HK40029700A

HK40029700A - Humanized anti-liv1 antibodies for the treatment of breast cancer

Info

Publication number: HK40029700A
Application number: HK62020019720.5A
Authority: HK
Inventors: D·肯尼迪; A·科斯蒂奇; E·科温; J·德拉克曼; P·豪伊; B·赵; P·加尔凡; C·帕兰切·韦塞尔斯; O·O·阿比多耶
Original assignee: 西雅图基因公司
Priority date: 2017-12-01
Filing date: 2018-11-30
Publication date: 2021-02-19

Description

Humanized anti-LIV 1 antibodies for the treatment of breast cancer

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 62/593,660, filed on 1/12/2017, the contents of which are incorporated herein by reference in their entirety.

Sequence Listing submitted in ASCII text files

The following is submitted in an ASCII text file and is incorporated herein by reference in its entirety: sequence Listing in Computer Readable Form (CRF) (filename: 761682001440SEQLIST. TXT, recording date: 2018, 11 months, 30 days, size: 3 KB).

Technical Field

The present invention relates to the field of antibody-based breast cancer therapy. In particular, the invention relates to the use of humanized anti-LIV 1 antibodies and antigen binding fragments or conjugates thereof (e.g., LIV1 antibody-drug conjugates (LIV 1-ADCs)) for the treatment of LIV-1 expressing cancers such as breast cancer (e.g., locally advanced or metastatic breast cancer).

Background

Breast cancer is classified based on three protein expression markers: overexpression of the Estrogen Receptor (ER), the progesterone receptor (PgR) and the growth factor receptor HER 2/neu. Hormone therapy, including tamoxifen (tamoxifen) and aromatase inhibitors, is effective in treating tumors that express the hormone receptors ER and PgR. HER 2-directed therapy is useful for HER 2/neu-expressing tumors; these tumors are currently the only breast cancer category for which immunotherapy is available. For these patients, unconjugated antibodies, such as Herceptin (Herceptin) or Perjeta, are often used in combination with chemotherapy.

Treatment options for triple negative breast tumors that do not express ER, PgR or HER2/neu are limited to chemotherapy, radiation therapy and surgery. In addition, effective treatment options are limited for patients with advanced disease where survival rates are relatively low in stage III patients (52%) and significantly poor in stage IV patients (15%).

Clearly, there is an urgent need for effective treatment of breast cancer, particularly advanced breast cancer.

LIV-1(SLC39A6) is a member of the solute carrier family, a multi-spanning transmembrane protein with putative zinc transporter and metalloprotease activities. LIV-1 was first identified as an estrogen-induced gene in the breast cancer cell line ZR-75-1. LIV-1 is expressed in most subtypes of metastatic breast cancer.

Summary of The Invention

The present disclosure is based on the surprising discovery that incurable, unresectable, locally advanced or metastatic breast cancer can be treated with the anti-LIV 1 antibodies and antigen binding fragments thereof described herein.

In one aspect, a method is provided for treating a subject having, or at risk of having, an LIV-1-associated cancer, comprising administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds human LIV-1, wherein the dose administered is less than about 200mg per treatment cycle of the antibody or antigen-binding fragment thereof, and wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having at least 95% identity to SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having at least 95% identity to SEQ ID NO: 2.

In another aspect, there is provided a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds human LIV-1, wherein the antibody or antigen-binding fragment thereof is administered at a dose of less than or equal to about 250mg per treatment cycle, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is identical to SEQ ID NO:1 and a Heavy Chain Variable Region (HCVR) having at least 95% identity to SEQ ID NO: 2a Light Chain Variable Region (LCVR) having at least 95% identity, and wherein if the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg per treatment cycle, the method further comprises administering to the subject Granulocyte Colony Stimulating Factor (GCSF). In certain exemplary embodiments, the GCSF is administered prophylactically, if at all.

In another aspect, there is provided a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising administering to the subject Granulocyte Colony Stimulating Factor (GCSF), administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the dose administered is greater than or equal to about 200mg per treatment cycle and less than or equal to about 250mg per treatment cycle of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having at least 95% identity to SEQ ID NO:1 and a Light Chain Variable Region (LCVR) having at least 95% identity to SEQ ID NO: 2. In certain exemplary embodiments, the GCSF is administered prophylactically.

In certain exemplary embodiments, the LIV-1 associated cancer is breast cancer, triple negative breast cancer, metastatic breast cancer, triple negative metastatic breast cancer, or hormone receptor positive metastatic breast cancer.

