to any one of claims 2 to 14.

16. a) the first antigen-binding domain comprises 1) the V Ｈ－１ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. Ｌ－１ and wherein the second antigen-binding domain comprises: 1) the V comprising the amino acid sequence set forth in SEQ ID NO:

4. Ｈ－２ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:

5. Ｌ－２ and wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody, b) the first antigen-binding domain comprises 1) the V Ｈ－１ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. Ｌ－１ and wherein the second antigen-binding domain comprises a full-length antibody comprising an Fc fragment, the second antigen-binding domain comprising: 1) the V domain comprising the amino acid sequence set forth in SEQ ID NO:

4. Ｈ－２ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:

5. Ｌ－２ and wherein the second antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody; or c) the first antigen-binding domain comprises 1) the V comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; Ｈ－１ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. Ｌ－１ and wherein the second antigen-binding domain comprises a full-length antibody comprising an Fc fragment comprising: 1) the V domain comprising an amino acid sequence set forth in SEQ ID NO: 10 or 58. Ｈ－２ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:

11. Ｌ－２ and wherein the second antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or d) the first antigen-binding domain comprises 1) the V comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; Ｈ－１ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. Ｌ－１ and the second antigen-binding domain comprises: 1) an scFv comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58; Ｈ－２ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:

11. Ｌ－２ and wherein the first antigen-binding domain is fused to the C-terminus of both heavy chains of the full-length antibody; or e) the first antigen-binding domain comprises 1) the V comprising the amino acid sequence set forth in SEQ ID NO: 7, 55, or 56; Ｈ－１ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO: 8 or 57. Ｌ－１ and the second antigen-binding domain comprises: 1) an scFv comprising the amino acid sequence set forth in SEQ ID NO: 10 or 58; Ｈ－２ and 2) the V comprising the amino acid sequence set forth in SEQ ID NO:

11. Ｌ－２ and a full-length antibody comprising an Fc fragment, wherein the first antigen-binding domain is fused to the N-terminus of both heavy chains of the full-length antibody. A construct according to any one of claims 2 to 15.

17. a) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 14 or a variant comprising an amino acid sequence having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant comprising an amino acid sequence having at least about 80% sequence identity; b) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having at least about 80% sequence identity; c) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 18 or a variant comprising an amino acid sequence having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 19 or a variant comprising an amino acid sequence having at least about 80% sequence identity; d) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 20 or a variant comprising an amino acid sequence having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 21 or a variant comprising an amino acid sequence having at least about 80% sequence identity; e) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 22 or a variant comprising an amino acid sequence having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 23 or a variant comprising an amino acid sequence having at least about 80% sequence identity; f) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 60 or a variant thereof having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having at least about 80% sequence identity; g) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 62 or a variant thereof having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof having at least about 80% sequence identity; h) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO:59 or a variant comprising an amino acid sequence having at least about 80% sequence identity, two light chains comprising the amino acid sequence set forth in SEQ ID NO:17 or a variant comprising an amino acid sequence having at least about 80% sequence identity, or i) two heavy chains comprising the amino acid sequence set forth in SEQ ID NO: 61 or a variant comprising an amino acid sequence having at least about 80% sequence identity, and two light chains comprising the amino acid sequence set forth in SEQ ID NO: 17 or a variant comprising an amino acid sequence having at least about 80% sequence identity. The construct according to any one of claims 1 to 16, comprising:

18. 18. The construct of any one of claims 1 to 17, wherein the construct comprises an antibody drug conjugate (ADC) having the formula: Ab-(L-D)n, where Ab is the bispecific antibody or binding fragment thereof, L is a linker or a direct bond, D is a therapeutic agent, and n is an integer from 1 to 10.

19. The construct of claim 18 , wherein the therapeutic agent is a cytotoxic agent.

20. A construct that specifically binds to human Trop2 and human HER2, said construct binding to human HER2 and / or Trop2 competitively with the construct according to any one of claims 2 to 19.

21. A pharmaceutical composition comprising the construct according to any one of claims 1 to 20 and a pharma- ceutically acceptable carrier.

22. An isolated nucleic acid encoding a construct according to any one of claims 1 to 20.

23. A vector comprising the isolated nucleic acid of claim 22.

24. 24. An isolated host cell comprising the isolated nucleic acid of claim 22 or the vector of claim 23.

25. 1. A method for producing a construct comprising the steps of: a) culturing the isolated host cell of claim 24 under conditions effective to express the construct or a portion thereof; b) obtaining the expressed construct or a portion thereof from the host cell; and The method comprising:

26. A composition comprising a construct according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 for use in a method of treating a disease or condition in a subject.

27. 27. The composition or pharmaceutical composition of claim 26, wherein the disease or condition is cancer.

28. 28. The composition or pharmaceutical composition of claim 27, wherein the cancer is Trop2 positive and / or HER2 positive.

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