Trophoblast cell surface antigen 2 (TROP-2) antibody

Abstract

The present application provides isolated antibodies and antigen-binding fragments thereof that specifically bind to trophoblast cell surface antigen 2 (Trop-2). These Trop-2 antibodies or antigen-binding fragments thereof have high binding affinity for Trop-2, can induce antibody-dependent cell-mediated cytotoxicity (ADCC), have good tumor penetrability, can be internalized by cells expressing Trop-2, and can be used to diagnose, predict, and treat human diseases associated with Trop-2 (e.g., cancer, infectious diseases, autoimmune diseases, asthma, transplant rejection, and inflammatory diseases).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 034,055, filed June 3, 2020, the contents of which are incorporated by reference in their entirety for all purposes. Technical Field

[0003] The present disclosure relates to anti-trophoblast cell surface antigen 2 (Trop-2) antibodies or antigen-binding fragments and uses thereof.

[0004] Submit a sequence listing as an ASCII text file

[0005] The following content submitted in the form of an ASCII text file is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 227362000140SEQLIST.TXT, Record Date: June 2, 2021, Size: 66KB). Background Art

[0006] 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. It transduces intracellular calcium signals and acts as a cell surface receptor. It has 323 amino acids and consists of a large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail.

[0007] Although originally discovered as a cell surface marker of trophoblasts, subsequent reports have shown that Trop-2 is also expressed at low levels in a limited number of normal tissues, such as nasal, breast, skin, and bronchial epithelial cells. Further studies have shown that Trop-2 is overexpressed in many different cancer types, including oral, head and neck, thyroid, lung, breast, gastric, colorectal, pancreatic, renal, prostate, ovarian, uterine, cervical, and gliomas. Trop-2 overexpression is associated with disease progression and poor prognosis in cancer patients. Studies have shown that overexpression of Trop-2 in cancer cells can stimulate tumor growth in vitro and in vivo. Similarly, inhibition of Trop-2 expression by siRNA inhibits tumor cell proliferation. In addition to being crucial for tumor growth, Trop-2 is also involved in tumor progression and metastasis. Trop-2 is implicated in at least six major signaling pathways, including IGF-1, ErbB3, ERK, MAPK, Notch-Wnt, and Raf. However, its precise role in these signaling pathways and which downstream signaling pathways are critical in different cancers and for different therapeutic approaches remain to be elucidated.

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

[0009] In one aspect of the present invention, isolated antibodies and antigen-binding fragments that specifically bind to trophoblast cell surface antigen-2 (Trop-2) are provided. These Trop-2 antibodies or antigen-binding fragments have high affinity for Trop-2 and good tumor penetrability, can induce antibody-dependent cell-mediated cytotoxicity (ADCC), are internalized by cells expressing Trop-2, and can be used to diagnose, prognose, and treat human diseases (e.g., cancer, infectious diseases, autoimmune diseases, asthma, transplant rejection, and inflammatory diseases).

[0010] In various embodiments, the antibody or antigen-binding fragment is selected from a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a single-chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, a Fab' fragment, a Fab2 fragment, a F(ab)'2 fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or an IgG4 antibody having at least one mutation in the hinge region that reduces the propensity to form intra-heavy chain disulfide bonds. In various embodiments, the antibody is a chimeric antibody. In various embodiments, the antibody is a humanized antibody. In various embodiments, the antibody is a fully human antibody. In various embodiments, isolated antibodies and antigen-binding fragments thereof having high affinity for the human Trop-2 protein of SEQ ID NO: 1 are provided.

[0011] In various embodiments, the dissociation constant (K) of the antibody or antigen-binding fragment and the Trop-2 protein D ) is at least about 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, or 1x10 -12 M.

[0012] In various embodiments, the isolated antibody or antigen-binding fragment thereof of the invention binds to human Trop-2 and comprises: (a) a light chain CDR3 sequence that is identical, or substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) to a CDR3 sequence selected from SEQ ID NOs: 13-14; (b) a heavy chain CDR3 sequence that is identical, or substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) to a CDR3 sequence selected from SEQ ID NOs: 7-8; (c) the light chain CDR3 sequence in (a) and the heavy chain CDR3 sequence in (b).

[0013] In various embodiments, the isolated antibody or antigen-binding fragment further comprises an amino acid sequence selected from the group consisting of: (d) a light chain CDR1 sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to a CDR1 sequence selected from SEQ ID NOs: 9-10; (e) a light chain CDR2 sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to a CDR2 sequence selected from SEQ ID NOs: 11-12; (f) a light chain CDR3 sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to a CDR3 sequence selected from SEQ ID NOs: 11-12; NO:3-4 CDR1 sequence sequence is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or a substantially similar heavy chain CDR1 sequence; (g) a CDR2 sequence sequence is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or a substantially similar heavy chain CDR2 sequence selected from SEQ ID NO:5-6; (h) the light chain CDR1 sequence of (d) and the heavy chain CDR1 sequence of (f); (i) the light chain CDR2 sequence of (e) and the heavy chain CDR2 sequence of (g).

[0014] In various embodiments, the isolated human monoclonal antibodies or antigen-binding fragments thereof of the present invention bind to human TROP-2 and comprise: (a) a light chain CDR1 sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or substantially similar to a CDR1 sequence selected from SEQ ID NOs: 9-10; (b) a light chain CDR2 sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or substantially similar to a CDR2 sequence selected from SEQ ID NOs: 11-12; (c) a light chain CDR3 sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or substantially similar to a CDR3 sequence selected from SEQ ID NOs: 11-12; NO: 13-14 CDR3 sequence is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) or substantially similar to a light chain CDR3 sequence selected from SEQ ID NO: 13-14; (d) a heavy chain CDR1 sequence is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) or substantially similar to a CDR1 sequence selected from SEQ ID NO: 3-4; (e) a heavy chain CDR2 sequence is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) or substantially similar to a CDR2 sequence selected from SEQ ID NO: 5-6; (f) a heavy chain CDR3 sequence is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) or substantially similar to a CDR2 sequence selected from SEQ ID NO: 5-6; The CDR3 sequences of NO:7-8 are identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity) or substantially similar.

[0015] In various embodiments, the isolated human monoclonal antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises: 1) a light chain variable region (V L), which comprises (a) a light chain CDR1 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 9; (b) a light chain CDR2 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 11; (c) a light chain CDR3 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 13; and 2) a heavy chain variable region (V H ), which comprises (a) a heavy chain CDR1 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 3; (b) a heavy chain CDR2 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 5; (c) a heavy chain CDR3 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 7. In some embodiments, the light chain CDRs 1-3 comprise up to 5, 4, 3, 2, or 1 amino acid substitution. In some embodiments, heavy chain CDR1-3 comprises up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 1 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 1 of the present application.

[0016] In some embodiments, the isolated human monoclonal antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and is a chimeric or humanized antibody derived from an anti-Trop-2 antibody, comprising 1) a light chain variable region (V L ), which comprises (a) the light chain CDR1 sequence shown in SEQ ID NO: 9; (b) the light chain CDR2 sequence shown in SEQ ID NO: 11; (c) the light chain CDR3 sequence shown in SEQ ID NO: 13; 2) a heavy chain variable region (V H), which comprises (a) the heavy chain CDR1 sequence shown in SEQ ID NO: 3; (b) the heavy chain CDR2 sequence shown in SEQ ID NO: 5; and (c) the heavy chain CDR3 sequence shown in SEQ ID NO: 7.

[0017] In various embodiments, the isolated human monoclonal antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises: 1) a light chain variable region (V L ), which comprises (a) a light chain CDR1 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 10; (b) a light chain CDR2 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 12; (c) a light chain CDR3 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 14; 2) a heavy chain variable region (V H ), which comprises (a) a heavy chain CDR1 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 4; (b) a heavy chain CDR2 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 6; (f) a heavy chain CDR3 sequence comprising a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or a substantially similar sequence to SEQ ID NO: 8. In some embodiments, the light chain CDRs 1-3 comprise up to 5, 4, 3, 2, or 1 amino acid substitution. In some embodiments, heavy chain CDR1-3 comprises up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 1 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 1 of the present application.

[0018] In some embodiments, the isolated human monoclonal antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and is a chimeric or humanized antibody derived from an anti-Trop-2 antibody, comprising 1) a light chain variable region (V L ), which comprises (a) the light chain CDR1 sequence shown in SEQ ID NO: 10; (b) the light chain CDR2 sequence shown in SEQ ID NO: 12; (c) the light chain CDR3 sequence shown in SEQ ID NO: 14; 2) a heavy chain variable region (V H ), which comprises (a) the heavy chain CDR1 sequence shown in SEQ ID NO: 4; (b) the heavy chain CDR2 sequence shown in SEQ ID NO: 6; and (c) the heavy chain CDR3 sequence shown in SEQ ID NO: 8.

[0019] In various embodiments, the isolated antibodies or antigen-binding fragments thereof of the present invention bind to human Trop-2 and comprise: (a) a heavy and / or light chain variable domain having a set of three light chain CDR1s, CDR2s, and CDR3s that are identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or substantially similar sequences to SEQ ID NOs: 9-10, 11-12, and 13-14, and / or a set of three heavy chain CDR1s, CDR2s, and CDR3s that are identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) or substantially similar sequences to SEQ ID NOs: 3-4, 5-6, and 7-8; and (b) a set of four variable region framework regions from a human immunoglobulin (IgG). In various embodiments, the antibodies may optionally include a hinge region. In various embodiments, the framework regions are selected from human germline exon X H 、J H , Vκ and Jκ sequences. In various embodiments, the antibody is a fully humanized antibody. In various embodiments, the antibody is a fully human antibody.

[0020] In various embodiments, the isolated antibody or antigen-binding fragment, when binding to human TROP-2: (a) binds to human TROP-2 with a Kd substantially the same as or greater than that of a reference antibody; (b) competes with the reference antibody for binding to human TROP-2; (c) is less immunogenic in a human subject than the reference antibody, wherein the reference antibody comprises a combination of heavy and light chain variable domain sequences selected from SEQ ID NOs: 15 / 17 and 16 / 18, respectively.

[0021] In various embodiments, the isolated murine monoclonal antibody or antigen-binding fragment thereof of the invention binds to human TROP-2 and comprises a heavy chain variable region having an identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or a substantially similar sequence to SEQ ID NO: 15, and comprises a light chain variable region having an identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or a substantially similar sequence to SEQ ID NO: 17.

[0022] In various embodiments, the isolated murine monoclonal antibody or antigen-binding fragment thereof of the invention binds to human TROP-2 and comprises a heavy chain variable region having an identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or a substantially similar sequence to SEQ ID NO: 16, and a light chain variable region having an identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or a substantially similar sequence to SEQ ID NO: 18.

[0023] In various embodiments, the isolated chimeric antibody or antigen-binding fragment thereof of the invention binds to human Trop-2 and comprises a heavy chain having a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or a substantially similar sequence to SEQ ID NO:47, and a light chain having a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or a substantially similar sequence to SEQ ID NO:48.

[0024] In various embodiments, the isolated chimeric antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises a heavy chain that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to SEQ ID NO:49, and a light chain that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to SEQ ID NO:50.

[0025] In various embodiments, the isolated humanized antibodies or antigen-binding fragments thereof of the invention bind to human TROP-2 and comprise a heavy chain variable region having a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to SEQ ID NOs: 23-26, 29-33, and 40-44, and a light chain variable region having a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or substantially similar to SEQ ID NOs: 27-28, 34-39, and 45-46.

[0026] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises a heavy chain variable region sequence as shown in SEQ ID NO:40 and a light chain variable region sequence as shown in SEQ ID NO:37.

[0027] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises a heavy chain variable region sequence as shown in SEQ ID NO:29 and a light chain variable region sequence as shown in SEQ ID NO:37.

[0028] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises a heavy chain variable region sequence as shown in SEQ ID NO:43 and a light chain variable region sequence as shown in SEQ ID NO:45.

[0029] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the invention binds to human Trop-2 and comprises a heavy chain having a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or a substantially similar sequence to SEQ ID NOs: 51, 53 and 55, and a light chain having a sequence that is identical, substantially identical (e.g., having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity), or a substantially similar sequence to SEQ ID NOs: 52, 54 and 56.

[0030] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises the heavy chain sequence set forth in SEQ ID NO:51 and the light chain sequence set forth in SEQ ID NO:52.

[0031] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises the heavy chain sequence set forth in SEQ ID NO:53 and the light chain sequence set forth in SEQ ID NO:54.

[0032] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises the heavy chain sequence set forth in SEQ ID NO:55 and the light chain sequence set forth in SEQ ID NO:56.

[0033] On the other hand, the antibodies or antigen-binding fragments disclosed herein can be used to produce bispecific antibodies or heterologous antibodies by chemical techniques, by "polydoma" technology or by recombinant DNA technology. In various embodiments, the bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes, at least one of which is a tumor-associated antigen. In various embodiments, antibodies and fragments can also be heterologous antibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having different specificities.

[0034] In another aspect, an isolated immunoconjugate or fusion protein is provided, comprising an antibody or antigen-binding fragment coupled, linked (or stably associated) to an effector molecule. In various embodiments, the effector molecule is an immunotoxin, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent. An antibody drug conjugate (ADC) according to one embodiment of the invention has the formula: Ab-(LD)n, wherein Ab is a Trop-2 binding antibody, L is a linker, D is a drug moiety, and n is an integer from 1 to 10.

[0035] In another aspect, the antibodies or antigen-binding fragments disclosed herein can be covalently linked (or otherwise stably associated) to additional functional moieties, such as labels or moieties that confer desired pharmacokinetic properties. In various embodiments, the label is selected from the group consisting of: a fluorescent label, a radioactive label, and a label with a unique nuclear magnetic resonance characteristic.

[0036] In another aspect, the present invention relates to a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment of the present invention mixed with a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition comprises an isolated human antibody mixed with a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition is formulated for administration by a route of administration selected from subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection, or by infusion.

[0037] In another aspect, the present invention relates to a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of an isolated antibody or antigen-binding fragment of the invention. In various embodiments, the cancer is a cancer associated with elevated expression of Trop-2. In various embodiments, the cancer is selected from colorectal cancer (CRC), renal cancer, non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, and glioma. In various embodiments, the subject previously responded to treatment with anticancer therapy but relapsed after cessation of treatment (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject has a resistant or refractory cancer.

[0038] In another aspect, the present invention relates to a combination therapy designed for treating cancer or 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 invention, and b) one or more additional therapies selected from immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, vaccination regimens, 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 other therapy when co-administered.

[0039] In another aspect, the present invention provides a method for detecting the presence of human Trop-2 antigen in a sample in vitro or in vivo, such as a method for diagnosing a human Trop-2-related disease.

[0040] In another aspect, an isolated nucleic acid comprising a polynucleotide sequence encoding an antibody or antigen-binding fragment disclosed herein is provided. Also provided are expression vectors comprising the nucleic acids of the present invention. Also provided are isolated cells comprising the expression vectors of the present invention. In various embodiments, the cell is a host cell comprising the expression vectors of the present invention. In various embodiments, the cell is a hybridoma, wherein the chromosome of the cell comprises the nucleic acids of the present invention. Further provided are methods for preparing the antibodies or antigen-binding fragments of the present invention, comprising culturing or incubating cells under conditions that allow the cells to express the antibodies or antigen-binding fragments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] As shown in the picture. Figure 1 Depicted are the binding specificities of selected chimeric IgGs #118-2-5 and #125-1-5 to Trop-2 antigens from different species.

[0042] As shown in the picture. Figure 2 The binding specificity of #118-2-5 humanized IgG to Trop-2 antigen from different species is described.

[0043] As shown in the picture. Figure 3 The binding specificity of humanized anti-Trop-2 antibodies to Trop-2 antigens from different species is depicted.

[0044] As shown in the picture. Figures 4A-4C Depicts the results of flow cytometric analysis of anti-Trop-2 antibody internalization. The results indicate that anti-Trop-2 antibodies can be internalized by cells expressing Trop-2.

[0045] As shown in the picture. Figure 5 Depicted are the percentage of specific lysis of SK-BR-3 target cells by A1X4 and A1,2X4 effector cells from peripheral blood mononuclear cells (PBMCs) from three different donors in an assay designed to measure antibody-dependent cell-mediated cytotoxicity (ADCC).

[0046] As shown in the picture. Figure 6 The in vitro cytotoxic activity of ADCs containing different DARs in tumor cells with different Trop-2 expression levels was shown. The results showed that ADCs containing DAR4 and DAR6 were more effective than the DAR2 ADC, and the difference was more obvious in cells with medium / high Trop-2 expression.

[0047] As shown in the picture. Figure 7 The in vivo efficacy of ADCs containing different DARs in the MDA-MB-468 xenograft model was shown.

[0048] As shown in the picture. Figures 8A-8B Shown is a correlation between Trop-2 expression levels and the in vitro potency of A1,2X4-MMAE.

[0049] As shown in the picture. Figure 9 Shown is the in vitro bystander killing effect of A1,2X4-MMAE under co-culture conditions.

[0050] As shown in the picture. Figures 10A-10B The in vivo efficacy of A1X4-MMAE and A1,2X4-MMAE in MDA-MB-468 and NCI-N87 xenograft models was shown.

[0051] As shown in the picture. Figure 11 In vivo antitumor activity following single or fractionated administration of A1X4-MMAE in the MDA-MB-468 xenograft model is shown. DETAILED DESCRIPTION

[0052] The present invention relates to antigen-binding proteins, such as antibodies or antigen-binding fragments thereof, that specifically bind to human Trop-2. In one aspect, isolated antibodies and antigen-binding fragments thereof are provided that specifically bind to Trop-2, have high or intermediate affinity for Trop-2, can induce antibody-dependent cell-mediated cytotoxicity (ADCC), have good tumor penetrability, are internalized by cells expressing Trop-2, and can be used to treat human diseases (such as cancer), infections, and other diseases mediated by Trop-2.

[0053] Also provided are nucleic acid molecules and derivatives and fragments thereof comprising a polynucleotide sequence encoding all or part of a polypeptide that binds to Trop-2, such as nucleic acids encoding all or part of an anti-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 preparing, identifying, or isolating antigen-binding proteins that bind to human Trop-2, such as anti-Trop-2 antibodies, methods for determining whether an antigen-binding protein binds to Trop-2, methods for preparing compositions, such as pharmaceutical compositions, comprising antigen-binding proteins that bind to human Trop-2, and methods for administering antibodies or antigen-binding fragments thereof that bind to Trop-2 to a subject, such as methods for treating conditions mediated by Trop-2.

[0054] Based on the differential expression of Trop-2 in tumors and normal tissues, its role in promoting tumor growth and metastasis, and its negative prognostic value, Trop-2 has been proposed as a promising diagnostic / therapeutic target. Blocking Trop-2 signaling may be a means of treating cancer. Trop-2-targeting antigen-binding fragments (Fabs) have been shown to induce apoptosis and have inhibitory effects on breast cancer cell proliferation. Although several anti-Trop-2 antibodies have been developed, none are suitable for treatment as naked antibodies. Trop-2-targeting antibody-drug conjugates (ADCs) have recently been developed. Sacituzumab govitecan (IMMU-132) is a conjugate of the humanized anti-Trop-2 antibody hRS7 and the active metabolite of irinotecan, SN-38.

[0055] The present application provides novel Trop-2 antibodies and antigen-binding fragments thereof. In some cases, the present application provides Trop-2 antibodies that exhibit higher affinity and / or higher internalization rate for Trop-2 compared to the benchmark hRS7 antibody. Such antibodies or fragments thereof show promising applications for diagnosis, prognosis, and treatment of conditions that would benefit from modulation of Trop-2 using the novel human anti-Trop-2 antibodies and antigen-binding fragments thereof described herein (e.g., cancer, persistent infectious diseases, autoimmune diseases, asthma, transplant rejection, and inflammatory diseases).

[0056] definition

[0057] Unless otherwise defined herein, the scientific and technical terms used in connection with the present invention should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural, and plural terms should include singular. Generally, the nomenclature and technology used in combination with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization as described herein are those commonly used and well known in the art. Unless otherwise indicated, the methods and technologies of the present invention are generally carried out according to conventional methods well known in the art, and as described in the 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 carried out according to the manufacturer's instructions, as generally completed in the art or as described herein. The nomenclature and laboratory procedures and technologies used in combination with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry as described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0058] Polynucleotide and polypeptide sequences are represented using standard one or three letter abbreviations. Unless otherwise indicated, the amino terminus of the polypeptide sequence is on the left, the carboxyl terminus is on the right, and the top chain of the single-stranded nucleic acid sequence and the double-stranded nucleic acid sequence has their 5' end on the left and their 3' end on the right. The specific portion of the polypeptide can be numbered by amino acid residues, such as amino acids 80 to 119, or by the actual residues at the site, such as Ser80 to Ser119. A specific polypeptide or polynucleotide sequence can also be described based on its differences from the reference sequence. The polynucleotide and polypeptide sequences of specific light chain and heavy chain variable domains are referred to as L1 ("light chain variable domain 1") and H1 ("heavy chain variable domain 1"). Antibodies comprising light chains and heavy chains are represented by combining the light chain name and the heavy chain variable domain name. For example, "L4H7" represents an antibody comprising, for example, a light chain variable domain of L4 and a heavy chain variable domain of H7.

[0059] The term "antibody" is used herein to refer to a protein comprising one or more polypeptides that are substantially or partially encoded by immunoglobulin genes or immunoglobulin gene fragments and are specific for antigens (e.g., tumor antigens or molecules overexpressed under pathological conditions). The protein need not be a full-length antibody and can be a fusion protein comprising an antibody portion that specifically recognizes an antigen and a second portion (e.g., a cytotoxic agent). In some embodiments, the protein is an antibody-drug conjugate. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as subtypes of these genes and countless immunoglobulin variable region genes. Light chains (LC) are divided into kappa or lambda. Heavy chains (HC) are divided into gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typical immunoglobulin (e.g., antibody) structural units comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition.

[0060] In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3 (in some cases, CH4). Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, C L The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs has been defined. The framework region sequences of different light or heavy chains are relatively conserved within a species, such as humans. The framework region of an antibody, that is, the combined framework regions of the light and heavy chains, is used to position and align the CDRs in three-dimensional space. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1, and IgA2), or subclass.

[0061] CDRs are primarily responsible for binding to the epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. They are also typically identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is located in the heavy chain variable domain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the light chain variable domain of the antibody in which it is found. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions in the CDRs are called specificity-determining residues (SDRs).