In certain exemplary embodiments, the treatment cycle is about every three weeks (once every three weeks).

In certain exemplary embodiments, the dose is about 2.5mg/kg of the subject's body weight.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof is conjugated to monomethyl reoxidine e (mmae):

in certain exemplary embodiments, the antibody or antigen-binding fragment thereof is conjugated to valine-citrulline-monomethyl reocetin e (vcmmae) ("reocetin" for Auristatin):

in certain exemplary embodiments, the ratio of vcMMAE to antibody or antigen-binding fragment thereof is from about 1 to about 8 or about 4.

In certain exemplary embodiments, the HCVR has at least 97% sequence identity to SEQ ID No. 1 and the LCVR has at least 97% sequence identity to SEQ ID No. 2.

In certain exemplary embodiments, the HCVR has at least 99% sequence identity to SEQ ID No. 1 and the LCVR has at least 99% sequence identity to SEQ ID No. 2.

In certain exemplary embodiments, the subject is a human.

In another aspect, there is provided a method of treating a subject having, or at risk of having, an LIV-1-associated cancer, comprising administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the administered dose is less than about 200mg per treatment cycle of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an HCVR having at least 95% identity to SEQ ID NO:1 and an LCVR having at least 95% identity to SEQ ID NO:2, and wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

in another aspect, there is provided a method of treating a subject having, or at risk of having, an LIV-1-associated cancer, comprising administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the administered dose is less than or equal to about 250mg per treatment cycle of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an HCVR with at least 95% identity to SEQ ID NO:1 and an LCVR with at least 95% identity to SEQ ID NO:2, wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

vcMMAE, and wherein if the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg per treatment cycle, the method further comprises administering Granulocyte Colony Stimulating Factor (GCSF) to the subject. In certain exemplary embodiments, the GCSF is administered prophylactically, if at all.

In another aspect, there is provided a method of treating a subject having or at risk of having an LIV-1-associated cancer, comprising administering to the subject Granulocyte Colony Stimulating Factor (GCSF), administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the dose administered is greater than or equal to about 200mg per treatment cycle and less than or equal to about 250mg per treatment cycle of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an HCVR having at least 95% identity to SEQ ID NO:1 and an LCVR having at least 95% identity to SEQ ID NO:2, and wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

vcMMAE. In certain exemplary embodiments, the GCSF is administered prophylactically.

In certain exemplary embodiments, the dose is administered at a concentration of about 2.5mg/kg of body weight of the subject.

In certain exemplary embodiments, each treatment cycle is administered to the subject once every three weeks.

In certain exemplary embodiments, the subject is a human.

In another aspect, there is provided a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the administered dose is less than about 200mg per treatment cycle of once every three weeks of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an HCVR of SEQ ID NO:1 and an LCVR of SEQ ID NO:2, and wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

in another aspect, there is provided a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the administered dose is less than or equal to about 250mg per treatment cycle of once every three weeks of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises an HCVR of SEQ ID NO:1 and an LCVR of SEQ ID NO:2, wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

In another aspect, there is provided a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising administering to the subject Granulocyte Colony Stimulating Factor (GCSF), administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds to human LIV-1, wherein the dose administered is greater than or equal to about 200mg per treatment cycle once every three weeks and less than or equal to about 250mg per treatment cycle once every three weeks, wherein the antibody or antigen-binding fragment thereof comprises an HCVR of SEQ ID NO:1 and an LCVR of SEQ ID NO:2, and wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

In certain exemplary embodiments, the ratio of vcMMAE to antibody or antigen-binding fragment thereof is about 4.

In certain exemplary embodiments, the subject is a human.

In another aspect, there is provided a method of treating a subject having, or at risk of having, LIV-1 associated breast cancer, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human LIV-1 at a dose of about 2.5mg/kg body weight of the subject, wherein the administered dose is less than about 200 mg/treatment cycle of the antibody or antigen-binding fragment thereof once every three weeks, wherein the antibody or antigen-binding fragment thereof comprises an HCVR of SEQ ID NO:1 and an LCVR of SEQ ID NO:2, and wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

in another aspect, there is provided a method of treating a subject having, or at risk of having, LIV-1 associated breast cancer, comprising administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human LIV-1 at a dose of about 2.5mg/kg body weight of the subject, wherein the administered dose is less than or equal to about 250 mg/treatment cycle of the antibody or antigen-binding fragment thereof once every three weeks, wherein the antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO:1 and the LCVR of SEQ ID NO:2, wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

In another aspect, there is provided a method of treating a subject having, or at risk of having, LIV-1-associated breast cancer, comprising administering to the subject Granulocyte Colony Stimulating Factor (GCSF), administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human LIV-1 at a dose of about 2.5mg/kg body weight of the subject, wherein the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg per treatment cycle of once every three weeks and less than or equal to about 250mg per treatment cycle of once every three weeks, wherein the antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO:1 and the LCVR of SEQ ID NO:2, and wherein the antibody or antigen-binding fragment thereof is conjugated to vcMMAE:

In certain exemplary embodiments, the breast cancer is triple negative breast cancer, metastatic breast cancer, triple negative metastatic breast cancer, or hormone receptor positive metastatic breast cancer.

In certain exemplary embodiments, the subject is a human.

The summary of the disclosure above is non-limiting and other features and advantages of the disclosed antibodies and methods of making and using them will be apparent from the detailed description, examples and claims.

Detailed description of the invention

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

I. Definition of

As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

"antibody-drug conjugate" or "ADC" refers to an antibody conjugated to a cytotoxic or cytostatic agent. Typically, the antibody-drug conjugate binds to a target antigen (e.g., LIV1) on the surface of a cell, then the antibody-drug conjugate is internalized into the cell, and then the drug is released into the cell. In certain exemplary embodiments, the antibody-drug conjugate is LIV 1-ADC.

A "polypeptide" or "polypeptide chain" is a polymer of amino acid residues, whether naturally occurring or synthetically produced, joined by peptide bonds. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

A "protein" is a macromolecule comprising one or more polypeptide chains. Proteins may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to a protein by the cell that produces the protein and will vary with the cell type. Proteins are defined herein in terms of their amino acid backbone structure. Substituents such as saccharide groups are not usually specified but may still be present.

The terms "amino-terminal" and "carboxy-terminal" refer to positions within a polypeptide. These terms are used to refer to a particular sequence or portion of a polypeptide to indicate proximity or relative position where context permits. For example, a sequence within a polypeptide that is located at the carboxy-terminus of a reference sequence is located near the carboxy-terminus of the reference sequence, but is not necessarily located at the carboxy-terminus of the entire polypeptide.

To classify amino acid substitutions as conservative or non-conservative, the following amino acid substitutions are considered conservative substitutions: serine is substituted with threonine, alanine or asparagine; threonine is substituted with proline or serine; asparagine is substituted with aspartic acid, histidine or serine; aspartic acid is substituted with glutamic acid or asparagine; glutamic acid is substituted with glutamine, lysine, or aspartic acid; glutamine substituted with arginine, lysine or glutamic acid; histidine by tyrosine or asparagine; arginine is substituted with lysine or glutamine; methionine is substituted with isoleucine, leucine or valine; isoleucine is substituted with leucine, valine, or methionine; leucine substituted with valine, isoleucine or methionine; phenylalanine is substituted with tyrosine or tryptophan; tyrosine is substituted by tryptophan, histidine or phenylalanine; proline is substituted by threonine; alanine is substituted with serine; lysine is substituted with glutamic acid, glutamine or arginine; valine by methionine, isoleucine or leucine; and tryptophan by phenylalanine or tyrosine. Conservative substitutions may also refer to substitutions between amino acids of the same class. The categories are as follows: group I (hydrophobic side chains): met, ala, val, leu, ile; group II (neutral hydrophilic side chains): cys, ser, thr; group III (acidic side chain): asp, glu; group IV (basic side chain): asn, gin, his, lys, arg; group V (residues affecting chain orientation): gly, pro; and group VI (aromatic side chains): trp, tyr, phe.

Two amino acid sequences have "100% amino acid sequence identity" if the amino acid residues of the two amino acid sequences are identical when aligned for maximum correspondence. Sequence comparisons can be performed using standard software programs, such as those contained in the LASERGENE bioinformatics computing suite, produced by DNASTAR (Madison, Wisconsin). Other methods of comparing two nucleotide or amino acid sequences by determining an optimal alignment are well known to those skilled in the art. (see, e.g., Peruski and Peruski, The Internet and The New Biology: Tools for genomic and Molecular Research (ASM Press, Inc. 1997); Wu et al (eds.), "information superior and Computer Databases of Nucleic Acids and Proteins," in methods Gene Biotechnology 123- "151 (CRC Press, Inc. 1997); Bishop (ed.)," Guide to human genome Computing (2nd ed., Academic Press, Inc. 1998)). Two amino acid sequences are considered "substantial sequence identity" if they have at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with respect to each other.