[0062] The Kabat definition is a standard for numbering residues in antibodies and is commonly used to identify CDR regions. The Kabat database is now maintained online and can determine CDR sequences, for example, see the IMGT / V-QUEST program version: 3.2.18, March 29, 2011, available on the Internet, and Brochet, X. et al., Nucl. Acids Res. 36, W503-508, 2008). The Chothia definition is similar to the Kabat definition, but takes into account the position of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17, 1986; Chothia et al., Nature, 342:877-83, 1989. The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group to simulate antibody structure. See, e.g., Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272, 1989; "AbM TM ", a computer program for modeling antibody variable regions," Oxford, UK; Oxford Molecular, Ltd. AbM definitions model the tertiary structure of antibodies from the primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, 1999. Contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol, 5:732-45, 1996.

[0063] The term "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, which can be produced by papain digestion of an intact antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of antibodies and antigen-antibody complexes (e.g., neonatal FcR (FcRn) binds to the Fc region of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to extending the serum half-life of IgG). Replacing amino acid residues in the Fc portion to change antibody effector functions is known in the art (see, for example, Winter et al., U.S. Patent Nos. 5,648,260 and 5,624,821).

[0064] Antibodies exist as intact immunoglobulins or in a number of well-characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab2, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv") that bind to the target antigen. ScFv proteins are fusion proteins in which the variable regions of the light chain and the variable regions of the heavy chain of an immunoglobulin are joined by a linker, while in dsFvs, these chains have been mutated to introduce disulfide bonds to stabilize the binding of the chains. While various antibody fragments are defined based on the digestion of intact antibodies, it will be appreciated by those skilled in the art that these fragments can be synthesized de novo chemically or using recombinant DNA methods. Thus, as used herein, the term antibody encompasses, for example, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit a desired biological activity, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments or antigen-binding fragments of any of the foregoing.

[0065] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. A "Fab fragment" contains one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule is unable to form disulfide bonds with another heavy chain molecule. A "Fab' fragment" contains one light chain and a portion of one heavy chain, including the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments, thereby forming a F(ab')2 molecule.

[0066] Pepsin treatment of antibodies produces F(ab')2 fragments that have two antigen-binding sites and are still capable of cross-linking antigen. A "F(ab')2 fragment" comprises two light chains and two heavy chains, each of which contains a portion of the constant region between the CH1 and CH2 domains, forming an interchain disulfide bond between the two heavy chains. Thus, a single F(ab')2 fragment consists of two Fab' fragments, which are held together by a disulfide bond between the two heavy chains.

[0067] The "Fv region" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

[0068] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain that forms the antigen binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649, U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0069] As used herein, the terms "antigen-binding fragment" and "antigen-binding protein" refer to any protein that binds to a specific target antigen. "Antigen-binding fragment" includes, but is not limited to, antibodies and binding portions thereof, such as immunologically functional fragments. Exemplary antigen-binding fragments of antibodies are heavy and / or light chain CDRs, or heavy and / or light chain variable regions.

[0070] As used herein, the term "immunologically functional fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain) antigen-binding protein is an antigen-binding protein that comprises an antibody that lacks a portion of at least some of the amino acids present in the full-length chain (regardless of how the portion is obtained or synthesized), but is still able to specifically bind to the antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding proteins, including intact antibodies, for binding to a given epitope. In some embodiments, the fragment is a neutralizing fragment. On the one hand, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy and / or light chain or a portion thereof. These biologically active fragments can be produced by recombinant DNA technology, or can be produced by enzymatic or chemical cleavage of antigen-binding proteins (including intact antibodies). Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, diabodies, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including but not limited to humans, mice, rats, camels, or rabbits. It is further contemplated that a functional portion of the antigen binding proteins disclosed herein, e.g., one or more CDRs, can be covalently bound to a second protein or small molecule to generate a therapeutic agent directed against a specific target in vivo, having bifunctional therapeutic properties, or having an extended serum half-life.

[0071] Diabodies are bivalent antibodies comprising two polypeptide chains, each comprising a VH and VL region connected by a linker that is too short to allow pairing between the two regions on the same chain, thereby allowing each region to pair with a complementary region on the other polypeptide chain (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-48, 1993; and Poljak et al., Structure, 2: 1121-23, 1994). If the two polypeptide chains of a diabody are identical, the diabody produced by their pairing will have two identical antigen-binding sites. Polypeptide chains with different sequences can be used to prepare diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which may be the same or different.

[0072] Bispecific antibodies or fragments can have several configurations. For example, a bispecific antibody may be similar to a single antibody (or antibody fragment), but has two different antigen binding sites (variable regions). In various embodiments, antibodies can be produced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci. USA, 78: 5807, 1981; by "polydoma" technology (see, e.g., U.S. Patent No. 4,474,893); or recombinant DNA technology. In various embodiments, the bispecific antibodies of the present disclosure can have binding specificities for at least two different epitopes, at least one of which is a tumor-associated antigen. In various embodiments, antibodies and fragments can also be heteroantibodies. Heteroantibodies are two or more antibodies, or antibody binding fragments (e.g., Fab) linked together, each antibody or fragment having different specificities.

[0073] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical, except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be interpreted as requiring the antibody to be produced by any particular method.

[0074] As used herein, the term "chimeric antibody" refers to an antibody having framework residues from one species, such as human, and CDRs (which typically confer antigen binding) from another species, such as a murine antibody that specifically binds a target antigen.

[0075] As used herein, the term "human antibody" is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), such as in CDR, particularly CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transplanted onto human framework sequences.

[0076] As used herein, the term "humanized antibody" refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions of amino acids extracted from the donor framework. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. In various embodiments, the framework regions are selected from human germline exon X H 、J H , Vκ and Jκ sequences. For example, for V H The humanized receptor sequence of the FR domain can be selected from the real V H Exon V H 1-18 (Matsuda et al., Nature Genetics 3:88-94, 1993) or V H 1-2 (Shin et al., EMBO J. 10: 3641-3645, 1991), for the hinge region (JH ) , exon JH-6 (Mattila et al., Eur. J. Immunol. 25: 2578-2582, 1995). In other examples, germline Vκ exon B3 (Cox et al., Eur. J. Immunol. 24: 827-836, 1994) and Jκ exon Jκ-1 (Hieter et al., J. Biol. Chem. 257: 1516-1522, 1982) can be selected as V L Humanized receptor sequences.

[0077] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells; antibodies isolated from recombinant combinatorial human antibody libraries; antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes; or antibodies prepared, expressed, produced or isolated by any other means involving splicing 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 transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis), and therefore, although derived from human germline VH and VL sequences and sequences related thereto, the amino acid sequences of the VH and VL regions of the recombinant human antibodies may not naturally occur in the human antibody germline library in vivo. All of these recombinant means are well known to those of ordinary skill in the art.

[0078] As used herein, the term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or T cell receptor or otherwise interact with a molecule. Epitope determinants are typically composed of chemically active surface groups of molecules, such as amino acids or carbohydrates or sugar side chains, and typically have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be "linear" or "conformational." In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, interaction points occur on amino acid residues separated from each other on the protein. Once the desired epitope on the antigen is determined, it is possible to produce antibodies against the epitope, for example, using the technology described in this disclosure. Alternatively, in the discovery process, the generation and characterization of antibodies can elucidate information about the desired epitope. Based on this information, antibodies that bind to the same epitope can be competitively screened. A method for achieving this goal is to conduct cross-competition studies to find antibodies that competitively bind to each other, such as antibodies competing for binding to antigens.

[0079] An antigen binding protein (including an antibody) "specifically binds" to an antigen if it binds to the antigen with a high binding affinity, as determined by the dissociation constant (K D or the corresponding Kb, as defined below) value, is at least 1x10 -6 M, or at least 1x10 -7 M, or at least 1x10 -8 M, or at least 1x10 -9 M, or at least 1x10 -10 M, or at least 1x10 -11 M. An antigen binding protein that specifically binds to a human target antigen may also be able to bind to the same target antigen from other species with the same or different affinity. As used herein, the term "K D ” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction and is defined as the ratio K off / K on Koff is the rate constant for the dissociation of the drug from the receptor, while Kon is the rate constant for the binding of the drug to the receptor.

[0080] The term "biolayer interferometry (BLI)" as used herein refers to a layer of molecules attached to the tip of an optical fiber that produces an interference pattern at a detector; any change in the number of bound molecules results in a change in the pattern. It allows real-time analysis to determine the affinity and kinetics of biomolecular interactions in microplates, for example using the Octet system (ForteBio, Frement, CA).

[0081] As used herein, the term "immune cell" refers to any cell of the hematopoietic lineage that participates in regulating an immune response to an antigen (e.g., an autoantigen). 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.

[0082] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. In various embodiments, "peptide", "polypeptide" and "protein" are amino acid chains whose α-carbons are connected by peptide bonds. Thus, the terminal amino acid at one end of the chain (amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (carboxyl terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated as N-terminus) refers to the free-amino group or α-amino group (imino group when participating in a peptide bond) of an amino acid at any other position within the peptide on the amino acid at the amino terminus of an α-peptide. Similarly, the term "carboxyl terminus" refers to the free carboxyl group on the carboxyl terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide. Peptides also include substantially any polyamino acid, including but not limited to peptide mimetics, such as amino acids connected by ether rather than amide bonds.

[0083] As used herein, the term "recombinant polypeptide" is intended to include all polypeptides, including fusion molecules, that are prepared, expressed, generated, derived or isolated by recombinant means, such as polypeptides expressed by transfecting a host cell using a recombinant expression vector.

[0084] The polypeptides of the present disclosure include polypeptides that have been modified in any manner and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in portions of the polypeptide outside of the domains that form intermolecular contacts). A "conservative amino acid substitution" refers to a substitution of an amino acid in a polypeptide with a functionally similar amino acid. Each of the following six groups includes amino acids that are conservative substitutes for each other:

[0085] Alanine (A), serine (S), and threonine (T)

[0086] Aspartic acid (D) and glutamic acid (E)

[0087] Asparagine (N) and glutamine (Q)

[0088] Arginine (R) and Lysine (K)

[0089] Isoleucine (I), leucine (L), methionine (M), and valine (V)

[0090] Phenylalanine (F), tyrosine (Y), and tryptophan (W)

[0091] "Non-conservative amino acid substitutions" refer to replacing a member of one of these classes with a member from another class. When making such changes, according to various embodiments, the hydropathic index of the amino acids can be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge properties. 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).

[0092] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on the hydropathic index, in various embodiments, substitution of amino acids whose hydropathic indices are within ±2 is included. In various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0093] As disclosed herein, it is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, particularly where the resulting biologically functional protein or peptide is intended for use in immunological embodiments. In various embodiments, the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

[0094] The following hydrophilicity values ​​have been assigned to these amino acid residues: 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 changes are made based on similar hydrophilicity values, in various embodiments, including hydrophilicity values ​​between +2 Amino acid substitutions within ±1, including those within ±1, and in various embodiments, including those within ±0.5 are encompassed. Exemplary amino acid substitutions are listed in Table 1.

[0095] Table 1

[0096] Exemplary substituted amino acids for the original amino acid residues

[0097] Preferred substituted amino acids

[0098] Ala Val,Leu,Ile Val

[0099] Arg Lys,Gln,Asn Lys

[0100] Asn Gln

[0101] Asp Glu

[0102] Cys Ser,Ala Ser

[0103] Gln Asn Asn

[0104] Glu Asp Asp

[0105] Gly Pro,Ala Ala

[0106] His Asn,Gln,Lys,Arg Arg

[0107] Ile Leu,Val,Met,Ala,Leu

[0108] Phe,Norleucine

[0109] Leu Norleucine,Ile,Ile

[0110] Val,Met,Ala,Phe

[0111] Lys Arg,1,4Diamino-butyric Arg

[0112] Acid,Gln,Asn

[0113] Met Leu,Phe,Ile Leu

[0114] Phe Leu,Val,Ile,Ala,Tyr Leu

[0115] Pro Ala Gly

[0116] Ser Thr,Ala,Cys Thr

[0117] Thr Ser

[0118] Trp Tyr,Phe Tyr

[0119] Tyr Trp,Phe,Thr,Ser Phe

[0120] Val Ile,Met,Leu,Phe,Leu

[0121] Ala, Norleucine

[0122] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to polypeptides having amino-terminal and / or carboxyl-terminal deletions 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, fragments can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids in length. A fragment can further comprise one or more additional amino acids at either or both ends, such as an amino acid sequence 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).

[0123] As used herein, the terms "polypeptide variant" and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues are inserted, deleted, and / or substituted into the amino acid sequence of another polypeptide. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted 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.

[0124] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, for example, by conjugation to another chemical moiety such as polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.

[0125] 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 compared using a sequence alignment program. For example, as used herein, 80% identity refers to something that is the same as 80% sequence identity determined by a defined algorithm, and refers to at least 80% identity between a given sequence and another length of another sequence. In various embodiments, % identity is selected from, for example, a sequence identity of 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 to a given sequence. In various embodiments, % identity is, for example, in the range of 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%.

[0126] The term "% sequence homology" is used interchangeably with the term "% homology" herein 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 compared using a sequence alignment program. For example, as used herein, 80% homology refers to the same thing as 80% sequence homology determined by a defined algorithm, so that homologs of a given sequence have greater than 80% sequence homology over the length of the given sequence. In various embodiments, the percentage of 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, % homology is, for example, in the range of 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%.

[0127] Exemplary computer programs that can be used to determine the identity between two sequences include, but are not limited to, the BLAST suite of programs, such as BLASTN, BLASTX and TBLASTX, BLASTP and TBLASTN, which are publicly available on the NCBI website on the Internet. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (with particular reference to the published default settings, i.e., parameters w=4, t=17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. The BLASTP program is typically used when evaluating a given amino acid sequence relative to amino acid sequences in GenBank protein sequences and other public databases. The BLASTX program is suitable for searching nucleic acid sequences that have been translated in all reading frames in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0, and using the BLOSUM-62 matrix. See ID.

[0128] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1, less than about 0.01, or less than about 0.001.

[0129] In the context of polypeptide sequences, the term "substantially similar" or "substantially similar" means that a region of the polypeptide has 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, one polypeptide is substantially similar to a second polypeptide where, for example, the two peptides differ by one or more conservative substitutions.

[0130] "Polynucleotide" refers to a polymer consisting of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include those that include non-natural bases, nucleotides bonded to other nucleotides other than naturally occurring phosphodiester bonds, or bases connected by bonds other than phosphodiester bonds. Thus, nucleotide analogs include, for example, but not limited to, phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates, borate phosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA), etc. For example, such polynucleotides can be synthesized using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, generally no more than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences (i.e., A, U, G, C) in which "U" replaces "T."

[0131] Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction in which nucleotides are added to the nascent RNA transcript from 5' to 3' is referred to as the direction of transcription. The DNA strand that has the same sequence as the mRNA is referred to as the "coding strand"; the sequence on the DNA strand that has the same sequence as the mRNA transcribed from that DNA and is located from the 5' end to the 5' end of the RNA transcript is referred to as the "upstream sequence"; and the sequence on the DNA strand that has the same sequence as the mRNA transcribed from that DNA and is located from the 3' end to the 3' end of the coding RNA transcript is referred to as the "downstream sequence."

[0132] "Complementary" refers to the topological compatibility or matching of the interacting surfaces of two polynucleotides. Thus, the two molecules are said to be complementary and the properties of the contact surfaces are complementary. 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.

[0133] "Specific hybridization" or "specific hybridization" or "selective hybridization" refers to the preferential binding, duplex formation or hybridization of a nucleic acid molecule to a specific nucleotide sequence under stringent conditions when the specific nucleotide sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target subsequence and to a lesser extent or not at all to other sequences. "Stringent hybridization" and "stringent hybridization wash conditions" in nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence-dependent and are different under different environmental parameters. For an extensive guide to nucleic acid hybridization, see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, “An Overview of Hybridization Principles and Strategies for Nucleic Acid Probe Assays,” Elsevier, NY; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3.sup.rd ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0134] Typically, highly stringent hybridization and wash conditions are selected to be about 5°C below the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to equal the Tm of the specific probe. In Southern or Northern blotting, an example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than about 100 complementary residues on a filter membrane is 50% formalin with 1 mg of heparin at 42°C overnight. An example of highly stringent wash conditions is a 0.15 M NaCl wash at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A low stringency wash may be performed before a high stringency wash to remove background probe signal. An exemplary moderate stringency wash for, for example, a duplex of more than about 100 nucleotides is 1x SSC at 45°C for 15 minutes. An exemplary low stringency wash for a duplex of, for example, more than about 100 nucleotides is 4-6xSSC for 15 minutes at 40° C. Generally, a signal-to-noise ratio of 2-fold (or greater) observed for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization.

[0135] " Primer " refers to the polynucleotide that can hybridize specifically with the polynucleotide template of appointment and provide the starting point of synthesis complementary polynucleotide.When polynucleotide primer is placed under the condition of inducing synthesis, namely in the presence of nucleotide, complementary polynucleotide template and polymerization reagent such as DNA polymerase, this synthesis can take place.Primer is normally single-stranded, but also can be double-stranded.Primer is normally deoxyribonucleic acid, but multiple synthetic and naturally occurring primers can be used for many applications.Primer is complementary to the template of its design hybridization, as the site of initial synthesis, but does not need to reflect the exact sequence of template.In this case, the specific hybridization of primer and template depends on the stringency of hybridization condition.Primer can be used as detectable part with for example developing color, radioactive or fluorescent moiety mark.

[0136] "Probe" when used for polynucleotides refers to a polynucleotide that is capable of specific hybridization with a specified sequence of another polynucleotide. The probe specifically hybridizes to the target complementary polynucleotide, but does not need to reflect the exact complementary sequence of the template. In this case, the specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. The probe can be labeled with, for example, a colorimetric, radioactive or fluorescent moiety and used as a detectable portion. In the case where the probe provides a starting point for the synthesis of a complementary polynucleotide, the probe can also be a primer.

[0137] "Vector" is a polynucleotide that can be used to introduce another nucleic acid connected thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid fragments can be connected. Another type of vector is a viral vector (for example, a replication-defective retrovirus, adenovirus, and adeno-associated virus) in which additional DNA fragments can be introduced into a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and episomal mammalian vectors comprising bacterial replication origins). Other vectors (for example, non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. "Expression vector" is a vector that can direct the expression of a selected polynucleotide.

[0138] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, time, or location of expression) of a nucleic acid to which it is operably linked. For example, a regulatory sequence can act directly on the regulated nucleic acid, or act through one or more other molecules (e.g., a polypeptide bound to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. If a regulatory sequence affects the expression (e.g., the level, time, or location of expression) of a nucleotide sequence, then the nucleotide sequence is "operably linked" to the regulatory sequence.

[0139] "Host cell" is a cell that can be used to express the polynucleotides of the present disclosure. The host cell can be a prokaryotic organism, such as Escherichia coli, or it can be a eukaryotic organism, such as a unicellular eukaryotic organism (such as yeast or other fungi), a plant cell (such as a tobacco or tomato plant cell), an animal cell (such as a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell or an insect cell), or a hybridoma cell. Generally, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to represent 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 it at a desired level, unless a regulatory sequence is introduced into the host cell so that it is operably linked to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell, but also to the offspring or potential offspring of such a cell. Because certain modifications may occur in offspring due to, for example, mutations or environmental influences, such offspring may actually be different from the parent cell, but are still included in the scope of the term as used herein.

[0140] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or polynucleotide) is a molecule that, by reason of its source or derivation (1) is not in an associated state with naturally associated components, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cells of its natural source will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a number of methods well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution can be provided by using HPLC or other purification means well known in the art.

[0141] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of a sample shows a single species of polypeptide. A polypeptide or protein can be monomeric or multimeric. A substantially pure polypeptide or protein will typically contain about 50%, 60%, 70%, 80%, or 90% w / w of a protein sample, more typically about 95%, and preferably greater than 99% pure. Protein purity or homogeneity can be indicated by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization of individual polypeptide bands after staining the gel with dyes well known in the art. For some purposes, higher resolution can be provided by using HPLC or other purification means well known in the art.

[0142] A "linker" refers to a molecule that connects two other molecules covalently or through ionic bonds, van der Waals bonds, or hydrogen bonds, for example, a nucleic acid molecule hybridizes to a complementary sequence at the 5' end and to another complementary sequence at the 3' end, thereby connecting two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise separated from the two components connected by the cleavable linker. Cleaved linkers are typically cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleaved linkers can also be cleaved by environmental factors, such as changes in temperature, pH, salt concentration, and the like. Non-cleavable linkers are linkers that release the attached payload upon internalization by lysosomal degradation of the antibody.

[0143] As used herein, the term "label" or "labeled" 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 radiolabeled amino acid or the binding of a biotinyl moiety to a polypeptide, which can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or 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 toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labels include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I), a fluorescent label (e.g., FITC, rhodamine, a lanthanide phosphor), an enzyme label (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), a chemiluminescent label, a biotin group, a predetermined polypeptide epitope recognized by a secondary reporter molecule (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag), a magnetic agent (e.g., a gadolinium chelate), a toxin such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. In some embodiments, tags are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0144] As used herein, the term "immunotherapy" refers to cancer treatment, including but not limited to treatment with depleting antibodies directed against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonist, antagonist, or blocking antibodies directed against co-stimulatory 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. treatment with chimeric antigen receptor (CAR)-T cells; treatment with CAR-NK cells; treatment with tumor infiltrating lymphocytes (TIL); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immunostimulants such as the Toll-like receptor (TLR) agonists CpG and imiquimod.

[0145] The term "immunoconjugate" or "fusion protein" refers to a molecule comprising an antibody or its antigen-binding fragment that is directly or indirectly conjugated (or connected) to an effector molecule. The effector molecule can be a detectable label, an immunotoxin, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent. The antibody or its antigen-binding fragment can be conjugated to the effector molecule through a peptide linker. The immunoconjugate and / or fusion protein retains the immunoreactivity of the antibody or antigen-binding fragment, for example, the antibody or antigen-binding fragment has approximately the same or only slightly reduced ability to bind to the antigen after conjugation as before conjugation. As used herein, immunoconjugates may also be referred to as antibody-drug conjugates (ADCs). Because immunoconjugates and / or fusion proteins are initially prepared from two molecules with different functions, such as antibodies and effector molecules, they are sometimes also referred to as "chimeric molecules."

[0146] "Pharmaceutical composition" means a pharmaceutical composition suitable for administration to an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" means an amount of an agent effective to produce the desired pharmacological result. "Pharmaceutically acceptable carrier" means any standard pharmaceutical carrier, vehicle, buffer, and excipient, 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, 21 st Ed. 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salts" are salts of a compound that can be formulated for pharmaceutical use and include, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0147] The terms "treat," "treatment," and "treatment" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, disorder, or condition refers to reducing the severity and / or frequency of the symptoms of a disease, disorder, or condition. As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of: alleviating one or more symptoms, alleviating the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, such as to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, and alleviating (whether partial or complete). "Treatment" also includes reducing the pathological consequences of proliferative diseases. The methods of the present invention contemplate any one or more of these therapeutic aspects.