Percent sequence identity is determined by the antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if the subject antibody region (e.g., the entire variable domain of a heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in the subject and reference antibody regions divided by the total number of aligned positions for the two regions (gaps not calculated) multiplied by 100 to convert to a percentage.

A composition or method that "comprises" one or more of the recited elements can include other elements not specifically recited. For example, a composition comprising an antibody may contain the antibody alone, or the antibody in combination with other components.

The specification of a range of values includes all integers within or defining the range.

In the antibodies or other proteins described herein, reference to amino acid residues corresponding to those specified in SEQ ID NOs includes post-translational modifications of such residues.

The term "antibody" refers to immunoglobulin proteins, antigen-binding fragments, and engineered variants thereof that are produced by the body in response to the presence of an antigen and bind to the antigen. Thus, the term "antibody" includes, for example, intact monoclonal antibodies (e.g., antibodies produced using hybridoma technology) and antigen-binding antibody fragments, such as F (ab')₂Fv fragment, diabody, single-chain antibody, scFv fragment or scFv-Fc. Genetically, engineered whole antibodies and fragments are also included, such as chimeric antibodies, humanized antibodies, single chain Fv fragments, single chain antibodies, diabodies, minibodies, linear antibodies, multivalent or multispecific (e.g., bispecific) hybrid antibodies, and the like. Thus, the term "antibody" is used broadly to encompass any protein that comprises the antigen-binding site of an antibody and is capable of specific binding to its antigen.

The term antibody or antigen-binding fragment thereof includes "conjugated" antibodies or antigen-binding fragments thereof or "antibody-drug conjugates (ADCs)", wherein the antibody or antigen-binding fragment thereof is covalently or non-covalently bound to an agent, e.g., to a cytostatic or cytotoxic drug.

The term "genetically engineered antibody" refers to an antibody in which the amino acid sequence differs from that of a native or parent antibody. There are many possible variations that range from changing only one or a few amino acids to complete redesign of, for example, the variable or constant regions. In general, the constant region is altered in order to improve or alter properties such as complement fixation and other effector functions. Typically, the variable region is altered in order to improve antigen binding properties, improve the stability of the variable region and/or reduce the risk of immunogenicity.

The term "chimeric antibody" refers to antibodies and fragments of such antibodies: wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species (e.g., human) or belongs to a particular antibody class or subclass, and the remaining portion of the chain is identical to or homologous to antibodies derived from another species (e.g., mouse) or belongs to another antibody class or subclass, so long as they exhibit the desired biological activity.

An "antigen-binding site of an antibody" is a portion of an antibody sufficient to bind its antigen. The smallest such region is typically a variable domain or a genetically engineered variant thereof. Single domain binding sites can be generated from camelid antibodies (see Muydermans and Lauwereys, mol. Recog.12: 131-. Typically, the antigen binding site of an antibody comprises a heavy chain Variable (VH) domain and a light chain Variable (VL) domain that bind to a common epitope. In the context of the present invention, an antibody may comprise one or more components other than an antigen binding site, for example, a second antigen binding site of an antibody (which may bind the same or a different epitope or the same or a different antigen), a peptide linker, an immunoglobulin constant region, an immunoglobulin hinge, an amphipathic helix (see Pack and Pluckthun, biochem.31: 1579-. Examples of molecules comprising the antigen binding site of an antibody are known in the art and include, for example, Fv, single chain Fv (scFv), Fab ', F (ab')2, F (ab) c, diabodies, minibodies, nanobodies, Fab-scFv fusions, bispecific (scFv)4-IgG and bispecific (scFv) 2-Fab. (see, e.g., Hu et al, Cancer Res.56: 3055-.

The term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by one or more immunoglobulin genes. One form of immunoglobulin constitutes the basic building block of a vertebrate natural (i.e., native or parent) antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain. In each pair, the light and heavy chain variable regions (VL and VH) are primarily responsible together for binding to antigen, while the constant regions are primarily responsible for antibody effector functions. Five classes of immunoglobulin proteins (IgG, IgA, IgM, IgD, and IgE) have been identified in higher vertebrates. IgG belongs to the main class, usually present as the second most abundant protein in plasma. In humans, IgG consists of four subclasses, designated IgG1, IgG2, IgG3, and IgG4, respectively. Each immunoglobulin heavy chain has a constant region consisting of a constant region protein domain (CH1, hinge, CH2 and CH 3; IgG3 also includes the CH4 domain) that is substantially invariant for a given subclass within a species.

DNA sequences encoding human and non-human immunoglobulin chains are known in the art. (see, for example, Ellison et al, DNA 1:11-18, 1981; Ellison et al, Nucleic Acids Res.10:4071-4079, 1982; Kenten et al, Proc. Natl. Acad. Set USA 79:6661-6665, 1982; Seno et al, Nucleic Acids Res.11: 1983; Riechmann et al, Nature 332:323-327, 1988; Amster et al, Nucleic Acids Res.8:2055-2065, 1980; Rusconi Kohler, Nature 314:330-334, 1985; Boss et al, Nucleic Acids sssssSRes.12: 3791-3806, 1984; thsen et al, Nature 298:380-382, 1985; Boss et al, Nucleic Acids J.245-195-19823; Evqgen J.22: 19823; Gene J.22: 19823: 1982; Gene J.22: 19823; Gene J.22: 19823; Gene J.32, 1983; Gene J.32). For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, VolV, Academic Press, Inc., 49-140, 1987; and Padlan, mol.Immunol.31:169-217, 1994. The term "immunoglobulin" is used herein in its general sense, which, depending on the context, is denoted as an intact antibody, a constituent chain thereof, or a fragment of a chain.

Full-length immunoglobulin "light chains" (about 25kDa or 214 amino acids) are encoded by amino-terminal variable region genes (encoding about 110 amino acids) and by carboxy-terminal kappa or lambda constant region genes. A full-length immunoglobulin "heavy chain" (about 50kDa or 446 amino acids) is encoded by a variable region gene (encoding about 116 amino acids) and a gamma, mu, alpha, or constant region gene (encoding about 330 amino acids), the latter defining the isotype of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. In both the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids (see generally Fundamental Immunology (Paul, ed., Raven Press, n.y., 2nd ed.1989), ch.7).

An immunoglobulin light or heavy chain variable region (also referred to herein as a "light chain variable domain" ("VL domain") or a "heavy chain variable domain" ("VH domain"), respectively) consists of a "framework" region interrupted by three "complementarity determining regions" or "CDRs". The framework regions help align the CDRs for epitope specific binding to an antigen. Thus, the term "CDR" refers to the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxy-terminus, both the VL and VH domains comprise the following Framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4.

The assignment of amino acids to each variable region is made according to the definition of Kabat, Sequences of Proteins of immunological Interest (national institute of health, Bethesda, Md., 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering), wherein corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. CDR1, 2 and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2 and CDR-L3, respectively. CDR1, 2 and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2 and CDR-H3, respectively. If so recorded, the allocation of CDRs may be in accordance with Kabat, instead of Kabat(Lefranc et al, development a)l&Synthetic Immunology 27: 55-77; 2003) the process is carried out.

The numbering of the heavy chain constant region is performed by the EU index as shown in Kabat (Kabat, Sequences of proteins of Immunological Interest, national institute of health, Bethesda, MD, 1987 and 1991).

Unless the context indicates otherwise, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" may include antibodies derived from a single clone, including any eukaryotic, prokaryotic, or phage clone. In particular embodiments, the antibodies described herein are monoclonal antibodies.

The term "humanized VH domain" or "humanized VL domain" refers to an immunoglobulin VH or VL domain: it comprises some or all CDRs entirely or substantially from a non-human donor immunoglobulin (e.g., mouse or rat) and variable domain framework sequences entirely or substantially from a human immunoglobulin sequence. The non-human immunoglobulin providing the CDRs is referred to as the "donor" and the human immunoglobulin providing the framework is referred to as the "acceptor". In some cases, humanized antibodies will retain some non-human residues within the human variable domain framework regions to enhance appropriate binding properties (e.g., when the antibody is humanized, mutations may be required in the framework to maintain binding affinity).

A "humanized antibody" is an antibody comprising one or both of a humanized VH domain and a humanized VL domain. Immunoglobulin constant regions need not be present, but if present, they are derived entirely or substantially from human immunoglobulin constant regions.

A humanized antibody is a genetically engineered antibody in which CDRs from a non-human "donor" antibody are grafted into human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence.

Human acceptor sequences with a high degree of sequence identity can be selected in the variable region framework with the donor sequence to match the canonical form between the acceptor and donor CDRs, as well as other criteria. Thus, a humanized antibody is an antibody having CDRs entirely or substantially from a donor antibody and constant regions (if present) entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain typically has all three CDRs entirely or substantially from the donor antibody heavy chain, as well as heavy chain variable region framework sequences and heavy chain constant regions (if present) substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain typically has all three CDRs entirely or substantially from the donor antibody light chain, as well as light chain variable region framework sequences and light chain constant regions (if present) substantially from human light chain variable region framework and constant region sequences.