[0148] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular condition, disorder, or disease, such as to improve, alleviate, mitigate, and / or delay one or more symptoms. With respect to NHL and other cancers or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow, and preferably prevent to some extent the infiltration of cancer cells into peripheral organs; (iv) inhibit (i.e., slow and preferably stop to some extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. An effective amount can be administered in one or more doses.

[0149] A "resistant or refractory cancer" refers to a tumor cell or cancer that has not responded 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 start of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment or that initially respond for a short period of time but do not respond to treatment. Refractory tumor cells also include tumors that respond to anti-cancer therapy but do not respond to subsequent rounds of therapy. For the purposes of this invention, refractory tumor cells also include tumors that appear to be suppressed by anti-cancer therapy treatment but recur up to five years, and sometimes up to ten years or more, after cessation of treatment. Anti-cancer therapy can use a chemotherapeutic agent alone, radiation therapy alone, targeted therapy alone, surgery alone, or a combination thereof. For ease of description and not limitation, it should be understood that refractory tumor cells are interchangeable with resistant tumors.

[0150] It should be understood that aspects and embodiments of the present invention described herein include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0151] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0152] As used herein and in the appended claims, the singular forms "a," "an," "or," and "the" include plural referents unless the context clearly dictates otherwise. It should be understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0153] Trop-2 antigen

[0154] 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 epitope with hTrop-2. In various embodiments, the hTrop-2 polypeptide as used herein may comprise the amino acid sequence set forth in NCBI Reference Sequence: NP_002344.2 (SEQ ID NO: 1):

[0155]

[0156] In various embodiments, the Trop-2 polypeptide comprises an amino acid sequence having an observed homology to the human Trop-2 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%. Polypeptide variants of Trop-2 can be described herein by reference to additions, deletions, or substitutions of amino acid residues at given positions within the 223 amino acid sequence of SEQ ID NO: 1. Thus, for example, the term "P21W" indicates that residue "P" (proline, in the standard single letter code) at position 21 of SEQ ID NO: 1 has been substituted with "W" (tryptophan, in the standard single letter code).

[0157] Antibody

[0158] Methods for generating novel antibodies that bind to a Trop-2 polypeptide are known to those skilled in the art. For example, a method for generating a monoclonal antibody that specifically binds to a Trop-2 polypeptide can include administering to a mouse an amount of an immunogenic composition comprising a Trop-2 polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) extracted from the mouse, 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 a Trop-2 polypeptide. Once obtained, the hybridomas can be propagated in cell culture, optionally under culture conditions in which the hybridoma-derived cells produce monoclonal antibodies that specifically bind to a Trop-2 polypeptide. The monoclonal antibodies can be purified from the cell culture. A variety of different techniques can then be used to test antibody:antigen interactions to identify particularly desirable antibodies.

[0159] Other suitable methods for producing or isolating antibodies with the desired specificity may be used, including, for example, methods that select recombinant antibodies from libraries or methods that rely on immunization of transgenic animals (e.g., mice) capable of producing the full repertoire of human antibodies. See, for example, Jakobovits et al., Proc. Natl. Am. Sci. USA, 90:2551-2555, 1993; Jakobovits et al., Nature, 362:255-258, 1993; Lonberg et al., U.S. Patent No. 5,545,806; Surani et al., U.S. Patent No. 5,545,807.

[0160] Antibodies can be engineered in a variety of ways. They can be made into single-chain antibodies (including small modular immunopharmaceuticals or SMIPs) TM ), Fab and F(ab')2 fragments, etc. Antibodies can be humanized, chimeric, deimmunized, or fully human. Numerous publications describe various types of antibodies and methods for modifying these antibodies. For example, see U.S. Patents 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.

[0161] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, the gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction endonucleases to remove the region encoding the murine Fc, and the equivalent portion of the gene encoding the human Fc constant region is substituted (see Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison et al., European Patent Application No. 173,494; Neuberger et al., International Application No. WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application No. 125,023; Better et al., Science, 240:1041-1043, 1988; Liu et al., PNAS USA, 84:3439-3443, 1987; Liu et al., PNAS USA, 84:3439-3443, 1987; Liu et al., PNAS USA, 84:3439-3443, 1987). al., J. Immunol. 139:3521-3526, 1987; Sun et al., PNAS USA, 84:214-218, 1987; Nishimura et al., Canc. Res. 47:999-1005, 1987; Wood et al., Nature 314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst., 80:1553-1559, 1988).

[0162] Methods for humanizing antibodies have been described in the art. In practice, humanized antibodies are generally human antibodies, in which some hypervariable region residues and possibly some framework region residues are replaced by residues from similar sites in rodent antibodies. Therefore, such "humanized" antibodies are chimeric antibodies, in which substantially less than complete human variable regions have been replaced by corresponding sequences from non-human species. To some extent, this can be achieved in combination with humanization techniques and display techniques using appropriate libraries. It should be understood that mouse antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art (see, for example, Winter et al., Immunol Today, 14: 43-46, 1993; and Wright et al., Crit. Reviews in Immunol., 12125-168, 1992). The antibody of interest can be engineered by recombinant DNA technology to replace the CH1, CH2, CH3, hinge domains, and / or framework domains with corresponding human sequences (see WO 92 / 02190 and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and 5,777,085). In addition, the use of Ig cDNA to construct chimeric immunoglobulin genes is known in the art (Liu et al., PNAS 84:3439, 1987; J. Immunol. 139:3521, 1987). mRNA is isolated from hybridomas or other antibody-producing cells and used to produce cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Patent Nos. 4,683,195 and 4,683,202). Alternatively, a library can be prepared and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to the human constant region sequence. The sequence of the human gene constant region can be found in Kabat et al. (1991) Sequence of Proteins of Immunological Interest, NIH publication no. 91-3242. Human C region genes are easily obtained from known clones. The choice of isotype will depend on the desired effector function, such as complement fixation, or antibody-dependent cellular cytotoxicity activity. In various embodiments, the isotype is selected from IgG1, IgG2, IgG3, and IgG4. Any human light chain constant region, κ or λ, can be used. The chimeric humanized antibody is then expressed by conventional methods.

[0163] U.S. Patent No. 5,693,761 to Queen et al. discloses an improvement to the method of Winter et al. for humanizing antibodies and is based on the premise that loss of affinity is attributed to problems with structural motifs in the humanized framework that interfere with the folding of the CDRs into a binding-competent conformation found in mouse antibodies due to steric or other chemical incompatibilities. To address this issue, Queen teaches the use of human framework sequences that are closely homologous in linear peptide sequence to the framework sequences of the mouse antibody to be humanized. Thus, Queen's approach focuses on comparing framework sequences between species. Typically, all available human variable region sequences are compared to a specific mouse sequence and the percent identity between the corresponding framework residues is calculated. The human variable region with the highest percentage is selected to provide the framework sequence for the humanization project. Queen also teaches that retaining certain amino acid residues from the mouse framework in the humanized framework is critical for supporting the CDRs in a binding-competent conformation. Potential criticality is assessed from molecular models. Candidate residues to be retained are typically those residues that are adjacent to the CDRs in the linear sequence or physically adjacent to any CDR residues. Residues within the range.

[0164] In other approaches, the importance of specific framework amino acid residues can be determined experimentally once a low-affinity humanized construct is obtained by restoring individual residues to the mouse sequence and assaying for antigen binding as described by Riechmann et al., 1988. Another example of a method for identifying important amino acids in framework sequences is disclosed in U.S. Pat. No. 5,821,337 to Carter et al. and U.S. Pat. No. 5,859,205 to Adair et al. These references disclose specific Kabat residue positions in the framework that may need to be substituted with the corresponding mouse amino acid in order to maintain affinity in a humanized antibody.

[0165] Another approach to humanizing antibodies, called "framework shuffling," relies on generating a combinatorial library in which non-human CDR variable regions are fused in-frame to a single human germline framework pool (Dall'Acqua et al., Methods, 36:43, 2005). The library is then screened to identify clones encoding humanized antibodies that retain good binding.

[0166] The selection of human variable regions, including light and heavy chains, for preparing the desired humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the variable region sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then accepted as the human framework region (framework region) of the humanized antibody (Sims et al., J. Immunol., 151: 2296, 1993; Chothia et al., J. Mol. Biol., 196: 901, 1987). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a specific light or heavy chain variable region subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285, 1992; Presta et al., J. Immunol., 151: 2623, 1993).

[0167] The selection of non-human residues in place of human variable regions may be affected by a variety of factors. These factors include, for example, the rarity of the amino acid at a particular position, the possibility of interacting with CDR or antigen, and the possibility of participating in the interface between the light chain and heavy chain variable domain interfaces. (See, for example, U.S. Patent numbers 5,693,761, 6,632,927 and 6,639,055). A method of analyzing these factors is to use a three-dimensional model of non-human and humanized sequences. Three-dimensional immunoglobulin models are typically available and are familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences. Checking these displays allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, such as analyzing the residues that influence the ability of candidate immunoglobulin to bind its antigen. In this way, non-human residues can be selected and substituted with human variable region residues to achieve desired antibody properties, such as increasing affinity to the target antigen.

[0168] Methods for preparing fully human antibodies have been described in the art. For example, a method for preparing an anti-Trop-2 antibody or antigen-binding fragment thereof comprises the steps of synthesizing a human antibody library on phage, screening the library with Trop-2 or an antibody-binding portion thereof, isolating phage that binds Trop-2, and obtaining antibodies from the phage. As another example, a method for preparing an antibody library for phage display technology comprises the steps of immunizing a non-human animal containing a human immunoglobulin locus with Trop-2 or an antigenic portion thereof to generate an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding the heavy and light chains of an antibody of the present invention from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector to express the antibody on phage. Recombinant anti-Trop-2 antibodies of the present invention can be obtained in this manner.

[0169] Recombinant human anti-Trop-2 antibodies can also be isolated by screening recombinant combinatorial antibody libraries. Preferably, the library is a scFv phage display library generated using human VL and VH cDNAs prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for generating phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP®). TMPhage Display Kit, Catalog No. 240612). Other methods and reagents are available for generating and screening antibody display libraries (see, e.g., U.S. Patent No. 5,223,409; PCT Publication Nos. WO 92 / 18619, WO 91 / 17271, WO 92 / 20791, WO 92 / 15679, WO 93 / 01288, WO 92 / 01047, WO 92 / 09690; Fuchs et al., Bio / Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; McCafferty et al., Nature 247:1375-1372 (1991). 348:552-554, 1990; Griffiths et al., EMBO J. 12:725-734, 1993; Hawkins et al., J. Mol. Biol. 226:889-896, 1992; Clackson et al., Nature 352:624-628, 1991; Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-3580, 1992; Garrad et al., Bio / Technology 9:1373-1377, 1991; Hoogenboom et al., Nuc. Acid Res. 19:4133-4137, 1991; and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991, each of which is incorporated herein by reference for the purpose of teaching the preparation and screening of phase display libraries.

[0170] Human antibodies are also produced by immunizing non-human transgenic animals containing some or all of the human immunoglobulin heavy and light chain loci in their genome, such as the XenoMouse, with human IgE antigens. TM Animals (Abgenix, Inc. / Amgen, Inc. - Fremont, CA). XenoMouse TM The mouse is an engineered mouse strain that contains large fragments of the human immunoglobulin heavy and light chain loci and lacks mouse antibody production. See,

[0171] For example, Green et al., Nature Genetics 7: 13-21, 1994; and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364, 6,162,963, and 6,150,584. See also WO 91 / 10741, WO 94 / 02602, WO 96 / 34096, WO 96 / 33735, WO 98 / 16654, WO 98 / 24893, WO 98 / 50433, WO 99 / 45031, WO 99 / 53049, WO00 / 09560 and WO 00 / 037504. XenoMouse TM Mice can produce a complete human antibody repertoire similar to that of adults and produce antigen-specific human antibodies. In some embodiments, XenoMouse TM The mouse contains 80% of the human antibody V gene repertoire by introducing megabase-sized germline-configured fragments of the human heavy chain locus and kappa light chain locus into a yeast artificial chromosome (YAC). In other embodiments, the XenoMouse TM Mice also contain approximately all of the human lambda light chain loci. See Mendez et al., Nature Genetics 15:146-156 (1997), Green and Jakobovits, J. Exp. Med. 188:483-495 (1998), and WO 98 / 24893 (each of which is incorporated by reference in its entirety for the purpose of teaching the production of fully human antibodies). In another aspect, the present invention provides a method for producing anti-Trop-2 antibodies from a non-human, non-mouse animal by immunizing a non-human transgenic animal containing a human immunoglobulin locus with a Trop-2 antigen. Such animals can be produced using the methods described in the aforementioned literature.

[0172] Characterization of antibody-antigen binding

[0173] Binding of the antibodies of the present invention to Trop-2 can be tested, for example, by standard ELISA. For example, a microtiter plate is coated with purified Trop-2 dissolved in PBS and then blocked with 5% bovine serum albumin dissolved in PBS. Antibody dilutions (e.g., plasma dilutions from Trop-2 immunized mice) are added to each well and incubated at 37°C for 1-2 hours. The plate is washed with PBS / Tween and then incubated with a secondary reagent conjugated to alkaline phosphatase (e.g., for human antibodies, a goat anti-human IgG Fc-specific polyclonal reagent) at 37°C for 1 hour. After washing, the plate is developed with pNPP substrate (1 mg / ml) and analyzed at an OD of 405-650. Preferably, the mouse that produces the highest titer will be used for fusion. ELISA assays can also be used to screen for hybridomas that react positively with the Trop-2 immunogen. Hybridomas that bind Trop-2 with high affinity are subcloned and further characterized. One clone from each hybridoma was selected that retained the reactivity of the parental cells (by ELISA) and was used to make a 5-10 vial bank of cells, stored at -140°C, and used for antibody purification.

[0174] To determine whether the selected anti-Trop-2 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using Trop-2-coated ELISA plates, as described above. Binding of the biotinylated mAb can be detected using a streptavidin-alkaline phosphatase probe. To determine the isotype of the purified antibodies, an isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated with 1 μg / ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plate can be reacted with 1 μg / ml or less of the test monoclonal antibody or a purified isotype control at ambient temperature for 1 to 2 hours. The wells can then be reacted with an alkaline phosphatase-conjugated probe specific for human IgG1 or human IgM. The plate can be developed and analyzed as described above.

[0175] Anti-Trop-2 human IgG can be further tested for reactivity with the Trop-2 antigen by Western blotting. Briefly, Trop-2 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed using BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0176] The binding affinity of human IgG to its antigen can be determined by the Octet system based on biolayer interferometry (BLI) technology. BLI is a layer of molecules attached to the tip of an optical fiber that produces an interference pattern at the detector, and any change in the number of bound molecules will cause the pattern to shift in measurement. The Octet system can analyze the affinity and kinetics of biomolecular interactions in a 96-well microplate in real time. In short, the target antigen can be diluted and loaded into a 96-well microplate. The antibody is then diluted and added to the designated wells. The plate is placed in the Octet system and the assay is started. The data can be analyzed using Octet Data Acquisition Software (fortebio data analysis 10.0) to calculate the association rate constant Kon, the dissociation rate constant Koff, and the dissociation constant Kd (Kd = Koff / Kon).

[0177] Binding of human IgG to antigens expressed on the cell surface can be tested by flow cytometry. Briefly, antibodies can be added to cells in FACS buffer and incubated at 4°C for 30 minutes. After incubation, cells are washed to remove unbound antibody. Cells are then dissociated and stained with a fluorescently conjugated secondary antibody on ice for 30 minutes before analysis using a Beckman flow cytometer system. Fluorescence intensity and percentage of cell binding can be analyzed using Beckman flow cytometer software.

[0178] Identification of Anti-Trop-2 Antibodies

[0179] The present invention provides a monoclonal antibody specifically binding to Trop-2 antigen and an antigen-binding fragment thereof.

[0180] The present invention also includes antibodies that bind to the same epitope as the anti-Trop-2 antibodies of the present invention. To determine whether an antibody can compete for binding to the same epitope as the anti-Trop-2 antibodies of the present invention, a cross-blocking assay, such as a competitive ELISA assay, can be performed. In an exemplary competitive ELISA assay, Trop-2 coated on microtiter plate wells is preincubated with or without a candidate competing antibody, followed by the addition of a biotin-labeled anti-Trop-2 antibody of the present invention. The amount of labeled anti-Trop-2 antibody bound to the Trop-2 antigen in the wells is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive or fluorescent label or some other detectable and measurable label. The amount of labeled anti-Trop-2 antibody bound to the antigen will indirectly correlate with the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope, i.e., the greater the affinity of the test antibody for the same epitope, the less labeled antibody will bind to the antigen-coated wells. Candidate competing antibodies are considered to bind to substantially the same epitope as, or compete for binding to the same epitope as, an anti-Trop-2 antibody of the invention if the candidate antibody can block binding of the Trop-2 antibody by at least 20%, at least 30%, at least 40%, or at least 50%, compared to a control performed in parallel without the candidate competing antibody. It should be understood that variations of this assay can be performed to achieve the same quantitative value. In various embodiments, antibodies of the invention include antibodies that bind to the same epitope as the monoclonal antibody designated #118-2-5. In various embodiments, antibodies of the invention include antibodies that bind to the same epitope as the monoclonal antibody designated #125-1-5.

[0181] The amino acid sequences of the heavy chain CDRs and light chain CDRs of two murine antibodies #118-2-5 and #125-1-5 generated as described herein are shown in Table 2 below.

[0182] Table 2

[0183]

[0184] In various embodiments of the present invention, the antibody or antigen-binding fragment is murine antibody #118-2-5, which comprises the heavy chain variable region sequence of SEQ ID NO: 15:

[0185] QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVNWIRQPPGKGLEWLGVMWAGGST NYNSALMRSRLSISKDNSKSQVFLKMNSLQTDDTGMYYCARDENWDGAWFAYWGQGT LVTVS(SEQ ID NO:15)

[0186] And the light chain variable region sequence of SEQ ID NO: 17:

[0187] DIQMTQSPSSLAVSAGEKVTMSCKSSQSLLNSGTRKNYLAWYQQKPGQSPKLLISWAS SRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGGGTKLELK(SEQ ID NO:17)

[0188] In various embodiments of the present invention, the antibody or antigen-binding fragment is murine antibody #125-1-5, which comprises the heavy chain variable region sequence of SEQ ID NO: 16:

[0189] QIQLVQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYINPNNDGT KYNEKFKGKATLISDKSSTTAYMEVRGLTSEDSAVYYCARPHFETHAMDYWGQGTSVT VSS(SEQ ID NO:16)

[0190] and the light chain variable region sequence of SEQ ID NO: 18:

[0191] DIQMTQSPSSFSVSLGDSVTITCKASEDIFNRLAWYQQKPGNAPRLLLISGATSLETGVP SRFSGGGSGKEYTLSITSLQNEDVATYYCQQYWNTWTFGGGTKLEIK(SEQ ID NO:18)

[0192] In various embodiments of the present invention, the antibody or antigen-binding fragment is a mouse-human chimeric antibody derived from mouse #118-2-5, comprising a heavy chain sequence of SEQ ID NO: 47:

[0193] QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVNWIRQPPGKGLEWLGVMWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTGMYYCARDENWDGAWFAYWGQGTLVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQAYICNVNHKPSNTKVDKKVGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIERTISKAKGQPREPQVYTLPPSRDELAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:47)

[0194] and the light chain sequence of SEQ ID NO:48:

[0195] DIQMTQSPSSLAVSAGEKVTMSCKSSQSLLNSGTRKNYLAWYQQKPGQSPKLLISWASSRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQ ID NO:48)

[0196] In certain alternative embodiments, the antibody is a murine-human chimeric antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence that is 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% identical to the amino acid sequence set forth in SEQ ID NO:47, and wherein the light chain comprises a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:48.

[0197] In various embodiments of the present invention, the antibody or antigen-binding fragment is a mouse-human chimeric antibody derived from mouse #125-1-5, comprising the heavy chain sequence of SEQ ID NO: 49:

[0198] QIQLVQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYINPNNDGTKYNEKFKGKATLISDKSSTTAYMEVRGLTSEDSAVYYCARPHFETHAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQAYICNVNHKPSNTKVDKKVGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIERTISKAKGQPREPQVYTLPPSRDELAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:49)

[0199] And the light chain sequence of SEQ ID NO:50:

[0200] DIQMTQSPSSFSVSLGDSVTITCKASEDIFNRLAWYQQKPGNAPRLLLISGATSLETGVPSRFSGGGSGKEYTLSITSLQNEDVATYYCQQYWNTWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQID NO:50)

[0201] In certain alternative embodiments, the antibody is a murine-human chimeric antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence that is 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% identical to the amino acid sequence set forth in SEQ ID NO:49, and wherein the light chain comprises a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in SEQ ID NO:50.

[0202] In various embodiments of the invention, the antibody is a humanized IgG A1, 2X4 comprising the heavy chain sequence of SEQ ID NO: 51:

[0203] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGST NYNSALMSRLTISKDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTL VTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQAYICNVNHKPSNTKVDKKVGPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIERTISKAKGQPR EPQVYTLPPSRDELAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:51)

[0204] and the light chain sequence of SEQ ID NO:52:

[0205] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Serial number:

[0206] In certain alternative embodiments, the antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO:51, and wherein the light chain comprises a sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO:52.

[0207] In various embodiments of the invention, the antibody is a humanized IgG A1X4 comprising the heavy chain sequence of SEQ ID NO: 53:

[0208] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGST NYNSALMSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQAYICNVNHKPSNTKVDKKVGPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIERTISKAKGQPR EPQVYTLPPSRDELAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:53)

[0209] And the light chain sequence of SEQ ID NO:54:

[0210] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWAS SRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSSGTASVVCLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLLSSTLTLSKADYEKHKVYACEVTHQGLSSPV54TKSFNRGEC*(SEQ)

[0211] In certain alternative embodiments, the antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an antibody 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 of SEQ ID NO: 53,

[0212] And wherein the light chain comprises an amino acid sequence that is 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% identical to that set forth in SEQ ID NO:54.

[0213] In various embodiments of the invention, the antibody is humanized IgG #125-1-5Hu3 / Lu5 comprising the heavy chain sequence of SEQ ID NO: 55:

[0214] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYVIHWVRQAPGQRLEWMGYINPNNDG TKYNEKFKGKATLTSDKSSTTAYMEVRGLTSEDSAVYYCARPHFETHAMDYWGQGTL VTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQAYICNVNHKPSNTKVDKKVGPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIERTISKAKGQP R EPQVYTLPPSRDELAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:55)

[0215] and the light chain sequence of SEQ ID NO:56:

[0216] DIQMTQSPSSLSASVGDRVTITCKASEDIFNRLAWYQQKPGKAPKLLLYGATSLETGVP SRF SGSGSGTDYTLTISSLQPEDFATYYCQQYWNTWTFGQGTKVEIK RTVAAPSVFIFP PSDEQLKSSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Serial number:

[0217] In certain alternative embodiments, the antibody is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO:55, and wherein the light chain comprises a sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO:56.

[0218] In various embodiments of the present disclosure, the antibody may be an anti-Trop-2 antibody having the same or higher antigen-binding affinity as an antibody comprising a heavy chain sequence as set forth in any one of SEQ ID NOs: 51, 53, and 55. In various embodiments, the antibody may be an anti-Trop-2 antibody that binds to the same epitope as an antibody comprising a heavy chain sequence as set forth in any one of SEQ ID NOs: 51, 53, and 55. In multiple embodiments, the antibody is an anti-Trop-2 antibody that competes with an antibody comprising a heavy chain sequence as set forth in any one of SEQ ID NOs: 51, 53, and 55. In various embodiments, the antibody may be an anti-Trop-2 antibody that comprises at least one (e.g., two or three) CDRs of a heavy chain sequence as set forth in any one of SEQ ID NOs: 51, 53, and 55.

[0219] In various embodiments, the antibody comprises a heavy chain amino acid sequence that is, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, or at least 90% identical to any one of SEQ ID NOs: 51, 53, or 55.

[0220] 5%, at least 96%, at least 97%, at least 98% or at least 99% observable homology.

[0221] In various embodiments of the present disclosure, the antibody may be an anti-Trop-2 antibody having the same or higher antigen-binding affinity as an antibody comprising a light chain sequence as set forth in any one of SEQ ID NOs: 52, 54, and 56. In various embodiments, the antibody may be an anti-Trop-2 antibody that binds to the same epitope as an antibody comprising a light chain sequence as set forth in any one of SEQ ID NOs: 52, 54, and 56. In multiple embodiments, the antibody is an anti-Trop-2 antibody that competes with an antibody comprising a light chain sequence as set forth in any one of SEQ ID NOs: 52, 54, and 56. In various embodiments, the antibody may be an anti-Trop-2 antibody that comprises at least one (e.g., two or three) CDRs of a light chain sequence as set forth in any one of SEQ ID NOs: 52, 54, and 56.

[0222] In various embodiments, the antibody comprises a light chain amino acid sequence having an observable homology to any one of SEQ ID NOs: 52, 54, 56, for example, 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%.

[0223] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises the heavy chain sequence set forth in SEQ ID NO:51 and the light chain sequence set forth in SEQ ID NO:52.

[0224] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises the heavy chain sequence set forth in SEQ ID NO:53 and the light chain sequence set forth in SEQ ID NO:54.

[0225] In various embodiments, the isolated humanized antibody or antigen-binding fragment thereof of the present invention binds to human Trop-2 and comprises the heavy chain sequence set forth in SEQ ID NO:55 and the light chain sequence set forth in SEQ ID NO:56.

[0226] In some embodiments, the anti-Trop-2 antibodies provided herein comprise an anti-Trop-2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23, or comprises a variant of an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises the amino acid sequence of SEQ ID NO: 27, or comprises a variant of an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0227] In some embodiments, the anti-Trop-2 antibodies provided herein comprise an anti-Trop-2 antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 23, or comprises a variant of an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises the amino acid sequence of SEQ ID NO: 28, or comprises a variant of an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0228] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 27, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0229] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V Hcomprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL An amino acid sequence comprising SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0230] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 27, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0231] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 25, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L An amino acid sequence comprising SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0232] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V Hcomprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 27, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0233] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L An amino acid sequence comprising SEQ ID NO: 28, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0234] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 29, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V Hcomprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The amino acid sequence of SEQ ID NO: 36 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LThe amino acid sequence of SEQ ID NO: 38 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L An amino acid sequence comprising SEQ ID NO: 39, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity.

[0235] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (VH ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 36 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 38 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL comprising the amino acid sequence of SEQ ID NO: 39, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0236] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (VL) of the anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The amino acid sequence of SEQ ID NO: 36 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (VH ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 38 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L An amino acid sequence comprising SEQ ID NO: 39, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity.

[0237] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V Hcomprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L ) of an anti-Trop2 antibody, wherein V H comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 36 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LThe invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The amino acid sequence of SEQ ID NO: 38 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 32, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L An amino acid sequence comprising SEQ ID NO: 39, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity.

[0238] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The amino acid sequence of SEQ ID NO: 34, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (VH ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 35, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The amino acid sequence of SEQ ID NO: 36 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 37, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region ( VL) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; andVL The amino acid sequence of SEQ ID NO: 38 is included, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the present application provides a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL An amino acid sequence comprising SEQ ID NO: 39, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%), 96%, 97%, 98% or 99%) sequence identity.

[0239] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 40, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L In some embodiments, the anti-Trop2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO: 52, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0240] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (VL) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L In some embodiments, the anti-Trop2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 53, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0241] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 45, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 41, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V LAn amino acid sequence comprising SEQ ID NO: 46, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0242] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 45, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and VL An amino acid sequence comprising SEQ ID NO: 46, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0243] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 43, or comprising a variant of an amino acid sequence having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity; and VLThe invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 45, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 43, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L An amino acid sequence comprising SEQ ID NO: 46, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0244] In some embodiments, the anti-Trop-2 antibodies provided herein comprise a heavy chain variable region (V H ) and light chain variable region (V L) , where V H comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V L The invention relates to a method for preparing an amino acid sequence comprising SEQ ID NO: 45, or a variant thereof comprising an amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, the invention provides a method for preparing an amino acid sequence comprising a heavy chain variable region (V H ) and light chain variable region (V L) Anti-Trop2 antibodies, of which V H comprising the amino acid sequence of SEQ ID NO: 44, or a variant thereof having at least about 80% (e.g., at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and V Lcomprising the amino acid sequence of SEQ ID NO: 46, or comprising a variant of the amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity. In some embodiments, the anti-Trop2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 55, or comprising a variant of the amino acid sequence having at least about 80% (e.g., at least about any one of 80%), 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and a light chain comprising the amino acid sequence of SEQ ID NO: 56, or comprising a variant of the amino acid sequence having at least about 80% (e.g., at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0245] The antibodies or antigen-binding fragments thereof of the present invention 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, such as a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an α-, δ-, ε-, γ- or mu-type heavy chain constant region, such as an IgA-, IgD-, IgE-, IgG- and IgM-type heavy chain constant region. In various embodiments, the light chain or heavy chain constant region is a fragment, derivative, variant or mutant protein of a naturally occurring constant region.

[0246] In some embodiments, the anti-Trop-2 antibodies described herein comprise an Fc constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof. In some embodiments, the Fc constant region has reduced effector function compared to a corresponding wild-type Fc constant region. In some embodiments, the Fc fragment has enhanced effector function compared to a corresponding wild-type Fc constant region. In some embodiments, the Fc constant region has an extended serum half-life. In some embodiments, the Fc constant region has a reduced serum half-life.

[0247] In some embodiments, an anti-Trop-2 antibody described herein comprises an IgG1 Fc constant region (eg, a wild-type IgG1 Fc constant region).

[0248] Known techniques for deriving antibodies of different subclasses or isotypes from an antibody of interest, i.e., subclass conversion. Thus, for example, an IgG antibody can be derived from an IgM antibody, and vice versa. Such techniques allow the preparation of new antibodies with the antigen-binding properties of a given antibody (parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA technology can be used. Cloned DNA encoding specific antibody polypeptides can be used for such programs, for example, DNA encoding a desired isotype antibody constant domain. See also Lanitto et al., Methods Mol. Biology. 178:303-16, 2002.

[0249] Antibodies of the invention may also be described or specified in terms of their cross-reactivity. The invention includes antibodies that bind to a Trop-2 polypeptide and have 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 Trop-2 (as calculated using methods known in the art and described herein).

[0250] The present invention also includes antibodies that bind to the same epitope as the anti-Trop-2 antibodies of the present invention. To determine whether an antibody can compete for binding to the same epitope as the anti-Trop-2 antibodies of the present invention, a cross-blocking assay, such as a competitive ELISA assay, can be performed. In an exemplary competitive ELISA assay, Trop-2 coated on microtiter plate wells is preincubated with or without a candidate competing antibody, followed by the addition of a biotin-labeled anti-Trop-2 antibody of the present invention. The amount of labeled anti-Trop-2 antibody bound to the Trop-2 antigen in the wells is measured using an avidin-peroxidase conjugate and an appropriate substrate. The antibody can be labeled with a radioactive or fluorescent label or some other detectable and measurable label. The amount of labeled anti-Trop-2 antibody bound to the antigen will indirectly correlate with the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope, i.e., the greater the affinity of the test antibody for the same epitope, the less labeled antibody will bind to the antigen-coated wells. A candidate competing antibody is considered to be an antibody that binds to substantially the same epitope as, or competes for binding to the same epitope as, an anti-Trop-2 antibody of the invention if the candidate antibody can block binding of the TRAPO-2 antibody by at least 20%, at least 30%, at least 40%, or at least 50%, as compared to a control performed in parallel without the candidate competing antibody. It should be understood that variations of this assay can be performed to achieve the same quantitative value.

[0251] In certain alternative embodiments, the antibodies of the present invention may be modified by modifying one or both variable regions (i.e., V Hand / or VL ) or by modifying residues within the constant region, for example, to alter the effector function of the antibody. In various embodiments, the variable region of the antibody will be modified by CDR grafting using framework sequences obtained from public DNA databases or published references including germline antibody gene sequences (e.g., Tomlinson, IM et al., J. Mol. Biol. 227:776-798, 1992; and Cox, JPL et al., Eur. J. Immunol. 24:827-836, 1994; the contents of each of which are expressly incorporated herein by reference). In various embodiments, antibodies can be modified using site-directed mutagenesis or PCR-mediated mutagenesis to introduce mutations in VH and / or VL to increase binding affinity and / or reduce immunogenicity. In various embodiments, antibodies can be modified in the Fc region to alter their serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In various embodiments, antibodies can be modified to modify their glycosylation. Methods for performing each of the modifications described herein, as well as other methods, are well known to those skilled in the art.

[0252] Pharmaceutical composition

[0253] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described above. The pharmaceutical compositions, methods and uses of the present invention therefore also include embodiments of combination with other active agents (co-administration), as described in detail below.

[0254] Typically, the antibodies or antigen-binding fragment antibodies of the present invention are suitable for administration as a formulation in combination with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the compounds of the present invention. The choice of excipient depends largely on factors such as the specific route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol such as mannitol, sorbitol, or sodium chloride, in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or small amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives, or buffers, which can increase the shelf life or effectiveness of the antibody. The pharmaceutical compositions of the present invention and their preparation methods will be 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.

[0255] The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk as a single unit dose or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject or a convenient fraction of such a dose, such as one-half or one-third of such a dose.

[0256] Any art-accepted method for administering peptides, proteins, or antibodies can be suitably employed with the antibodies and portions of the invention.

[0257] The pharmaceutical composition of the present invention is generally suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by the physical disruption of individual tissues and administration of the pharmaceutical composition through the disruption in the tissue, thus typically resulting in direct administration into the blood, into muscle, or into internal organs. Therefore, parenteral administration includes, but is not limited to, administering the composition by injection, administering the composition through a surgical incision, administering the composition through a tissue-penetrating non-surgical wound, etc. In particular, parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial injection or infusion; and renal dialysis infusion techniques. Various embodiments include intravenous and subcutaneous routes.

[0258] The preparation of the pharmaceutical composition suitable for parenteral administration generally comprises an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such preparations can be prepared, packaged or sold in a form suitable for push administration or continuous administration. Injectable preparations can be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers containing preservatives. Preparations for parenteral administration include but are not limited to emulsions, pastes, etc. in suspensions, solutions, oily or aqueous excipients. Such preparations may also include one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment of the preparation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) before the composition reconstructed for parenteral administration. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), but for some applications, they may be more suitable for being formulated into sterile non-aqueous solutions or for use as dry forms with suitable carriers (e.g., sterile, pyrogen-free water). Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol solutions or glucose solutions. If desired, this dosage form can be appropriately buffered. Other available parenteral administration formulations include those comprising the active ingredient in microcrystalline form or liposomal formulations. Preparations for parenteral administration can be formulated to release immediately and / or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0259] For example, in one aspect, sterile injectable solutions can be prepared by incorporating the desired amount of an anti-Trop-2 antibody into an appropriate solvent having one or a combination of ingredients listed above, as desired, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other desired ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof. Proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. Absorption of the injectable composition can be prolonged by including in the composition an agent that delays absorption, such as monostearate and gelatin.

[0260] The antibodies of the invention can also be administered intranasally or by inhalation, usually in the form of a dry powder (alone, as a mixture or as mixed component particles, e.g., in admixture with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aerosol spray from a pressurized container, pump, sprayer, nebulizer (preferably one that uses electrohydrodynamics to produce a fine mist) or atomizer, with or without a suitable propellant, or as nasal drops.

[0261] The pressurized container, pump, spray, atomizer, or nebulizer typically contains a solution or suspension of the antibody of the invention containing, for example, suitable agents for dispersing, dissolving, or prolonging the release of the active substance, a propellant such as a solvent.

[0262] Prior to use in dry powder or suspension formulations, the drug product is typically micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This can be achieved by any suitable comminution method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[0263] Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base and a performance enhancer.

[0264] Suitable flavoring agents, such as menthol and levomenthol, or sweetening agents, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled / intranasal administration.

[0265] Formulations for inhaled / intranasal administration can be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0266] In the case of dry powder inhalers and aerosols, the dosage unit is determined by a valve that delivers a metered amount. Units according to the invention are typically arranged to administer metered doses or "bursts of the antibody of the invention." The total daily dose is typically administered in a single dose or, more typically, as divided doses throughout the day.

[0267] The antibodies and antibody portions of the invention can also be formulated for oral administration. Oral administration can include swallowing, so that the compound enters the gastrointestinal tract, and / or buccal, lingual or sublingual administration, whereby the compound enters the bloodstream directly from the mouth.

[0268] Formulations suitable for oral administration include solid, semisolid, and liquid systems, such as tablets; soft or hard capsules containing multi- or nanoparticulates, liquids, or powders; lozenges (including liquids); chewables; gels; rapidly dispersing dosage forms; films; ovules; sprays; and buccal / mucoadhesive patches.

[0269] Pharmaceutical compositions intended for oral administration can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may include one or more agents selected from sweeteners to provide pharmaceutically elegant and palatable preparations. For example, to prepare tablets that can be delivered orally, the antibody or its antigen-binding fragment is mixed with at least one pharmaceutical excipient, and the solid preparation is compressed into tablets according to known methods for delivery to the gastrointestinal tract. Tablet compositions are typically formulated with additives such as sugar or cellulose carriers, adhesives such as starch paste or methylcellulose, fillers, disintegrants, or other additives commonly used in the manufacture of pharmaceutical preparations. To prepare capsules that can be delivered orally, DHEA is mixed with at least one pharmaceutical excipient, and the solid preparation is placed in a capsule container suitable for delivery to the gastrointestinal tract. Compositions comprising antibodies or their antigen-binding fragments can be prepared according to the general description in Remington's Pharmaceutical Sciences, 18th edition. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, incorporated herein by reference.

[0270] In various embodiments, the pharmaceutical composition is formulated as an orally deliverable tablet containing the antibody or antigen-binding fragment thereof and mixed with non-toxic pharmaceutically acceptable excipients suitable for preparing tablets. These excipients can be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants such as corn starch, gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. The tablets can be uncoated, or they can be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be used alone or with a wax.

[0271] In various embodiments, the pharmaceutical compositions are formulated into 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 into soft gelatin capsules in which the antigen-binding fragment thereof is mixed with an aqueous or oily medium, such as peanut oil, peanut oil, liquid paraffin, or olive oil.

[0272] Liquid preparations include suspensions, solutions, syrups and elixirs. Such preparations can be used as fillers in soft or hard capsules (e.g., made of gelatin or hydroxypropyl methylcellulose) and typically include carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or suitable oils, and one or more emulsifiers and / or suspending agents. Liquid preparations can also be prepared by, for example, reconstructing a solid from a pouch.

[0273] Therapeutic uses

[0274] In another aspect, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention (as a monotherapy or in a combination therapy regimen). Such diseases include, but are not limited to, solid tumors, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin 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, chronic or acute leukemia, Leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, renal cancer or ureteral cancer, renal cancer renal pelvis, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, and the combination of the cancer. In various embodiments, the subject has previously responded to the treatment of anticancer therapy, but after stopping treatment, relapse (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject suffers from resistance or refractory cancer. In various embodiments, the cancer cell is an immunogenic tumor (for example, those tumors using the tumor itself to carry out vaccination can lead to immunity to tumor attack). In various embodiments, the cancer is selected from colorectal cancer (CRC), renal cancer, non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, and glioma.

[0275] In various embodiments, the antibodies and antigen-binding fragments thereof of the present invention directly kill or ablate cancer cells in vivo. Direct killing comprises administering the antibody (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 comparable tissue. Because the antibody recognizes Trop-2 on cancer cells, any such cells bound by the antibody will be destroyed. Where the antibody is used alone to kill or ablate cancer cells, such killing or ablation can be effected by activating endogenous host immune functions such as CDC and / or ADCC. Assays to determine whether an antibody kills cells in this manner are within the capabilities of those skilled in the art.

[0276] In various embodiments, the antibodies and antigen-binding fragments thereof of the present invention can be used to promote growth inhibition and / or proliferation 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, where 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%.

[0277] Inhibition of cancer cell proliferation can be measured by cell-based assays such as bromodeoxyuridine (BRDU) incorporation (Hoshino et al., Int. J. Cancer 38, 369, 1986; Campana et al., J. Immunol. Meth. 107: 79, 1988; [ 3 [H]-thymidine incorporation (Chen, J., Oncogene 13:1395-403, 1996; Jeoung, J., J. Biol. Chem. 270:18367-73, 1995; the dye Alamar Blue (available from Biosource International) (Voytik-Harbin et al., In Vitro Cell Dev Biol Anim 34:239-46, 1998). Anchorage-independent growth of cancer cells is assessed by colony formation assay in soft agar, for example, by counting the number of cancer cell colonies formed on top of the soft agar (see Examples and Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989).

[0278] Inhibition of cancer cell growth in a subject can be assessed by monitoring cancer growth in the subject, for example in an animal model or in a human subject. An exemplary monitoring method is a tumorigenicity assay. In one example, a xenograft comprises human cells from a pre-existing tumor or from a 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, a hollow fiber assay is used to monitor tumorigenicity, which is described in U.S. Patent No. 5,698,413, which is incorporated herein by reference in its entirety.

[0279] The percentage of inhibition can be calculated by comparing the proliferation, anchorage-independent growth or growth of cancer cells under modulator treatment with that under negative control conditions (usually without modulator treatment). For example, when the number of cancer cells or cancer cell colonies (colony formation assay) or PRDU or [ 3 [H]-thymidine incorporation is shown as A (under modulator treatment) and C (under negative control conditions) with inhibition as (CA) / C x 100%.

[0280] Examples of tumor cell lines that are derived from human tumors and can be used for in vitro and in vivo studies include, but are not limited to, leukemia cell lines (e.g., CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-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-H522, and LXFL 529); small cell lung cancer cell lines (e.g., DMS 114 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 XF 498); melanoma cell lines (e.g., LOX 1 MVI, 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); renal 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).

[0281] In another aspect, the present invention provides a method for treating an infectious disease in a subject, comprising administering to the subject an isolated antibody or antigen-binding fragment of the present invention of a therapeutically effective amount (as a monotherapy or in a combination therapy regimen). In various embodiments, the subject suffers from an infectious disease caused by a pathogenic virus. In various embodiments, the subject suffers from an infectious disease caused by a pathogenic bacterium. In various embodiments, the subject suffers from an infectious disease caused by a pathogenic fungus. In various embodiments, the subject suffers from an infectious disease caused by a pathogenic parasite. In various embodiments, the subject suffers from an infectious disease that is resistant to treatment with conventional vaccines or is ineffective with conventional vaccines.

[0282] "Therapeutically effective amount" or "therapeutically effective dose" refers to that amount of the administered therapeutic agent which will relieve to some extent one or more of the symptoms of the disease being treated.

[0283] The therapeutically effective dose can be determined initially from cell culture assays using the IC 50 The dose can then be formulated in animal models to achieve the IC, including that determined in cell culture. 50 The circulating plasma concentration range of the drug can be determined more accurately. Such information can be used to more accurately determine a useful dose in humans. Levels in plasma can be measured, for example, by HPLC. The individual physician can select the exact composition, route of administration, and dosage based on the subject's condition.

[0284] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time (multiple or repeated or maintenance), and the dose can be proportionally reduced or increased according to the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form to facilitate administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dose for a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and the required pharmaceutical carrier. The specifications of the dosage unit forms of the present disclosure will be primarily determined by the unique characteristics of the antibody and the specific therapeutic or prophylactic effect to be achieved.

[0285] Therefore, it will be understood by those skilled in the art that, based on the disclosure provided herein, dosages and administration regimens are adjusted according to methods well known in the therapeutic art. That is, the maximum tolerable dose can be readily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, as can the time requirement for administering each agent to provide a detectable therapeutic benefit to the subject. Therefore, although certain dosages and administration regimens are exemplified herein, these examples in no way limit the dosages and administration regimens that can be provided to a subject when practicing this disclosure.

[0286] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated, and may include single or multiple doses. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen of the compositions of the present disclosure can be based on a variety of factors, including disease type, age, weight, sex, the subject's medical condition, the severity of the condition, the route of administration, and the specific antibody used. Therefore, the dosage regimen can vary greatly, but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, such as toxic effects and / or laboratory values. Therefore, the present disclosure includes intra-subject dose escalation determined by a technician. Determining appropriate dosages and regimens is well known in the relevant art, and once provided with the teachings disclosed herein, those skilled in the art will understand.

[0287] For administration to human subjects, the total monthly dosage of the antibodies or antigen-binding fragments thereof of the present disclosure can be in the range of 0.5-1200 mg per subject, 0.5-1100 mg per subject, 0.5-1000 mg per subject, 0.5-900 mg / subject, 0.5-800 mg / subject, 0.5-700 mg / subject, 0.5-600 mg / subject, 0.5-500 mg / subject, 0.5-400 mg / subject, 0.5-300 mg / subject, 0.5-200 mg per subject, 0.5-400 mg / subject. The dosage of the antibody of the present invention can be 0.5-100 mg per experimenter, 0.5-50 mg per experimenter, 1-1200 mg per experimenter, 1-1100 mg per experimenter, 1-1000 mg per experimenter, 1-900 mg per experimenter, 1-800mg / experimenter, 1-700mg / experimenter, 1-600mg / experimenter, 1-500mg / experimenter, 1-400mg / experimenter, 1-300mg / experimenter, 1-200mg / experimenter, 1-100mg per experimenter, or 1-50 mg per experimenter, of course, depends on the mode of administration. For example, intravenous monthly dosage may need about 1-1000mg / experimenter. In various embodiments, antibody or its Fab of the present invention can be used with about 1-200mg / experimenter, 1-150mg / experimenter or 1-100mg / experimenter. Monthly total dose can be single or divided administration, and can fall outside the typical range provided herein according to the judgment of the doctor.

[0288] Exemplary non-limiting daily dosing ranges for a therapeutically or prophylactically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure can be 0.001 to 100 mg / kg, 0.001 to 90 mg / kg, 0.001 to 80 mg / kg, 0.001 to 70 mg / kg, 0.001 to 60 mg / kg, 0.001 to 50 mg / kg, 0.001 to 40 mg / kg, 0.001 to 30 mg / kg, 0.001 to 20 mg / kg, 0.001 to 10 mg / kg, 0.001 to 5 mg / kg, 0.001 to 4 mg / kg, 0.001 to 3 mg / kg, 0.001 to 2 mg / kg, 0.001 to 1 mg / kg, 0.010 to 50 mg / kg, 0.010 to 40 mg / kg, 0.010 to 30 mg / kg, 0.010 to 20 or 1 to 1 mg / kg body weight. It should be noted that dosage values ​​may vary with the type and severity of the condition to be alleviated. It should be further understood that 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 supervising the administration of the composition, and that the dosage ranges described herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0289] In various embodiments, the total dose administered will achieve 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, about 30 to 250 μg / ml.

[0290] Toxicity and therapeutic index of the pharmaceutical compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (The dose has a therapeutic effect on 50% of the population). The ratio of the toxic dose to the therapeutically effective dose is the therapeutic index, which can be expressed as LD50. 50 / ED 50比值表示 Compositions that exhibit large therapeutic indices are generally preferred.

[0291] In various embodiments, the pharmaceutical composition is administered in single or multiple doses depending on the dose and frequency required and tolerated by the subject. In any case, the composition should provide a sufficient amount of at least one antibody or antigen-binding fragment thereof disclosed herein to effectively treat the subject. The dose can be administered once, but can be applied periodically until a therapeutic effect is achieved or until side effects require cessation of treatment.

[0292] The frequency of administration of the antibody or antigen-binding fragment thereof pharmaceutical composition depends on the nature of the treatment and the specific disease being treated. Subjects can be treated regularly, for example weekly or monthly, until the desired therapeutic outcome is achieved, or treated with a loading dose followed by a maintenance dose on a regular basis. Exemplary dosing frequencies include, but are not limited to: once a week without interruption; once a week, once every other week; once every two weeks; once every three weeks; weakly weekly without interruption for 2 weeks, then monthly; once a week without interruption for 3 weeks, then monthly; monthly; every other month; every three months; every four months; every five months; or every six months, or annually.

[0293] Combination therapy

[0294] As used herein, the terms "co-administration," "co-administration," and "combination" are intended to refer to an antibody or antigen-binding fragment thereof of the present disclosure and one or more additional therapeutic agents, and do mean and include the following: simultaneous administration of such combination of an antibody or antigen-binding fragment thereof of the present disclosure and a therapeutic agent to a subject in need of treatment that, when such components are formulated together into a single dosage form, release said components to said subject substantially simultaneously; substantially simultaneous administration of such combination of an antibody or antigen-binding fragment thereof of the present disclosure and a therapeutic agent to a subject in need of treatment that, when such components are formulated separately from one another into separate dosage forms, release said components to said subject substantially simultaneously; Such combinations of an antibody or antigen-binding fragment thereof of the present disclosure and a therapeutic agent are administered sequentially to a subject in need of treatment, when such components are formulated separately from one another into separate dosage forms, by being taken successively to the subject with a significant time interval between each administration so that the components are released to the subject at substantially different times; when these components are formulated together into a single dosage form that releases the components in a controlled manner, such combinations of an antibody or antigen-binding fragment thereof of the present disclosure and a therapeutic agent are administered sequentially to a subject in need of treatment so that they are released to the subject simultaneously, successively and / or overlappingly at the same and / or different times, wherein each part can be administered by the same or different routes.

[0295] In another aspect, the present invention relates to a combination therapy designed for treating cancer or 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 invention, and b) one or more additional therapies selected from immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, vaccination regimens, and stem cell transplantation, wherein the combination therapy provides increased tumor cell killing, i.e., there is a synergistic effect between the isolated antibody or antigen-binding fragment and the other therapy when co-administered.

[0296] In various embodiments, the immunotherapy is selected from the group consisting of: treatment with agonistic, antagonistic, or blocking antibodies directed against co-stimulatory 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 Treatment with blinatumomab: treatment involving the administration of biologic response modifiers such as IL-2, IL-12, IL-15, IL-21, GM-CSF, and 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); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment with TALL-104 cells; and treatment with immunostimulants such as the Toll-like receptor (TLR) agonists CpG and imiquimod.

[0297] Many conventional chemical compounds have been shown to have anti-tumor activity. These compounds have been used as agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemias or myeloid malignancies. Although chemotherapy is effective in treating various types of malignancies, many anti-tumor compounds can cause adverse side effects. It has been shown that when two or more different treatments are combined, these treatments can work synergistically and allow for a reduction in the dose of each treatment, thereby reducing the harmful side effects that can occur at higher doses of each compound. In other cases, a malignancy that is refractory to treatment may respond to a combination of two or more different treatments.

[0298] When the antibodies or antigen-binding fragments disclosed herein are administered in combination with another conventional anti-tumor agent, whether administered simultaneously or sequentially, such antibodies or antigen-binding fragments can enhance the therapeutic effect of the anti-tumor agent or overcome cellular resistance to such anti-tumor agent. This allows for a reduction in the dose of the anti-tumor agent, thereby reducing undesirable side effects, or restoring the effectiveness of the anti-tumor agent in resistant T cells.

[0299] Drug compounds that can be used in combination anti-tumor therapy include, for illustration only: aminoglutamine, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestilbestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, goserelin, hydroxyurea, idarubicin astrocytes, ifosfamide, imatinib, interferon, irinotecan, ferrocephalosporin, letrozole, folinic acid, leuprorelin, levamisole, lomustine, meclofenac, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, niconazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentosamine statins, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozotocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vinorelbine.

[0300] These chemotherapeutic antitumor compounds can be classified into the following groups according to their mechanism of action: antimetabolites / anticancerosine agents, such as pyrimidine analogs (5-fluorouracil, fluorouracil, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimiotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and naviribine, epipodophyllotoxins (etoposide, teniposide), DNA damaging drugs (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cyclophosphamide, cytosine, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylpyridamole oxaliplatin, ifosfamide, melphalan, methylclophanamine, mitomycin, mitoxantrone, promycin, nitrourea-paclitaxel, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; the enzyme (L-asparaginase, which systemically metabolizes L-asparagine and deprives asparagine from cells that are unable to synthesize their own antiplatelet drugs; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozotocin), triazine (dacarbazine (DTIC)); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutamine; hormones, hormone analogs (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 (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP-470, genistein) and growth factor 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 (retinoic acid); 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 prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0301] In various embodiments, chemotherapy comprises a chemotherapeutic agent selected from the group consisting of daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, mechlorethamine, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), fluorouracil (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fluadabine, ifosfamide, hydroxyurea taxanes (e.g., paclitaxel and docetaxel) and / or anthracycline antibiotics, and combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP, or FOLFOX.

[0302] In various embodiments, the small molecule kinase inhibitor targeted therapy comprises a small molecule kinase inhibitor selected from Bruton's tyrosine kinase (BTK) inhibitors, phosphatidylinositol-3-kinase (PI3K) inhibitors, SYK inhibitors (e.g., entospletinib), AKT inhibitors, mTOR inhibitors, Src inhibitors, JAK / STAT inhibitors, Ras / Raf / MEK / ERK inhibitors, and Aurora inhibitors (see D'Cruz et al., Expert Opin Pharmacother, 14(6):707-21, 2013).

[0303] In various embodiments, the combination therapy comprises simultaneous administration of an antibody or antigen-binding fragment thereof and one or more additional therapies. In 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 before or after the one or more additional therapies.

[0304] In various embodiments, administration of the antibody or antigen-binding fragment thereof composition and one or more additional therapies is concurrent, ie, the administration periods of the antibody or antigen-binding fragment thereof composition and one or more additional therapies overlap with each other.

[0305] In various embodiments, the administration of the antibody or antigen-binding fragment thereof composition and the one or more additional therapies is non-simultaneous. For example, in various embodiments, administration of the antibody or antigen-binding fragment thereof composition is terminated prior to administration of the one or more additional therapies. In various embodiments, administration of the one or more additional therapies is terminated prior to administration of the antibody or antigen-binding fragment thereof composition.

[0306] When the antibodies or antigen-binding fragments thereof disclosed herein are administered in combination with one or more additional therapies, concomitantly or sequentially, such antibodies or antigen-binding fragments thereof can enhance the therapeutic effect of the one or more additional therapies or overcome cellular resistance to the one or more additional therapies. This allows for a reduction in the dose or duration of the one or more additional therapies, thereby reducing undesirable side effects, or restoring the effectiveness of the one or more additional therapies.

[0307] Diagnostic uses

[0308] In another aspect, the present invention provides a method for detecting the presence of a human Trop-2 antigen in a sample in vitro or in vivo, for example, for diagnosing a human Trop-2-associated disease. In some methods, this is achieved by contacting a test sample and a control sample with a human sequence antibody or human monoclonal antibody of the invention, or an antigen-binding portion thereof (or bispecific or multispecific molecule), under conditions that allow for the formation of a complex between the antibody and human Trop-2. Complex formation is then detected in both samples (e.g., using an ELISA), and any statistically significant difference in complex formation between the samples indicates the presence of a human Trop-2 antigen in the test sample.

[0309] In various embodiments, a method for detecting cancer or confirming a cancer diagnosis in a subject is provided. The method comprises contacting a biological sample from the subject with an antibody or its Fab of the present invention and detecting the binding of the isolated human monoclonal antibody or its Fab to the sample. Compared with the binding of the isolated human monoclonal antibody or its Fab to the control sample, the binding of the isolated human monoclonal antibody or its Fab to the sample increases the detection of cancer in the subject or confirms the diagnosis of the subject's cancer. The control can be a sample or a standard value from a known subject who does not suffer from cancer. The sample can be any sample, including but not limited to tissue from a biopsy, autopsy, and pathological specimen. Biological samples also include tissue sections, for example, frozen sections for histological purposes. Biological samples also include body fluids, such as blood, serum, plasma, sputum, and spinal fluid.

[0310] In one embodiment, a kit for detecting Trop-2 in a biological sample, such as a blood sample, is provided. The kit for detecting the polypeptide generally comprises a human antibody that specifically binds to Trop-2, such as any of the antibodies disclosed herein. In some embodiments, the kit includes an antibody fragment, such as an Fv fragment. For in vivo use, the antibody can be a scFv fragment. In another embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzymatic label).

[0311] In one embodiment, the kit includes instructional materials describing methods for using an antibody that specifically binds to Trop-2. The instructional materials may be written in electronic form (e.g., on a computer floppy disk or CD) or may be visual (e.g., a video file). The kit may also include additional components to facilitate the specific application for which the kit is designed. Thus, for example, the kit may additionally include means for detecting the label (e.g., an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, a suitable secondary label such as a secondary antibody, etc.). The kit may additionally include buffers and other reagents conventionally used to practice a particular method. Such kits and appropriate contents are well known to those skilled in the art.

[0312] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassay may vary depending on the particular format employed, methods for detecting TROP-2 in a biological sample generally comprise the steps of contacting the biological sample with an antibody that specifically reacts with TROP-2 under immunoreactive conditions. The antibody is allowed to specifically bind under immunoreactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0313] In various embodiments, the antibody or antigen-binding fragment can be labeled or unlabeled for diagnostic purposes. Typically, diagnostic assays require detection of the formation of a complex resulting from the binding of the antibody to Trop-2. The antibody can be directly labeled. A variety of labels can be used, including but not limited to radionuclides, fluorescent agents, 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, e.g., U.S. Patent Nos. 3,817,827; 3,850,752; 3,901,654; and 4,098,876). When unlabeled, the antibody can be used in assays, such as agglutination assays. The unlabeled antibody can also be used in combination with another (one or more) suitable reagents that can be used to detect the antibody, such as a labeled antibody (e.g., a secondary antibody) that reacts with a primary antibody (e.g., an anti-idiotypic antibody or other antibody specific for an unlabeled immunoglobulin) or other suitable reagent (e.g., labeled protein A).

[0314] The antibodies or antigen-binding fragments provided herein can also be used in methods for detecting a mammal's susceptibility to certain diseases. For illustration, this method can be used to detect a mammal's susceptibility to a disease that progresses based on the amount of Trop-2 present on cells and / or the number of Trop-2-positive cells in the mammal. In one embodiment, the present application provides a method for detecting a mammal's susceptibility to a tumor. In this embodiment, a sample to be tested is contacted with an antibody that binds to Trop-2 or a portion thereof under conditions suitable for binding to the antibody, wherein the sample comprises cells expressing Trop-2 in a normal individual. Binding of the antibody and / or the amount of binding is detected, which indicates the individual's susceptibility to the tumor, wherein higher levels of the receptor correlate with increased susceptibility to the tumor in the individual.

[0315] In various embodiments, the antibody or antigen-binding fragment is linked to a label that can be detected (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, for example, 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 Tc. Binding agents attached to such moieties can be used as imaging agents and administered in an amount effective for diagnostic use in mammals, such as humans, and the localization and accumulation of the imaging agent can be detected. The localization and accumulation of the imaging agent can be detected by radioscintigraphy, magnetic resonance imaging, computed tomography, or positron emission tomography.

[0316] Immunoscintigraphy using antibodies or antigen-binding fragments directed against Trop-2 can be used to detect and / or diagnose cancer and vasculature. 99 technetium, 111 Indium or 125Iodine-labeled Trop-2 labeled monoclonal antibodies can be effectively used for such imaging. As will be apparent to those skilled in the art, the amount of radioisotope to be administered depends on the radioisotope. One of ordinary skill in the art can easily formulate the amount of imaging agent to be administered based on the specific activity and energy of the given radionuclide used as the active portion. Typically, 0.1-100 millicuries, or 1-10 millicuries, or 2-5 millicuries are administered per dose of imaging agent. Thus disclosed compositions can be used as imaging agents, comprising a 0.1-100 millicuries targeting portion coupled to a radioactive portion, in some embodiments 1-10 millicuries, in some embodiments 2-5 millicuries, and in some embodiments 1-5 millicuries.

[0317] Immunoconjugates

[0318] The application further provides immunoconjugates comprising antibodies of the present invention or their antigen-binding fragments that are directly or indirectly coupled (or connected) to effector molecules. In this regard, the term "coupled" or "connected" refers to making two polypeptides into a continuous polypeptide molecule. The connection can be carried out by chemical or recombinant means. In one embodiment, the connection is chemical, wherein the reaction between the antibody portion and the effector molecule produces a covalent bond formed between the two molecules to form a molecule. A peptide linker (short peptide sequence) can optionally 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 bound to the effector molecule is further bound to a lipid, protein or peptide to increase its half-life in vivo. Therefore, in various embodiments, the antibodies of the present disclosure can be used to deliver a variety of effector molecules.

[0319] The effector molecule can be a detectable label, an immunotoxin, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent.

[0320] Specific non-limiting examples of immunotoxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, bile toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells.

[0321] "Cytokines" are a class of proteins or peptides released by one cell population that act as intercellular mediators on another cell. Cytokines can act as immunomodulators. Examples of cytokines include lymphokines, monokines, growth factors, and traditional polypeptide hormones. Thus, embodiments can utilize interferons (e.g., IFN-α, IFN-β, and IFN-γ); members of the tumor necrosis factor superfamily (TNFSF); human growth hormone; thyroxine; insulin; proinsulin; relaxin; relaxin precursor; follicle-stimulating hormone (FSH); thyroid-stimulating hormone (TSH); luteinizing hormone (LH); liver growth factor; prostaglandins, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α; tumor necrosis factor-β; integrins; thrombopoietin (TPO); nerve growth factors, such as NGF-β. Platelet growth factor; transforming growth factor-α; transforming growth factor-β; insulin-like growth factor-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 to 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 native sequence cytokines.

[0322] Chemokines can also be coupled to the antibodies disclosed herein. Chemokines are a superfamily of small (about 4 to about 14 kDa), inducible and secreted proinflammatory cytokines that primarily serve as chemoattractants and activators for specific leukocyte subtypes. The production of chemokines is induced by inflammatory cytokines, growth factors, and pathogenic stimuli. Chemokine proteins are divided into subfamilies (α, β, and δ) based on conserved amino acid sequence motifs and are divided into four highly conserved groups, CXC, CC, C, and CX3C, based on the positions of the first two cysteines adjacent to the amino terminus. To date, more than 50 chemokines have been discovered, including at least 18 human seven-transmembrane domain (7TM) chemokine receptors. The chemokines used include, but are not limited to, RANTES, MCAF, MCP-1, and fractalkine.

[0323] The therapeutic agent can be a chemotherapeutic agent. Those skilled in the art can readily identify the chemotherapeutic agent used (e.g., see Slapak and Kufe, Princes of Cancer Therapy, Chapter 86, Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Chapter 17, Abeloff, Clinical Oncology 2nd edition). .2000Churchill Livingstone, Inc; Baltzer L., Berkery R. (eds): Oncology PocketGuide to Chemotherapy, 2nd Edition St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-YearBook, 1993). Useful chemotherapeutic agents for preparing immunoconjugates include auristatin, dolatin, MMAE, MMAF, AFP, DM1, AEB, doxorubicin, daunorubicin, methotrexate, melphalan, chlorambucil, vinca alkaloids, 5-fluorouridine, mitomycin-C, paclitaxel, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, mechlorethamine, cyclophosphamide, etoposide, BCNU, irinotecan, camptothecin, bleomycin, idarubicin, actinomycin, plicamycin, mitoxantrone, asparagine, vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel, and their salts, solvents and derivatives. In various embodiments, the chemotherapeutic agent is auristatin E (also known as dolastatin-10 in the art) or its derivatives and pharmaceutical salts or solvates thereof. Typical auristatin derivatives include DM1, AEB, AEVB, AFP, MMAF and MMAE. The synthesis and structure of auristatin E and its derivatives and linkers are described in, for example, 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 herein by reference in its entirety). In various embodiments, the therapeutic agent is auristatin or an auristatin derivative. In different embodiments, the auristatin derivative is dovaline-valine-dolaisoleunine-dolaproine-phenylalanine (MMAF) or monomethyauristatin E (MMAE). In various embodiments, the therapeutic agent is a maytansinoid or a maytansinol analog. In various embodiments, the maytansinoid is DM1.

[0324] The effector molecule can be connected to the antibody or antigen-binding fragment of the present invention using any number of means known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. The process of attaching the effector molecule to the antibody varies depending on the chemical structure of the effector molecule. Polypeptides typically contain a variety of functional groups; for example, carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause the effector molecule to bind. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. Derivatization can involve attaching any one of many linker molecules, such as those available from Pierce Chemical Company in Rockford, Illinois. A linker can be any molecule used to attach the antibody to the effector molecule. A linker can form a covalent bond with 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 carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and effector molecule are polypeptides, the linker can be attached to the α-carbon amino and carboxyl groups of the constituent amino acids or to the terminal amino acids through its side groups (e.g., through a disulfide bond with cysteine).

[0325] In some cases, when the immunoconjugate reaches its target site, it is desirable to release the effector molecule from the antibody. Therefore, in these cases, the immunoconjugate will contain a bond that is cleavable near the target site. Cleavage of the linker to release the effector molecule from the antibody can be promoted by enzymatic activity or by conditions to which the immunoconjugate is subjected inside the target cell or near the target site.

[0326] Procedures for coupling antibodies to effector molecules have been previously described and are within the scope of those skilled in 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. Pat. No. 5,057,313; Shih Cancer Research Center. 51:4192, International Publication WO 02 / 088172; U.S. Pat. No. 6,884,869; International Patent Publication WO 2005 / 081711; U.S. Published Application 2003-0130189A; and U.S. Patent Application No. 20080305044, each of which is incorporated herein by reference for the purpose of teaching this technology.

[0327] The immunoconjugates of the present invention retain the immunoreactivity of the antibody or antigen-binding fragment, for example, the antibody or antigen-binding fragment has approximately the same or only slightly reduced ability to bind antigen after conjugation as before conjugation. As used herein, immunoconjugates are also referred to as antibody-drug conjugates (ADCs). An antibody-drug conjugate (ADC) according to one embodiment of the present invention has the formula: Ab-(LD)n, wherein Ab is a Trop-2 binding antibody, L is a linker, D is a drug moiety, and n is an integer of 2, 4, 6, or 7.

[0328] In another aspect, an isolated immunoconjugate or fusion protein is provided, comprising an antibody or antigen-binding fragment coupled, linked (or stably associated) to an effector molecule. In various embodiments, the effector molecule is an immunotoxin, a cytokine, a chemokine, a therapeutic agent, or a chemotherapeutic agent.

[0329] Bispecific molecules

[0330] In another aspect, the present invention features a bispecific molecule comprising an anti-Trop-2 antibody or antigen-binding fragment thereof of the present invention. The antibody or antigen-binding fragment thereof of the present invention can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand) to generate binding to at least two different binding sites or target molecules. The antibodies of the present invention can actually be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To generate the bispecific molecules of the present invention, the antibodies of the present invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, thereby generating a bispecific molecule. In various embodiments, the present invention includes bispecific molecules capable of binding to 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 and trigger Fc receptor-mediated effector cell activity, such as phagocytosis of Trop-2 expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anions. Methods for preparing the bispecific molecules of the invention are well known in the art.

[0331] In various embodiments, another functional molecule linked to the anti-Trop-2 antibody, such as another antibody or ligand for a receptor, can be selected from: agonistic, antagonistic or blocking antibodies against signaling molecules, such as Her-2, Her-3, EGFR, IGF-R, c-Met, EphA2, EphB2 and MUC16; agonistic, antagonistic or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as PD-1, PD-L1, OX-40, CS137, GITR, LAG3, TIM-3 and VISTA; CD3 found on T cells.

[0332] Polynucleotide and antibody expression

[0333] The present application further provides a polynucleotide comprising a nucleotide sequence encoding an anti-Trop-2 antibody or antigen-binding fragment thereof (eg, any anti-Trop-2 antibody or antigen-binding fragment thereof described herein).

[0334] In some embodiments, the present application provides polynucleotides encoding any of SEQ ID Nos: 15-18 and 23-56.

[0335] In some embodiments, the present application provides a polynucleotide comprising a nucleic acid sequence set forth in any one of SEQ ID Nos: 19-22.

[0336] Due to the degeneracy of the genetic code, various nucleic acid sequences encode each antibody amino acid sequence. The present application further provides polynucleotides that hybridize to a polynucleotide encoding an antibody that binds human Trop-2 under stringent or less stringent hybridization conditions, such as those defined herein.

[0337] Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6xSSC at about 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C, highly stringent conditions such as hybridization to filter-bound DNA in 6xSSC at about 45°C, followed by a wash at about 60°C, or any other stringent hybridization conditions (see, e.g., Ausubel, FM et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY, pages 6.3.1 to 6.3.6 and 2.10.3).

[0338] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotides is determined. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)). In short, this involves synthesizing overlapping oligonucleotides comprising a sequence encoding a portion of the antibody, annealing and connection of these oligonucleotides, and then amplifying the connected oligonucleotides by PCR. In one embodiment, the codons used include typical codons for humans or mice (see, for example, Nakamura, Y., Nucleic Acids Res. 28:292 (2000)).

[0339] The polynucleotide encoding the antibody can also be produced by nucleic acid from a suitable source. If the clone containing the nucleic acid encoding a specific antibody is unavailable, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or from a suitable source (e.g., an antibody cDNA library, or a cDNA library, or nucleic acid, preferably polyA+RNA, isolated from any tissue or cell expressing the antibody, such as a hybridoma cell that selects to express the antibody) by PCR amplification using synthetic primers that can hybridize with 3' and 5' or by using oligonucleotide probes specific for a particular gene sequence to clone, for example, to identify a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid produced by PCR can then be cloned into a reproducible cloning vector using any method well known in the art.

[0340] The present invention also relates to host cells that express the Trop-2 polypeptides and / or anti-Trop-2 antibodies of the present invention. A variety of host expression systems known in the art can be used to express the antibodies of the present invention, including prokaryotic (bacterial) and eukaryotic expression systems (e.g., yeast, baculovirus, plant, mammalian and other animal cells, transgenic animals, and hybridoma cells), as well as phage display expression systems.

[0341] The antibodies of the present invention can be prepared by recombinantly expressing immunoglobulin light chain and heavy chain genes in host cells. In order to recombinantly express antibodies, host cells are transformed, transduced, infected or similar with one or more recombinant expression vectors, and the one or more recombinant expression vectors carry DNA fragments of immunoglobulin light chain and / or heavy chain encoding antibodies so that light chain and / or heavy chain are expressed in host cells. The heavy chain and light chain can be expressed independently from different promoters and operably connected in one vector, or the heavy chain and light chain can be expressed independently from different promoters and operably connected in two vectors, one expressing the heavy chain and the other expressing the light chain. Optionally, the heavy chain and light chain can be expressed in different host cells.

[0342] In addition, the recombinant expression vector can encode a signal peptide that promotes secretion of the antibody light chain and / or heavy chain from the host cell. The antibody light chain and / or heavy chain gene can be cloned into the vector so that the signal peptide is operably linked to the amino terminus of the antibody chain gene in the reading frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Preferably, the recombinant antibody is secreted into the culture medium of the host cell, and the antibody can be recovered or purified from the culture medium.

[0343] 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 the heavy chain constant region. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments comprising these regions can be obtained, for example, by standard PCR amplification. The heavy chain constant region can be any type (e.g., IgG, IgA, IgE, IgM, or IgD), class (e.g., IgG1, IgG2, IgG3, and IgG4), or subclass constant region and any allotypic variants thereof as described in Kabat (supra).

[0344] The isolated DNA encoding the LCVR region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the LCVR to another DNA molecule encoding the light chain constant region. The sequences of human and other mammalian light chain constant region genes are known in the art. DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0345] In addition to the antibody heavy chain and / or light chain genes, the recombinant expression vectors of the present invention carry regulatory sequences that control the expression of the antibody chain genes in the host cell. As needed, 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 genes. The design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level expression of proteins in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)) and / or polyomavirus.

[0346] In addition, the recombinant expression vectors of the present invention can carry additional sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., origin of replication) and one or more selectable marker genes. Selectable marker genes help select host cells into which the vector has been introduced. For example, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate on host cells into which the vector has been introduced. Preferred selectable marker genes include dihydrofolate reductase (dhfr) genes (for dhfr-negative host cells with methotrexate selection / amplification), neo genes (for G418 selection), and glutamine synthetase (GS) (e.g., NSO) in GS negative cell lines for selection / amplification.

[0347] For expression of the light and / or heavy chains, expression vectors 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, and the like. Although the antibodies of the present invention can theoretically be expressed in prokaryotic or eukaryotic host cells, eukaryotic cells are preferred, and mammalian host cells are most preferred, because such cells are more likely to assemble and secrete properly folded and immunologically active antibodies. Preferred mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO cells) [including DHFR-minus CHO cells, such as Urlaub and Chasin, Proc. Natl. Am. Sci. USA 77:4216-20, 1980, used with a DHFR selection marker, such as 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 cells in a growth medium under appropriate conditions known in the art for a period of time sufficient to allow expression or, more preferably, secretion of the antibody into the host cells. The antibody can be recovered from the host cells and / or culture medium using standard purification methods.

[0348] The present invention provides host cells comprising nucleic acid molecules of the present invention. Preferably, the host cells of the present invention comprise one or more vectors or constructs comprising nucleic acid molecules of the present invention. For example, a host cell of the present invention is a cell into which a vector of the present invention has been introduced, wherein the vector comprises a polynucleotide encoding the LCVR of an antibody of the present invention and / or a polynucleotide encoding the HCVR of the present invention. The present invention also provides a host cell into which two vectors of the present invention have been introduced; one comprising a polynucleotide encoding the LCVR of an antibody of the present invention and one comprising a polynucleotide encoding the HCVR present in an antibody of the present invention and each operably linked to an enhancer / promoter regulatory element (e.g., derived from SV40, CMV, adenovirus and such as a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of gene transcription.

[0349] Once expressed, the complete antibodies of the present invention, individual light and heavy chains, or other immunoglobulin forms 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, and the like. For example, Feng Li, Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee describe methods for purifying antibodies in an article entitled "Current Therapeutic Antibody Production and Process Optimization" (BioProcessing Journal, September / October 2005, incorporated herein by reference in its entirety). In addition, 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). The effectiveness of known filtration in removing viruses from therapeutic antibody preparations depends, at least in part, on the concentration of protein and / or antibody in the solution to be filtered. The antibody purification method of the present invention may include a filtration step to remove viruses from the main stream of one or more chromatographic operations. Preferably, the chromatographic main stream containing the antibody of the present invention is diluted or concentrated to produce a total protein and / or total 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 pharmaceutical 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 is most preferred. Once partially purified or purified to homogeneity as needed, the sterile antibodies can then be used for treatment, as described herein.

[0350] In view of the above discussion, the present invention further relates to antibodies obtainable by a method comprising the steps of culturing a host cell, including but not limited to a mammalian, plant, bacterial, transgenic animal or transgenic plant cell, transformed with a polynucleotide or vector comprising a nucleic acid molecule encoding an antibody of the present invention, thereby expressing the nucleic acid, and optionally recovering the antibody from the host cell culture medium.

[0351] In certain aspects, the present application provides hybridoma cell lines and monoclonal antibodies produced by these hybridoma cell lines. The disclosed cell lines have uses other than for producing monoclonal antibodies. For example, the cell lines can be fused with other cells (e.g., appropriately drug-labeled human myeloma, mouse myeloma, human-mouse heteromyeloma, or human lymphoblastoid cells) to produce additional hybridomas, thereby providing transfer of the encoding gene. Monoclonal antibodies. In addition, the cell lines can be used as a source of nucleic acids encoding anti-Trop-2 immunoglobulin chains, which can be isolated and expressed (e.g., when transferred to other cells using any suitable technique (see, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Winter, U.S. Patent No. 5,225,539)). For example, clones containing rearranged anti-Trop-2 light or heavy chains can be isolated (e.g., by PCR), or cDNA libraries can be prepared from mRNA isolated from the cell lines, and cDNA clone chains encoding anti-Trop-2 immunoglobulins can be isolated. Therefore, it is possible to obtain and use the nucleic acid encoding the heavy chain and / or light chain of an antibody or its part according to recombinant DNA technology in a variety of host T cells or in an in vitro translation system, to produce specific immunoglobulin, immunoglobulin chain or its variant (for example, humanized immunoglobulin).For example, nucleic acid, including cDNA, or its coding variant such as a derivative of a humanized immunoglobulin or immunoglobulin chain, can be placed in a suitable protokaryotic or eukaryotic vector (for example, expression vector), and by a suitable method (for example, conversion, transfection, electroporation, infection), so that nucleic acid is operably connected to one or more expression control elements (for example, in a vector or integrated into the host T cell genome).For production, host T cells can be maintained under conditions suitable for expression (for example, in the presence of an inducing agent, being supplemented with a suitable culture medium of suitable salts, growth factors, antibiotics, nutritional supplements, etc.), thus producing the encoded polypeptide. If necessary, the encoded protein (for example, from host T cells or culture medium) can be recovered and / or separated. It will be appreciated that production methods include expression in host T cells of transgenic animals (see, e.g., WO 92 / 03918, GenPharm International, published March 19, 1992) (incorporated by reference in its entirety).

[0352] 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 necessary to transfect host cells with DNA encoding the light or heavy chains of an antibody of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that are not essential for binding to human Trop-2. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the invention.

[0353] Methods for expressing single-chain antibodies from bacteria such as E. coli and / or refolding into a suitable active form, including single-chain antibodies, have been described and are well known and 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, each incorporated herein by reference).

[0354] Functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using a strong denaturant before refolding. During the solubilization step, a reducing agent must be present to separate disulfide bonds, as is well known in the art. An exemplary buffer with 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 forms of low molecular weight thiol reagents, as described in Saxena et al., Biochemistry 9:5015-5021, 1970, incorporated herein by reference, and particularly as described in Buchner et al., supra.

[0355] Renaturation is typically accomplished by diluting (e.g., 100-fold) the denatured and reduced protein into 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.

[0356] As a modification of the diabody purification protocol, the heavy and light chain regions are solubilized and reduced separately and then combined in the refolding solution. Exemplary yields were obtained when the two proteins were mixed in a molar ratio such that one protein did not exceed a 5-fold molar excess of the other. After redox shuffling is complete, excess oxidized glutathione or other oxidizing low molecular weight compounds can be added to the refolding solution.

[0357] 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 and then sequentially adding the remaining amino acids in the sequence. Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology describes solid-phase synthesis techniques. Vol. 2: Special Methods in Peptide Synthesis, Part A, pp. 3-284; Merrifield et al., J. Am. Chem. Soc. Soc. Am. 85: 2149-2156, 1963 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, IL., 1984. Longer proteins can be synthesized by condensing the amino and carboxyl termini of shorter fragments. Methods for forming peptide bonds by activating the carboxyl terminus (eg, by using the coupling agent N,N'-dicyclohexylcarbodiimide) are well known in the art.

[0358] The following examples are provided to more fully illustrate the present invention but are not to be construed as limiting the scope thereof.

[0359] Example 1

[0360] Generation of monoclonal antibodies specifically targeting human Trop-2

[0361] Human Trop-2 (NP_002344.2, Met 1 -Ala 275 ) with a 6x His tag at the C-terminus (referred to as Trop-2-ECD-6His fusion protein), expressed in a human 293 cell line, purified on a Protein A column, and used as an immunogen. The estimated purity of the Trop-2-ECD-6His protein was greater than 95% based on SDS-PAGE under reducing conditions and visualized by silver staining. The amino acid sequence of Trop-2-ECD-6His is shown in SEQ ID NO: 2.

[0362] Female Balb / c and C57BI / 6 mice were immunized intraperitoneally (ip) three times (biweekly) at week 0 with 100 μg of Trop-2-ECD-6His per mouse and 50 μg of Trop-2-ECD-6His at weeks 2 and 4. In the first immunization, the antigen was injected in a 1:1 mixture with complete Freund's adjuvant (Sigma, St. Louis, MO). The second and third doses were injected in a 1:1 mixture with incomplete Freund's adjuvant (Sigma, St. Louis, MO). Mice received a final boost of 50 μg of Trop-2-ECD-6His at week 6. Seven days later, spleen cells were harvested and fused with the myeloma cell line SP2 / 0. Hybridomas were generated using electrofusion, and hybridoma supernatants were screened for antigen binding, ligand blocking, IgG binning, reference antibody binding, and FACS binding. Ten mAbs were ultimately selected from this initial screen for subcloning (limiting dilution) and further evaluation. BD Cell MAb medium was used to grow hybridomas in roller bottles to collect supernatants for antibody production. mAbs were purified using protein A affinity chromatography. The estimated mAb purity was greater than 90% based on Coomassie staining of SDS-PAGE. hRS7 is an anti-Trop2 antibody developed for sacituzumab govitecan (IMMU-132), an FDA-approved antibody-drug conjugate for the treatment of triple-negative breast cancer. It was used as the benchmark antibody for our studies. Secondary screening of the 20 purified mAbs against the benchmark hRS7 mAb included a human Trop-2-ECD-6His binding assay (ELISA) and an internalization assay using flow cytometry (FACS) analysis.

[0363] Based on the cumulative results of the analysis, two mAbs were selected for sequencing. Total RNA was extracted from frozen hybridoma cells according to the Reagent technical manual. Total RNA was analyzed by agarose gel electrophoresis. TM The 1st Strand cDNA Synthesis Kit technical manual allows for reverse transcription of total RNA into cDNA using isotype-specific antisense primers or universal primers. PCR is then performed to amplify the variable regions (heavy and light chains) of the antibody, which are then cloned into standard cloning vectors and sequenced.

[0364] mAb #118-2-5 comprises the heavy chain variable region sequence shown in SEQ ID NO: 15 and the light chain variable region sequence shown in SEQ ID NO: 17. mAb #125-1-5 comprises the heavy chain variable region sequence shown in SEQ ID NO: 16 and the light chain variable region sequence shown in SEQ ID NO: 18.

[0365] Example 2

[0366] Generation of chimeric IgG specifically targeting human Trop-2

[0367] Using the HCVR and LCVR sequences of mAbs #118-2-5 and #125-1-5, two mouse-human chimeric IgG1s (hereinafter referred to as "chimeric IgG1s") were prepared, comprising the heavy chain sequences listed in SEQ ID NOs: 47 and 49, and the light chain sequences listed in SEQ ID NOs: 48 and 50. The nucleic acids encoded by SEQ ID NOs: 19, 20 and 21, 22 were amplified and inserted into pTT5 to produce a plasmid expressing full-length IgG. The heavy and light chain expression plasmids were used to co-transfect 100 mL of HEK293-6E cells. The recombinant IgG secreted into the culture medium was purified using protein A affinity. Under non-reducing conditions, the purified IgG migrated as a ~150 kDa band in SDS-PAGE, and under reducing conditions, as ~55 kDa and ~30 kDa bands. The purity of the chimeric IgG was >90%. The binding affinity between chimeric IgG and human Trop-2-ECD-6His fusion protein (SEQ ID NO: 2) was determined by ELISA. The Trop-2-ECD-6His fusion protein was immobilized on the surface of a microplate well and incubated with the chimeric IgG. A secondary enzyme-labeled goat anti-mouse IgG-AP was then added. After washing, substrate was added to measure the activity of the microplate-bound enzyme. Color was developed and the absorbance was read at 405 nm.

[0368] Table 3 below summarizes the antigen binding assay data. As shown, two antibodies (#118-2-5 and #125-1-5) bind to Trop 2 with higher affinity than the benchmark reference antibody hRS7. See OD values ​​in Table 3. The data demonstrate that chimeric IgG #118-2-5 and #125-1-5 mAbs recognize the Trop-2-ECD-6His fusion protein better than the benchmark hRS7 IgG.

[0369] Table 3

[0370]

[0371]

[0372] Chimeric IgG1 #118-2-5 and #125-1-5 were further evaluated in an internalization assay using the Trop-2-positive breast cancer cell line MDA-MB-468. MDA-MB-468 cells were seeded at 1 x 10 cells / well. Next, chimeric or benchmark IgG was added to the cells in FACS buffer and incubated at 4°C for 2 hours. After incubation, the cells were washed to remove unbound antibody. The cells were then incubated at 37°C. After 2 hours, the cells were dissociated and stained with a fluorescently conjugated goat anti-mouse IgG secondary antibody on ice in the dark for 30 minutes and then analyzed using a Beckman flow cytometer system. Fluorescence intensity and percentage of cell binding at each time point were analyzed using Beckman flow cytometer software. The amount of chimeric IgG internalized into the cells was determined using the following formula: % internalization = 100 x (MFI 4°C – MFI 37°C) / MFI 4°C.

[0373] #118-2-5 showed better internalization rate than the benchmark IgG.The internalization assay data are summarized in Table 4 below.

[0374] Table 4

[0375] <![CDATA[ clone ]]> <![CDATA[ Internalization rate ]]> <![CDATA[ #118-2-5 ]]> <![CDATA[ 40.44％ ]]> <![CDATA[ #125-1-5 ]]> <![CDATA[ 4.70％ ]]> <![CDATA[ Benchmark ]]> <![CDATA[ 17.62％ ]]>

[0376] Internalization Rat

[0377] ELISA evaluates the cross-reactivity of chimeric IgG #118-2-5 and #125-1-5 to Trop-2 antigens from different species. In this experiment, 1 μg / mL human Trop-2 ECD-6 His, mouse Trop-2-His, cynomolgus monkey Trop-2-His, human EPCAM-His, and unrelated proteins were coated on microtiter plates at 50 μL / well and incubated at 4°C overnight. The plates were washed and blocked at 37°C for 1 hour and then washed with wash buffer. Purified chimeric IgG appropriately diluted in PBS was added to the wells and incubated at 37°C for 1 hour. After washing, bound antibodies were detected using HRP-conjugated goat anti-human IgG Ab. The color development and absorbance at OD450nm were read. Nonspecific mock mouse IgG and nonspecific mock human IgG were included as negative controls, while anti-His antibody was used as a positive control.

[0378] #118-2-5, #125-1-5, and benchmark IgGs bind to human and cynomolgus monkey Trop-2 antigens. They do not recognize mouse Trop-2, hEPCAM, and non-related proteins. Cross-reactivity binding assay data are summarized in Figure 1 middle.

[0379] Example 3

[0380] Generation of humanized Abs specifically targeting human Trop-2

[0381] A humanized anti-Trop-2 mAb derived from mAb #118-2-5 was prepared using a CDR grafting and backmutation approach. Briefly, the HCVR and LCVR sequences of the murine monoclonal antibody were BLAST-searched against human germline databases. The human receptors selected for VH and VL were GenBank 28401 and 28908, respectively. The CDRs and HV loops of the human receptors were replaced with their mouse counterparts (CDR grafting), which gave the sequence of the grafted antibody.

[0382] Typical residues in the CDRs, framework regions, and VH-VL interfaces of the grafted antibody that are considered important for binding activity were selected for replacement with the corresponding regions of the parent antibody. Homology modeling of the Trop-2 antibody Fv fragment was performed. A BLAST search of the Trop-2 sequence against the PDB_Antibody database was performed to identify the best template for the Fv fragment, particularly for constructing the domain interface. The structural template 1I3G (crystal structure of ampicillin single-chain Fv, version 1) was selected with an identity of 48.78%. A total of 34 amino acids, 21 of which from the HCVR (K5Q, Q6E, A13K, Q16E, S17T, I20L, L48I, S68T, K71V, N73T, S76N, V78F, F79S, M82L, N83S, L85V, Q86T, T87A, D88A, G91A, M92V) and 13 from the LCVR (Q3V, S9D, A15L, K18R, V19A, M21I, S22N, S49P, T68S, V84L, L89V, L109V, L111I) were identified for replacement. The reversed antibodies were then expressed in HEK293 cells and evaluated. Based on the evaluation, the humanized heavy chains constructed for screening lead humanized antibodies were named #118-2-5HuA, #118-2-5HuB, #118-2-5HuC and #118-2-5HuD and contained the heavy chain variable region sequences shown in SEQ ID NOs: 23-26, respectively, and the obtained humanized light chains were named #118-2-5LuX and #118-2-5LuY and contained the light chain variable region sequences listed in SEQ ID NOs: 27-28, respectively.

[0383] Eight humanized antibodies were expressed in HEK 293-6E cells using various combinations of HA-HD and LX-LY. Briefly, the amino acid sequence of any one of the heavy chain variable region amino acid sequences of SEQ ID NO: 23 (HuA), 24 (HuB), 25 (HuC) or 26 (HuD) and the amino acid sequence of any one of the light chain variable regions of SEQ ID NO: 27 (LuX) or 28 (LuY) were amplified and inserted into pTT5 to prepare expression plasmids for full-length IgG. The heavy and light chain expression plasmids were used to co-transfect 100 mL of HEK293-6E cells. The recombinant IgG secreted into the culture medium was purified using protein A affinity. The purified antibody was buffer-exchanged into PBS using a PD-10 desalting column. The purified IgG migrated as a ~150 kDa band in SDS-PAGE under non-reducing conditions, and the yield of 100 mL of culture exceeded 20 mg / L. The amino acid sequences of the HC and LC variable regions of the eight humanized Trop-2 antibodies are summarized in Table 5:

[0384] Table 5

[0385] Humanized IgG Heavy chain (HC) Light chain (LC) 1(HuA / LuX) Serial Number: 23 Serial Number: 27 2(HuB / LuX) Serial number: 24 Serial Number: 27 3(HuC / LuX) Serial number: 25 Serial Number: 27 4(HuD / LuX) Serial number: 26 Serial Number: 27 5(HuA / LuY) Serial Number: 23 Serial number: 28 6(HuB / LuY) Serial number: 24 Serial number: 28 7(HuC / LuY) Serial number: 25 Serial number: 28 8(HuD / LuY) Serial number: 26 Serial number: 28

[0386] Eight humanized IgGs were evaluated in the antigen binding assay and internalization assay described in Example 3. The results are summarized in Tables 6 and 7.

[0387] Table 6

[0388]

[0389] Table 7

[0390]

[0391] The cross-reactivity of eight humanized IgGs to Trop-2 antigens from different species was evaluated using the ELISA described in Example 3. All eight IgGs and the benchmark IgG were able to bind to human and cynomolgus monkey Trop-2 antigens. They did not recognize mouse Trop-2, hEPCAM, and non-related proteins. The cross-reactivity binding assay data are summarized in Figure 2 middle.

[0392] Based on the ranking of binding affinity and internalization rate, IgG HuA / LuX was selected for the second round of humanization. Five separate mutations were made on the heavy chain HuA Framework 3 to pair with the light chain Lux. These five new heavy chains were named #118-2-5Hu(A1), #118-2-5Hu(A2), #118-2-5Hu(A3), #118-2-5Hu(A4) and #118-2-5Hu(A5), and respectively contained the variable region sequences listed in SEQ ID Nos: 29-33. Six separate mutations were made on the light chain Lux Framework 1 / 2 / 3 to pair with the heavy chain HuA. These six new light chains were designated #118-2-5Lu(X1), #118-2-5Lu(X2), #118-2-5Lu(X3), #118-2-5Lu(X4), #118-2-5Lu(X5), and #118-2-5Lu(X6), and respectively comprised the variable region sequences set forth in SEQ ID Nos: 34-39. The resulting 11 humanized antibodies were evaluated in the antigen binding assay and internalization assay described in Example 3. The results are summarized in Tables 8 and 9, respectively.

[0393] Table 8

[0394] Antibody EC50nM HuD / LuY 0.06 HuA / LuX 0.39 HuA / Lu(X1) 0.26 HuA / Lu(X2) 0.36 HuA / Lu(X3) 0.45 HuA / Lu(X4) 0.12 HuA / Lu(X5) 0.31 HuA / Lu(X6) 0.26 Hu(A1) / LuX 0.28 Hu(A2) / LuX 0.37 Hu(A3) / LuX 0.31 Hu(A4) / LuX 0.39 Hu(A5) / LuX 0.34 Benchmark 0.09

[0395] Table 9

[0396] Antibody Internalization rate HuD / LuY 57.34％ HuA / LuX 96.77％ HuA / Lu(X1) 48.93％ HuA / Lu(X2) 43.24％ HuA / Lu(X3) 13.22％ HuA / Lu(X4) 61.53％ HuA / Lu(X5) 19.66％ HuA / Lu(X6) 46.43％ Hu(A1) / LuX 56.01％ Hu(A2) / LuX 14.59％ Hu(A3) / LuX 55.81％ Hu(A4) / LuX 52.81％ Hu(A5) / LuX 11.48％ Benchmark 10.87％

[0397] The #118-2-5 antibody was subjected to a third round of humanization, in which two mutations were simultaneously introduced at the X and Y points of the heavy chain variable region HuA framework 3 (designated #118-2-5HuA(1,2) (SEQ ID NO: 40), and then paired with the light chain variable region LuX4 (SEQ ID NO: 37) to obtain IgG HuA(1,2) / Lu(X4) (hereinafter also referred to as "A1,2X4") and IgG Hu(A1) / Lu(X4) (comprising SEQ ID NO: 29 / 37) (hereinafter also referred to as "A1X4") generated from the second round of humanization were selected for expression in HEK293 cell culture. Recombinant IgGs were secreted into the culture medium and purified using protein A affinity chromatography. The purified IgG migrated as a ~150 kDa band in SDS-PAGE under non-reducing conditions, and the yield from 100 mL of culture exceeded 20 mg / L. As assessed by SDS-PAGE, the purity of the humanized IgGs was above 90%.

[0398] Humanized anti-Trop-2 mAbs derived from mAb #125-1-5 were prepared using the same CDR grafting and backmutation methods described above. The humanized heavy chains constructed for screening lead humanized antibodies were designated #125-1-5Hu1, #125-1-5Hu2, #125-1-5Hu3, and #125-1-5Hu4 and comprised the sequences set forth in SEQ ID NOs: 41-44, respectively, while the resulting humanized light chains were designated #125-1-5Lu5 and #125-1-5Lu6 and comprised the sequences set forth in SEQ ID NOs: 45-46, respectively.

[0399] Humanized #125-1-5 antibody was evaluated in the antigen binding assay and internalization assay described in Example 3. The results are summarized in Tables 10 and 11, respectively.

[0400] Table 10

[0401]

[0402] Table 11

[0403]

[0404] Based on the ranking of binding affinity and internalization rate, IgG Hu3 / Lu5 was selected as the final humanized #125-1-5 antibody. The HC and LC sequences of Hu3 / Lu5 are summarized in Table 12:

[0405] Table 12

[0406]

[0407] Example 4

[0408] Production and characterization of humanized Trop-2 IgG

[0409] Two humanized Trop-2 binders, including A1, 2X4, A1X4 and benchmark hRS7, were used for full-length IgG expression, purification and affinity measurement.

[0410] The DNA sequences encoding A1, 2X4 and A1X4, including the leader sequence, were amplified and inserted into a vector to produce an expression plasmid for full-length IgG. The heavy and light chain expression plasmids were used to co-transfect 293 cells (100 ml cell culture). The recombinant IgG was secreted into the culture medium and purified using protein A affinity chromatography. Under non-reducing conditions, the purified IgG migrated as an approximately 150 kDa band in SDS-PAGE and migrated as approximately 55 kDa and approximately 30 kDa bands under reducing conditions.

[0411] The HC and LC sequences of A1X4 and A1,2X4 are summarized in Table 13:

[0412] Table 13

[0413] IgG Heavy chain (HC) Light chain (LC) A1X4 Serial number: 53 Serial number: 54 A1,2X4 Serial number: 51 Serial number: 52

[0414] Example 5

[0415] Binding affinity of humanized Trop-2 IgG

[0416] The binding affinity of purified anti-Trop-2 IgG for human Trop-2 protein was determined using the Octet system, which is based on biolayer interferometry (BLI) technology. BLI involves a layer of molecules attached to the tip of an optical fiber that produces an interference pattern at the detector; any change in the number of bound molecules results in a measured shift in the pattern. The Octet system allows real-time analysis of the affinity and kinetics of biomolecular interactions in a 96-well microplate. Briefly, Trop-2 antigen (human Trop-2 ECD-6His) was diluted to concentrations of 50, 25, 12.5, 6.25, and 3.125 nM and loaded into a 96-well microplate. A1X4 or A1,2X4 antibodies were diluted and added to the designated wells at a final concentration of 8 μg / ml. The plate was placed in the Octet system and the assay was started. Data were analyzed using OctetData Acquisition Software (fortebio data analysis 10.0) to calculate the association rate constant, Kon, the dissociation rate constant, Koff, and the dissociation constant, Kd (Kd = Koff / Kon).

[0417] Table 14 summarizes the Kd and Koff of selected anti-Trop-2 antibodies A1X4, A1,2X4, and the benchmark hRS7 from the Octet kinetic assay. The results show that the A1X4 and A1,2X4 antibodies of the present invention can bind to the human Trop-2 antigen and have intermediate binding affinity for huTrop-2 compared to the benchmark hRS7 antibody.

[0418] Table 14

[0419] IgG Kd(nM) Koff(1 / S) hRS7 0.78 2.44E-04 A1X4 2.11 5.69E-04 A1,2X4 4.09 8.44E-04

[0420] Previous studies have shown that high-affinity antibodies with slower Koff rates are more likely to interact bivalently with target cells, occupying two antigens with one Fc. In contrast, antibodies with faster Koff rates may dissociate each binding arm more quickly, thus resulting in monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved effector cell recruitment and ADDC activity. Similarly, another study also showed that antibodies with high Kd affinity have rapid internalization and degradation, which may limit their tumor penetration, while low-affinity antibodies with faster Koff are more effective in penetrating tumors because the rate of antibody-antigen dissociation is higher than the rate of antigen internalization. See, for example, Rudnick et al., Cancer Res. 2011 Mar 15;71(6):2250-2259. Given that the A1X4 and A1,2X4 antibodies have relatively low affinities but faster dissociation rates than the benchmark hRS7, these two antibodies, especially A1,2X4, may have more effective ADCC and better tumor penetration than hRS7.

[0421] Example 6

[0422] Specificity of humanized Trop-2 IgG

[0423] Purified IgG was tested for cross-reactivity with Trop-2 antigens from different species by ELISA. In this experiment, 1 μg / mL human Trop-2 ECD-6His, mouse Trop-2-His, cynomolgus monkey Trop-2-His, human EPCAM-His, and an unrelated protein were applied to the surface of a microtiter plate at 50 uL / well and incubated overnight at 4°C. The plate was washed and blocked at 37°C for 1 hour, then washed with wash buffer. Purified anti-Trop-2 antibody appropriately diluted in PBS was added to the wells and incubated at 37°C for 1 hour. After washing, bound antibody was detected using an HRP-conjugated goat anti-human IgG antibody. The color development and absorbance at OD450nm were read. Nonspecific human IgG1 was included as a negative control, while hRS7 was used as a positive control.

[0424] As shown in the picture. Figure 3 Results from experiments are shown demonstrating that the A1X4 and A1,2X4 antibodies can bind to human and cynomolgus monkey Trop-2 antigens with high efficiency similar to that of the hRS7 antibody ("BM"). It does not recognize mouse Trop-2, hEPCAM, and non-related proteins, whereas the benchmark hRS7 antibody shows some nonspecific binding to mouse Trop-2, hEPCAM, and non-related proteins.

[0425] Example 7

[0426] Internalization of Trop-2 mAb

[0427] Receptor-mediated antibody internalization can provide cell-specific drug delivery. Internalization is essential for some targeted therapies using ADCs. The speed and extent of ADC internalization are crucial for their effectiveness. Previous studies have shown that in some cases, unconjugated antibodies and their corresponding ADCs are internalized at the same rate, while in other cases, ADC internalization is affected by payload conjugation.

[0428] Internalization and degradation of anti-Trop-2 antibodies were measured by flow cytometry. In this study, a fluorescently labeled secondary antibody was used to detect the primary antibody that remained after internalization.

[0429] Trop-2 positive breast cancer cells MDA-MB-468 were cultured at 1×10 5 Cells / well were seeded. Next, hRS7, A1X4, or A1,2X4 antibodies were added to the cells at a concentration of 400, 1000, 3000, or 10,000 ng / ml in FACS buffer, respectively, and incubated at 4°C for 30 minutes. After incubation, the cells were washed three times with FACS buffer to remove unbound antibodies. The cells were then incubated at 37°C and 5% CO 2下孵育以 Antibody internalization was performed. After 2 hours, cells were dissociated with trypsin and stained with a fluorescently conjugated goat anti-human IgG secondary antibody for 30 minutes on ice in the dark, and then analyzed using a Beckman flow cytometer system. The fluorescence intensity and cell binding percentage at each time point were analyzed by Beckman flow cytometer software. The amount of anti-Trop-2 antibody internalized into cells at each concentration was determined using the following formula: % internalization = 100 x (MFI 4℃ –MFI 37℃ ) / MFI 4℃

[0430] As shown in the figure. Figure 4 depicts the results of the study. The results demonstrate that the anti-Trop-2 antibodies of the present invention can be internalized by cells expressing Trop-2, with the A1X4 antibody being the most potent. Both the A1X4 and A1,2X4 antibodies exhibited higher internalization rates compared to the benchmark antibody hRS7. Given that the A1X4 and A1,2X4 antibodies have slightly lower binding affinities than the hRS7 benchmark, our observations are consistent with published results from earlier studies showing that antibodies with intermediate affinities exhibited the highest levels of tumor accumulation, indicating good tumor penetration.

[0431] Example 8

[0432] ADCC activity of Trop-2 mAb

[0433] The antibody-dependent cell-mediated cytotoxicity (ADCC) assay was designed to test the ability of anti-Trop-2 antibodies to mediate lysis of target cells by effector cells. Peripheral blood mononuclear cells (PBMCs) were purified from pooled healthy human blood (from over 20 healthy donors) and used as effector cells. The Trop-2-positive human ovarian cancer cell line SK-BR-3 was used as the target cell line. SK-BR-3 target cells were incubated with serially diluted A1X4 antibody, A1,2X4 antibody, or A1,2X4 ADC at room temperature for 0.5 hours before the addition of effector cells. The effector-to-target (E:T) ratio was 25:1. After incubation at 37°C for 6 hours, the supernatant was transferred to another plate and incubated with lactate dehydrogenase (LDH) working solution at room temperature for 30 minutes. LDH activity was measured using PHERAStarPlus. Three replicates were performed for each data point. Data were fitted to a 4-parameter logistic (4PL) nonlinear regression model to obtain dose-response curves, and the EC50 was calculated using GraphPad Prism 6 software. Error bars: ±SEM. Herceptin antibody was used as a positive control, and human IgG1 was used as a negative control.

[0434] Calculate the percentage of cell lysis according to the following formula:

[0435] % cell lysis = 100 x (OD 样品 -OD 靶细胞加效应细胞 ) / (OD 最大释放 -OD 最小释放 )

[0436] As shown in the figure. Figure 5 As shown, the A1,2X4 antibody ADC exhibited robust ADCC activity against SK-BR-3 cells in an antibody dose-dependent manner. The ADCC activity was comparable to that observed with the positive control, Herceptin. The negative control showed no activity.

[0437] Example 9

[0438] Preparation of Anti-Trop-2-vc-MMAE and Anti-Trop-2-SMCC-DM1 Conjugates

[0439] Humanized anti-Trop-2 antibodies, including A1, 2X4, A1X4, and the benchmark hRS7, were conjugated to MMAE to form ADCs and evaluated for their ability to inhibit the growth of various cancer cell lines expressing varying levels of Trop-2. Monomethyl auristatin E (MMAE) is an anti-tumor agent that inhibits cell division by blocking the polymerization of tubulin. It is derived from a peptide found in marine shellless molluscs (algal toxins). MMAE has been shown to be a useful payload for ADCs.

[0440] The linker in an ADC can have a significant impact on biological activity. For example, in vivo studies have shown that peptide-linked conjugates induce regression and cure of established tumor xenografts, with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency, and conjugation methods in developing safe and effective ADCs for cancer therapy.

[0441] Some embodiments of the present invention relate to MMAE linked to antibodies via a lysosomal cleavable dipeptide, valine-citrulline (vc), or DM1 linked to antibodies via a non-cleavable amine-thiol crosslinker, which have been shown to improve ADC efficacy. In this example, anti-Trop-2 antibodies 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 buffer was exchanged into the conjugation reaction buffer. mc-vc-PABA-MMAE auristatin or SMCC-DM1 maytasinoid payload dissolved in DMSO was added to a reduced anti-Trop-2 monoclonal antibody (A1, 2X4, or A1X4) solution at a payload / antibody ratio of 5:1-7:1 to achieve different drug-to-antibody ratios (DARs). The payload / antibody solution was incubated at 20°C for 1-2 hours. After the coupling is complete, the reaction mixture is desalted and concentrated to produce the anti-Trop-2-vc-MMAE ADC. The biochemical characterization of the resulting ADC was performed using size exclusion chromatography high-pressure liquid chromatography (SEC-HPLC) to determine purity and aggregate content, and hydrophobic interaction chromatography HPLC (HIC-HPLC) to determine the drug:antibody ratio (DAR). The final ADC product, A1,2X4-MMAE or A1X4-MMAE or A1,2X4-DM1, is composed of two, four, or six MMAE-linked molecules.

[0442] Example 10

[0443] In vitro cytotoxicity and in vivo antitumor activity of ADCs with different DARs

[0444] The ADC containing a cytotoxic payload of the present invention can be used to kill target cells, such as cancer cells. For in vitro assays, MDA-MB-468, SK-BR-3, NCI-N87, Colo205, and A549 were tested. All cell lines were cultured in appropriate culture medium at 37°C in a humidified incubator atmosphere of 5% CO2. The cells were seeded in 96-well flat-bottom plates. The cell seeding number ranged from 500 cells / 100 μl / well to 6,000 cells / 100 μl / well. The cells were allowed to adhere overnight at 37°C in a humidified atmosphere of 5% CO2. A1X4-MMAE was prepared from a stock solution with DAR2, DAR4, or DAR6 and diluted to an appropriate working concentration 24 hours after cell seeding. Seven points were serially diluted tenfold with culture medium. The final concentration range was 1000 nM to 0.001 nM. The cells were incubated with the ADC for 72 hours. Cell Counting Kit-8 solution (Dojindo China Co., Ltd, lot #PL701) was added to the wells, placed at 37°C for 1-4 hours, and the absorbance at 450 nm was measured using a microplate reader (SpectraMax M5, Molecular Devices) and SoftMax Pro 5.4.1 software. The percentage of inhibition was calculated by the following formula: (mean absorbance of treated samples / mean absorbance of control samples) × 100. For all cell assays, dose-response curves were generated using GraphPad Prism 7 three-parameter curve fitting.

[0445] To compare the in vivo antitumor activity of ADCs with different DARs, A1X4-MMAE DAR2, A1X4-MMAE DAR4, and A1X4-MMAE DAR6 were tested in the MDA-MB-468 xenograft model. MDA-MB-468 cells were harvested from culture flasks and mixed with matrigel (1:1). Five million cells were subcutaneously implanted into the right flask of 6- to 7-week-old BALB / c nude mice. After tumor establishment, mice were randomized into groups based on tumor volume. ADCs were administered intravenously at a single bolus dose of 1.5 mg / kg. Tumors were measured twice a week throughout the study, and tumor volume was calculated using the following formula: Tumor volume (mm 3 )=(length×width 2 ) / 2. Data were analyzed by GraphPad Prism 7 and presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance.

[0446] The results of these measurements are shown in Figure 6 and Figure 7The results showed that DAR4 and DAR6 ADCs were more cytotoxic in vitro than DAR2 ADC and showed stronger anti-tumor activity in cells with medium / high Trop-2 expression (i.e., MDA-MB-468, SK-BR-3, NCI-N87). In tumor models with medium / high Trop-2 levels, ADCs with DAR4 and DAR6 were able to achieve durable tumor regression, while DAR2 ADC achieved regression for a period of time, but then the tumor began to regrow.

[0447] Example 11

[0448] Correlation between Trop-2 expression level and ADC cytotoxicity in vitro

[0449] To determine whether Trop-2 expression levels affect ADC activity, A1, 2X4 ADC, A1X4 ADC, and the benchmark hRS7 ADC were tested against BxPC-3 pancreatic cancer cells and MDA-MB-468 breast cancer cells, which represent high Trop-2 expression (MFI 161823, 74943), SK-BR-3 ovarian cancer cells and N87 gastric cancer cells, which represent moderate Trop-2 expression (MFI 53526, 40400, respectively), Colo205 colon cancer cells, which represent low Trop-2 expression (MFI 19444), and MDA-MB-231 breast cancer cells, which have undetectable Trop-2 levels. In vitro cytotoxicity assays were performed according to the procedures described in Example 9.

[0450] As shown in the picture. Figure 8A Trop-2 expression levels in BxPC-3, MDA-MB-468, NCI-N87, SK-BR-3, Colo205, and MDA-MB-231 cells were summarized, along with the maximum inhibition percentage and IC50 for each ADC tested. The results demonstrated a clear overall correlation between Trop-2 expression levels and the in vitro potency of anti-Trop-2 ADCs. A1X4-MMAE was most effective against Trop-2-high BxPC-3 cells, producing an IC50 value of less than 1 nM. It also induced potent cytotoxicity against moderately expressing MDA-MB-468, SK-BR-3, and NCI-N87 cells (IC50 values ​​in the single-digit nM range), and was least active against Colo-205 cells, which have low Trop-2 levels. On the other hand, A1,2X4-MMAE was highly effective against all cells expressing Trop-2 at varying levels (IC50 values ​​less than 1 nM). The in vitro activity of A1X4-MMAE and A1,2X4-MMAE was comparable to that observed with the benchmark hRS7 ADC. In Trop-2-negative MDA-MB-231 cells, no IC50 could be calculated for any of the three antibodies, indicating that specific killing activity was not achieved.

[0451] As shown in the picture. Figure 8B Shown is a correlation between Trop-2 expression levels and the in vitro potency of A1,2X4-MMAE.

[0452] Example 12

[0453] Bystander Killing Effect of Anti-Trop-2-vc-MMAE ADC

[0454] Bystander killing effects have been observed for some ADCs in preclinical studies. By transferring the released payload from antigen-expressing cells to adjacent antigen-negative cells, it can affect not only antigen-expressing tumor cells but also adjacent antigen-negative cells. To confirm whether A1,2X4-MMAE induces bystander killing, a transwell co-culture cell killing assay was performed. Trop-2-positive MDA-MB-468 cells were plated at a density of 3,000 cells / 500μl / well on the bottom of the plate. Cell culture inserts (Thermo, cat#140627) were inserted, and Trop-2-negative MDA-MB-231 cells were seeded in the inserts at the same density as MDA-MB-468. After incubation for 24 hours, A1,2X4-MMAE at concentrations of 1, 10, and 100nM was added to the MDA-MB-468 cells in the bottom wells and incubated for 72 hours. The cytotoxicity of MDA-MB-468 and MDA-MB-231 cells was measured by Cell Counting Kit-8 as described in Example 7. The inhibition percentage was calculated by the following formula: (mean absorbance of treated samples / mean absorbance of control samples)×100.

[0455] like Figure 9 As expected, A1,2X4-MMAE effectively inhibited the proliferation of Trop-2-positive MDA-MB-468 cells but had no effect on Trop-2-negative MDA-MB-231 cells. In in vitro co-culture conditions, A1,2X4-MMAE killed both MDA-MB-468 and MDA-MB-231 cells. Given the Trop-2-specific cytotoxicity of A1,2X4-MMAE, the killing of MDA-MB-231 cells is likely due to the released payload, suggesting that A1,2X4-MMAE exhibits a bystander killing effect. The results of this assay suggest that A1,2X4-MMAE exhibits potent bystander killing due to its highly membrane-permeable payload and the potential of A1,2X4 to target Trop-2-heterogeneous tumors.

[0456] Example 13

[0457] In vivo characterization of Anti-Trop-2-vc-MMAE ADC

[0458] The anti-tumor activity of the anti-Trop-2-vc-MMAE ADC was evaluated in mouse subjects using Trop-2-positive MDA-MB-468 and NCI-N87 xenograft models. Five million NCI-N87 cells were harvested from culture flasks and implanted subcutaneously into the right flask of 6- to 7-week-old BALB / c nude mice. Tumors were measured twice weekly throughout the experiment, and tumor volume was calculated using the following formula: Tumor volume (mm 3 )=(length x width 2 ) / 2.

[0459] The ADCs tested in the NCI-N87 model had different DAR values: the benchmark hRS7-MMAE had a DAR of 4.33, the A1X4-MMAE had a DAR of 6.1, and the A1,2X4-MMAE had a DAR of 5.8. Therefore, the dose of each ADC was adjusted to ensure the same amount of payload. Therefore, the benchmark hRS7-MMAE, A1X4-MMAE, or A1,2X4-MMAE was dosed at 0.5 mg / kg, 1.0 mg / kg, and 1.5 mg / kg. The ADCs were administered intravenously as a single bolus dose.

[0460] Figure 10A Results from an in vivo N87 xenograft study are shown. As shown, a single 5.0 mg / kg administration of the ADCs of the invention (A1X4-MMAE, A1,2X4-MMAE) induced durable tumor regression. An ADC dose of 1.5 mg / kg resulted in significant tumor growth inhibition, while the lowest dose of 0.5 mg / kg resulted in only modest tumor growth inhibition. Both A1X4-MMAE and A1,2X4-MMAE exhibited similar antitumor activity to the benchmark hRS-7-MMAE (BM-MMAE) in this model.

[0461] To further compare the antitumor activity of different ADCs, A1X4-MMAE and A1,2X4-MMAE were tested head-to-head in the MDA-MB-468 xenograft model. All ADCs were administered at a single dose of 1.5 mg / kg.

[0462] Figure 10B Depicts the results of an in vivo MDA-MB-468 xenograft study. Results demonstrate that all ADCs were able to achieve durable tumor regressions following a single 1.5 mg / kg dose. A1,2X4-MMAE was more potent than A1X4-MMAE.

[0463] Example 14

[0464] Comparison of antitumor activity of different dosing regimens of Anti-Trop-2-vc-MMAE ADC

[0465] The impact of dosing schedule on the anti-Trop-2 ADC's antitumor activity was evaluated by comparing single and split-dose schedules of A1,2X4-MMAE in the Trop-2-positive MDA-MB-468 xenograft model. Three dosing schedules were tested, including 1) a single dose of 1.5 mg / kg; 2) two doses of 0.75 mg / kg every 7 days; and 3) four doses of 0.375 mg / kg twice weekly.

[0466] like Figure 11 As shown, the results show that a single dose of 1.5 mg / kg is the most effective and more effective than the split-dose regimen, resulting in significant anti-tumor activity that lasted until day 28 of the study. A twice-weekly split dose of 0.375 mg / kg produced better tumor growth inhibition than a weekly split dose of 0.75 mg / kg. The fact that a shorter dosing interval (e.g., 3-4 days) produced better results at the same total dose was unexpected. This may indicate that for anti-Trop-2-vc-MMAE ADC, a higher AUC may be more important than a higher Cmax.

[0467] In view of the present disclosure, all articles and methods disclosed and claimed herein can be made and executed without undue experimentation. Although the articles and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles and methods without departing from the spirit and scope of the invention. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are considered to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes, and to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The invention illustratively described herein can be practiced without any element not specifically disclosed herein. Therefore, it should be understood that although the present invention has been specifically disclosed with respect to preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0468] Sequence Listing

[0469] The nucleic acid and amino acid sequences set forth in the accompanying sequence listing use standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822.

[0470] SEQ ID NO: 1 is the amino acid sequence of human TROP-2

[0471] SEQ ID NO: 2 is the amino acid sequence of human TROP-2-ECD-6His antigen

[0472] SEQ ID NO: 3 and 4 are the amino acid sequences of the heavy chain CDR1 of a murine monoclonal antibody that specifically binds to Trop-2

[0473] SEQ ID NO: 5 and 6 are the amino acid sequences of the heavy chain CDR2 of a murine monoclonal antibody that specifically binds to Trop-2

[0474] SEQ ID NOs: 7 and 8 are the amino acid sequences of the heavy chain CDR3 of a murine monoclonal antibody that specifically binds to Trop-2

[0475] SEQ ID NOs: 9 and 10 are the amino acid sequences of the light chain CDR1 of a murine monoclonal antibody that specifically binds to Trop-2

[0476] SEQ ID NOs: 11 and 12 are the amino acid sequences of the light chain CDR2 of a murine monoclonal antibody that specifically binds to Trop-2.

[0477] SEQ ID NOs: 13 and 14 are the amino acid sequences of the light chain CDR3 of a murine monoclonal antibody that specifically binds to Trop-2

[0478] SEQ ID NO: 15 is the amino acid sequence of the heavy chain variable region of mouse monoclonal antibody #118-2-5

[0479] SEQ ID NO: 16 is the amino acid sequence of the heavy chain variable region of mouse monoclonal antibody #125-1-5

[0480] SEQ ID NO: 17 is the amino acid sequence of the light chain variable region of mouse monoclonal antibody #118-2-5

[0481] SEQ ID NO: 18 is the amino acid sequence of the light chain variable region of mouse monoclonal antibody #125-1-5

[0482] SEQ ID NO: 19 is the nucleic acid sequence of the heavy chain variable region of chimeric antibody #118-2-5

[0483] SEQ ID NO: 20 is the nucleic acid sequence of the light chain variable region of chimeric antibody #118-2-5

[0484] SEQ ID NO: 21 is the nucleic acid sequence of the heavy chain variable region of chimeric antibody #125-1-5

[0485] SEQ ID NO: 22 is the nucleic acid sequence of the light chain variable region of chimeric antibody #125-1-5

[0486] SEQ ID NO: 23 is the amino acid sequence of the heavy chain HuA variable region of humanized antibody #118-2-5

[0487] SEQ ID NO: 24 is the amino acid sequence of the heavy chain HuB variable region of humanized antibody #118-2-5

[0488] SEQ ID NO: 25 is the amino acid sequence of the heavy chain HuC variable region of humanized antibody #118-2-5

[0489] SEQ ID NO: 26 is the amino acid sequence of the heavy chain HuD variable region of humanized antibody #118-2-5

[0490] SEQ ID NO: 27 is the amino acid sequence of the light chain HuX variable region of humanized antibody #118-2-5

[0491] SEQ ID NO: 28 is the amino acid sequence of the light chain HuY variable region of humanized antibody #118-2-5

[0492] SEQ ID NO: 29 is the amino acid sequence of the variable region of the heavy chain Hu(A1) of the humanized antibody #118-2-5

[0493] SEQ ID NO: 30 is the amino acid sequence of the variable region of the heavy chain Hu(A2) of humanized antibody #118-2-5

[0494] SEQ ID NO: 31 is the amino acid sequence of the variable region of the heavy chain Hu(A3) of humanized antibody #118-2-5

[0495] SEQ ID NO: 32 is the amino acid sequence of the variable region of the heavy chain Hu(A4) of humanized antibody #118-2-5

[0496] SEQ ID NO: 33 is the amino acid sequence of the variable region of the heavy chain Hu(A5) of humanized antibody #118-2-5

[0497] SEQ ID NO: 34 is the amino acid sequence of the light chain Lu (X1) variable region of humanized antibody #118-2-5

[0498] SEQ ID NO: 35 is the amino acid sequence of the light chain Lu (X2) variable region of humanized antibody #118-2-5

[0499] SEQ ID NO: 36 is the amino acid sequence of the light chain Lu (X3) variable region of humanized antibody #118-2-5

[0500] SEQ ID NO: 37 is the amino acid sequence of the light chain Lu (X4) variable region of humanized antibody #118-2-5

[0501] SEQ ID NO: 38 is the amino acid sequence of the light chain Lu (X5) variable region of humanized antibody #118-2-5

[0502] SEQ ID NO: 39 is the amino acid sequence of the light chain Lu (X6) variable region of humanized antibody #118-2-5

[0503] SEQ ID NO: 40 is the amino acid sequence of the heavy chain HuA (1,2) variable region of humanized antibody #118-2-5

[0504] SEQ ID NO: 41 is the amino acid sequence of the heavy chain Hu1 variable region of humanized antibody #125-1-5

[0505] SEQ ID NO: 42 is the amino acid sequence of the heavy chain Hu2 variable region of humanized antibody #125-1-5

[0506] SEQ ID NO: 43 is the amino acid sequence of the heavy chain Hu3 variable region of humanized antibody #125-1-5

[0507] SEQ ID NO: 44 is the amino acid sequence of the heavy chain Hu4 variable region of humanized antibody #125-1-5

[0508] SEQ ID NO: 45 is the amino acid sequence of the light chain Lu5 variable region of humanized antibody #125-1-5

[0509] SEQ ID NO: 46 is the amino acid sequence of the light chain Lu6 variable region of humanized antibody #125-1-5

[0510] SEQ ID NOs: 47 and 49 are the amino acid sequences of the heavy chains of chimeric antibodies that specifically bind to Trop-2.

[0511] SEQ ID NOs: 48 and 50 are the amino acid sequences of the light chains of chimeric antibodies that specifically bind to Trop-2.

[0512] SEQ ID NOs: 51, 53, and 55 are the amino acid sequences of the heavy chains of humanized antibodies that specifically bind to Trop-2.

[0513] SEQ ID NOs: 52, 54, and 56 are the amino acid sequences of the light chains of humanized antibodies that specifically bind to Trop-2.

[0514] Sequence Listing

[0515] SEQ ID NO: 1—Human TROP-2 amino acid sequence

[0516] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRP TAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL*

[0517] SEQ ID NO:2—Amino acid sequence of human TROP-2-ECD-6His antigen

[0518] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALG SGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQ CNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAHHHHHH

[0519] SEQ ID NO:3—Amino acid sequence of heavy chain CDR1 of murine monoclonal antibody #118-2-5

[0520] SYGVN

[0521] SEQ ID NO:4—Amino acid sequence of heavy chain CDR1 of murine monoclonal antibody #125-1-5

[0522] TYVIH

[0523] SEQ ID NO:5—Rat monoclonal antibody #118-2-5 heavy chain CDR2 amino acid sequence

[0524] VMWAGGSTNYNSALMS

[0525] SEQ ID NO:6—Rat monoclonal antibody #125-1-5 heavy chain CDR2 amino acid sequence

[0526] YINPNNDGTKYNEKFKG

[0527] SEQ ID NO:7—Rat monoclonal antibody #118-2-5 heavy chain CDR3 amino acid sequence

[0528] DENWDGAWFAY

[0529] SEQ ID NO:8—Rat monoclonal antibody #125-1-5 heavy chain CDR3 amino acid sequence

[0530] PHETHAMDY

[0531] SEQ ID NO:9—Rat monoclonal antibody #118-2-5 light chain CDR1 amino acid sequence

[0532] KSSQSLLNSGTRKNYLA

[0533] SEQ ID NO:10—Rat monoclonal antibody #125-1-5 light chain CDR1 amino acid sequence

[0534] KASEDIFNRLA

[0535] SEQ ID NO: 11 - Murine monoclonal antibody #118-2-5 light chain CDR2 amino acid sequence

[0536] WASSRES

[0537] SEQ ID NO:12 - Mouse monoclonal antibody #125-1-5 light chain CDR2 amino acid sequence

[0538] GATSLET

[0539] SEQ ID NO:13 - Mouse monoclonal antibody #118-2-5 light chain CDR3 amino acid sequence

[0540] KQSYNLFT

[0541] SEQ ID NO:14 - Mouse monoclonal antibody #125-1-5 light chain CDR3 amino acid sequence

[0542] QQYWNTWT

[0543] SEQ ID NO:15—Amino acid sequence of the heavy chain variable region of murine monoclonal antibody #118-2-5

[0544] QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVNWIRQPPGKGLEWLGVMWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTGMYYCARDENWDGAWFAYWGQGTLVTVS

[0545] SEQ ID NO:16—Amino acid sequence of the heavy chain variable region of murine monoclonal antibody #125-1-5

[0546] QIQLVQSGPELVKPGASVKMSCKASGYTFTTYVIHWVKQKPGQGLEWIGYINPNNDGTKYNEKFKGKATLISDKSSTTAYMEVRGLTSEDSAVYYCARPHFETHAMDYWGQGTSVTVSS

[0547] SEQ ID NO: 17 - Mouse monoclonal antibody #118-2-5 light chain variable region amino acid sequence

[0548] DIQMTQSPSSLAVSAGEKVTMSCKSSQSLLNSGTRKNYLAWYQQKPGQSPKLLISWASSRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGGGTKLELK

[0549] SEQ ID NO:18 - Amino acid sequence of the light chain variable region of mouse monoclonal antibody #125-1-5

[0550] DIQMTQSPSSFSVSLGDSVTITCKASEDIFNRLAWYQQKPGNAPRLLLISGATSLETGVPSRFSGGGSGKEYTLSITSLQNEDVATYYCQQYWNTWTFGGGTKLEIK

[0551] SEQ ID NO:19 - Chimeric antibody #118-2-5 heavy chain variable region nucleic acid sequence

[0552] caggtgcagctgcaggagtccggccccggcctggtgaagccctccgagaccctgtccctgacctgcaccgtgtccggcggctccatctcctcctacggcgtgaactggatccgccagccccccggcaagggcctggagtggatcggcgtgatgtgggccggcggctccaccaactacaactccgccctgatgtcccgcgtgaccatctccgtggacacctccaagaaccagttctccctgaagctgtcctccgtgaccgccgccgacaccgccgtgtactactgcgcccgcgacgagaactgggacggcgcctggttcgcctactggggccagggcaccctggtgaccgtctcgagt

[0553] SEQ ID NO: 20 - Nucleic acid sequence of the light chain variable region of chimeric antibody #118 - 2 - 5

[0554] gacatcgtgatgacccagtcccccgactccctggccgtgtccctgggcgagcgcgccaccatcaactgcaagtcctcccagtccctgctgaactccggcacccgcaagaactacctggcctggtaccagcagaagcccggccagccccccaagctgctgatctactgggcctcctcccgcgagtccggcgtgcccgaccgcttctccggctccggctccggcaccgacttcaccctgaccatctcctccctgcaggccgaggacgtggccgtgtactactgcaagcagtcctacaacctgttcaccttcggccagggcaccaagctcgaggtcgacataaaa

[0555] SEQ ID NO: 21 - Nucleic acid sequence of the heavy chain variable region of chimeric antibody #125 - 1 - 5

[0556] caggtgcagctggtgcagtccggcgccgaggtgaagaagcccggcgcctccgtgaaggtgtcctgcaaggcctccggctacaccttcaccacctacgtgatccactgggtgcgccaggcccccggccagcgcctggagtggatgggctacatcaaccccaacaacgacggcaccaagtacaacgagaagttcaagggccgcgtgaccatcacccgcgacacctccgcctccaccgcctacatggagctgtcctccctgcgctccgaggacaccgccgtgtactactgcgcccgcccccacttcgagacccacgccatggactactggggccagggcaccctggtgaccgtctcgagt

[0557] SEQ ID NO: 22 - Nucleotide sequence of the light chain variable region of chimeric antibody #125 - 1 - 5

[0558] gacatccagatgacccagtccccctcctccctgtccgcctccgtgggcgaccgcgtgaccatcacctgcaaggcctccgaggacatcttcaaccgcctggcctggtaccagcagaagcccggcaaggcccccaagctgctgctgtacggcgccacctccctggagaccggcgtgccctcccgcttctccggctccggctccggcaccgactacaccctgaccatctcctccctgcagcccgaggacttcgccacctactactgccagcagtactggaacacctggaccttcggcgccggcaccaaggtcgacataaaa

[0559] SEQ ID NO: 23 - Amino acid sequence of HuA of the heavy chain variable region of humanized antibody #118 - 2 - 5

[0560] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0561] SEQ ID NO:24 - Humanized antibody #118-2-5 heavy chain variable region HuB amino acid sequence

[0562] QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0563] SEQ ID NO:25 - Humanized antibody #118-2-5 heavy chain variable region HuC amino acid sequence

[0564] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTGMYYCARDENWDGAWFAYWGQGTLVTVSS

[0565] SEQ ID NO:26 - Humanized antibody #118-2-5 heavy chain variable region HuD amino acid sequence

[0566] QVQLKESGPGLVAPSETLSITCTTVSGFSLTSYGVNWIRQPPGKGLEWLGVMWAGGSTNYNSALMSRLTISKDTSKNQVSLKLSSVQAADTAMYYCARDENWDGAWFAYWGQGTLVTVSS

[0567] SEQ ID NO:27 - Humanized antibody #118-2-5 light chain variable region Lux amino acid sequence

[0568] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIK

[0569] SEQ ID NO:28—Amino acid sequence of the light chain variable region LuY of humanized antibody #118-2-5

[0570] DIVMTQSPSSLAVSLGERATMSCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFLTLTISSVQAEDLAVYYCKQSYNLFTFGGGTKVEIK

[0571] SEQ ID NO:29—Humanized antibody #118-2-5 heavy chain Hu(A1) variable region amino acid sequence

[0572] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0573] SEQ ID NO:30—Humanized antibody #118-2-5 heavy chain Hu(A2) variable region amino acid sequence

[0574] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNY NSALMSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0575] SEQ ID NO:31—Humanized antibody #118-2-5 heavy chain Hu(A3) variable region amino acid sequence

[0576] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRVTISVDTSKNQVSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0577] SEQ ID NO:32—Humanized antibody #118-2-5 heavy chain Hu(A4) variable region amino acid sequence

[0578] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRVTISVDTSKNQFSLKLSSVQAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0579] SEQ ID NO:33—Humanized antibody #118-2-5 heavy chain Hu(A5) variable region amino acid sequence

[0580] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAMYYCARDENWDGAWFAYWGQGTLVTVSS

[0581] SEQ ID NO:34—Amino acid sequence of the light chain Lu(X1) variable region of humanized antibody #118-2-5

[0582] DIVMTQSPSSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIK

[0583] SEQ ID NO:35—Amino acid sequence of the light chain Lu(X2) variable region of humanized antibody #118-2-5

[0584] DIVMTQSPDSLAVSLGERATMNCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIK

[0585] SEQ ID NO:36—Amino acid sequence of the light chain Lu(X3) variable region of humanized antibody #118-2-5

[0586] DIVMTQSHKFISTSVGDRVSITCKASQDVDTAVAWYQQKPGQSSPKLLIHWASTRHTGVPDRFTGGGSGTDFTLTLSNVQSEDLADYFCQQYSTFPWTFGGGTRLEIK

[0587] SEQ ID NO:37—Amino acid sequence of the light chain Lu(X4) variable region of humanized antibody #118-2-5

[0588] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLISWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYNLFTFGGGTKVEIK

[0589] SEQ ID NO:38—Amino acid sequence of the light chain Lu (X5) variable region of humanized antibody #118-2-5

[0590] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLIYWASSRE SGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCKQSYNLFTFGGGTKVEIK

[0591] SEQ ID NO:39—Amino acid sequence of the light chain Lu(X6) variable region of humanized antibody #118-2-5

[0592] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGTRKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCKQSYNLFTFGGGTKVEIK

[0593] SEQ ID NO:40—Humanized antibody #118-2-5 heavy chain HuA(1,2) variable region amino acid sequence

[0594] QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGVNWIRQPPGKGLEWIGVMWAGGSTNYNSALMSRLTISKDTSKNQFSLKLSSVTAADTAVYYCARDENWDGAWFAYWGQGTLVTVSS

[0595] SEQ ID NO:41—Amino acid sequence of the heavy chain Hu1 variable region of humanized antibody #125-1-5

[0596] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYVIHWVRQAPGQRLEWMGYINPNNDGTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARPHFETHAMDYWGQGTLVTVSS

[0597] SEQ ID NO:42—Amino acid sequence of the Hu2 variable region of the heavy chain of humanized antibody #125-1-5

[0598] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYVIHWVKQKPGQGLEWIGYINPNNDGTKYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARPHFETHAMDYWGQGTLVTVSS

[0599] SEQ ID NO:43—Amino acid sequence of the Hu3 variable region of the heavy chain of humanized antibody #125-1-5

[0600] QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYVIHWVRQAPGQRLEWMGYINPNNDGTKYNEKFKGKATLTSDKSSTTAYMEVRGLTSEDSAVYYCARPHFETHAMDYWGQGTLVTVSS

[0601] SEQ ID NO:44—Amino acid sequence of the Hu4 variable region of the heavy chain of humanized antibody #125-1-5

[0602] QVQLVQSGAEVKKPGASVKMSCKASGYTFTTYVIHWVRQAPGQRLEWIGYINPNNDGTKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCARPHFETHAMDYWGQGTLVTVSS

[0603] SEQ ID NO:45—Amino acid sequence of the light chain Lu5 variable region of humanized antibody #125-1-5

[0604] DIQMTQSPSSSLSASVGDRVTITCKASEDIFNRLAWYQQKPGKAPKLLLYGATSLETGVPSRF SGSGSGTDYTLTISSLQPEDFATYYCQQYWNTWTFGQGTKVEIK

[0605] SEQ ID NO:46—Amino acid sequence of the light chain Lu6 variable region of humanized antibody #125-1-5

[0606] DIQMTQSPSSSLSASVGDRVTITCKASEDIFNRLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWNTWTFGQGTKVEIK

[0607] SEQ ID NO: 47 - Amino Acid Sequence of the Heavy Chain of Chimeric Antibody #118-2-5

[0608] QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVNWIRQPPGKGLEWLGVMWAGGSTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTGMYYCARDENWDGAWFAYWGQGTLVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQAYICNVNHKPSNTKVDKKVGPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK...

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to human Trop-2 and comprises: a heavy chain sequence as shown in SEQ ID NO: 51 and a light chain sequence as shown in SEQ ID NO: 52; Or, the heavy chain sequence shown in SEQ ID NO:53 and the light chain sequence shown in SEQ ID NO:54; Or, the heavy chain sequence shown in SEQ ID NO:55 and the light chain sequence shown in SEQ ID NO:

56.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has a concentration of at least 1×10 -12 The dissociation constant KD of M binds to Trop-2 protein.

3. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment is selected from the group consisting of a human antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a Fab fragment, a Fab' fragment, a Fab2 fragment, a F(ab)'2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.

4. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, wherein the antibody or antigen-binding fragment is further selected from a humanized antibody and a chimeric antibody.

5. A pharmaceutical composition comprising a mixture of the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. An isolated immunoconjugate or fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 coupled to an effector molecule.

7. An isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. A recombinant expression vector comprising the isolated nucleic acid according to claim 7.

9. A host cell comprising the vector according to claim 8.

10. An antibody-drug conjugate ADC that specifically binds to Trop-2, comprising: The antibody or antigen-binding fragment thereof, and the therapeutic agent according to any one of claims 1 to 4, wherein the ADC has the formula: Ab-(LD)n, wherein Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, L is a linker or a direct bond, D is a cytotoxic warhead, and n is an integer from 2 to 6.

11. The ADC according to claim 10, wherein The antibody is a monoclonal antibody comprising the IgG1 isotype.

12. The ADC according to claim 10, wherein The antibody is a humanized antibody.

13. The ADC according to claim 10, wherein The therapeutic agent is a cytotoxic agent.

14. The ADC according to claim 13, wherein The cytotoxic agent is monomethyl auristatin.

15. The ADC of claim 13, wherein the cytotoxic agent is maytansinoid DM1.

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