A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the corresponding residues (as defined by Kabat numbering) or wherein about 100% of the corresponding residues (as defined by Kabat numbering) are identical between the individual CDRs. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, when at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the corresponding residues (as defined by Kabat numbering for the variable region and EU numbering for the constant region) or about 100% of the corresponding residues (as defined by Kabat numbering for the variable region and EU numbering for the constant region) are identical.

Although humanized antibodies typically incorporate all six CDRs from a mouse antibody (preferably by Kabat orDefinitions) but alsoLess than all six CDRs (e.g., at least 3, 4, or 5) from a mouse antibody can be made (e.g., Pascales et al, J.Immunol.169:3076, 2002; Vajdos et al, Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al, mol.Immunol.36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).

A CDR in a humanized antibody is "substantially from" a corresponding CDR in a non-human antibody when at least 60%, at least 85%, at least 90%, at least 95%, or 100% of the corresponding residues (as defined by Kabat (or IMGT)) are identical between the individual CDRs. In particular variants in which the CDRs are substantially from a humanized VH or VL domain of a non-human immunoglobulin, the CDRs of the humanized VH or VL domain have no more than six (e.g., no more than five, no more than four, no more than three, no more than two, or no more than one) amino acid substitutions (preferably conservative substitutions) in all three CDRs relative to the corresponding non-human VH or VL CDRs. A variable region framework sequence of an antibody VH or VL domain or a sequence of an immunoglobulin constant region (if present) is "substantially derived from" a human VH or VL framework sequence or a human constant region, respectively, when at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the corresponding residues (as defined by Kabat numbering for the variable region and EU numbering for the constant region) or about 100% of the corresponding residues (as defined by Kabat numbering for the variable region and EU numbering for the constant region) are identical. Thus, all parts of a humanized antibody, except for the CDRs, are typically derived entirely or substantially from the corresponding parts of a natural human immunoglobulin sequence.

The antibody is typically provided in an isolated form. This means that the antibody is typically at least about 50% w/w pure from interfering proteins and other contaminants that it produces or purifies, but does not exclude the possibility that the antibody will be associated with an excess of a pharmaceutically acceptable carrier or other vehicle to facilitate its use. Sometimes the antibody is at least about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% w/w pure from the interfering protein and contaminants produced or purified. Antibodies, including isolated antibodies, can be conjugated to cytotoxic agents and provided as antibody drug conjugates.

Specific binding of an antibody to its target antigen generally means at least about 10⁶About 10⁷About 10⁸About 10⁹Or about 10¹⁰M^-1The affinity of (a). Specific binding is detectable by a higher order of magnitude and is distinguishable from the presence of non-specific binding to at least one non-specific target. Specific binding may be the result of the formation of bonds between specific functional groups or a specific spatial fit (e.g., lock and key types), while non-specific binding is typically the result of van der waals forces.

The term "epitope" refers to the site of an antigen that binds to an antibody. Epitopes may be formed of contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing agents such as solvents, whereas epitopes formed from tertiary folding are typically lost after treatment with denaturing agents such as solvents. Epitopes typically comprise at least about 3, and more typically at least about 5, at least about 6, at least about 7, or about 8-10 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol.66, Glenn E.Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the ability of one antibody to compete with another for binding to the target antigen. Epitopes of antibodies can also be determined by X-ray crystallography of the antibody bound to its antigen to identify contact residues.

Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody (provided that such mutations do not produce an overall change in the structure of the antigen). Two antibodies have overlapping epitopes if certain amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

Competition between antibodies can be determined by assays in which a test antibody inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al, Cancer res.50:1495, 1990). If an excess of test antibody inhibits binding of the reference antibody, the test antibody competes with the reference antibody.

Antibodies identified by competition assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to a nearby epitope sufficiently close to the epitope bound by the reference antibody to undergo steric hindrance. Antibodies identified by competition assays also include those that compete indirectly with the reference antibody by causing conformational changes in the target protein thereby preventing the reference antibody from binding to an epitope different from that bound by the test antibody.

Antibody effector functions refer to functions contributed by the Fc region of Ig. Such functions may be, for example, Antibody Dependent Cellular Cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP) or Complement Dependent Cytotoxicity (CDC). Such functions can be affected, for example, by binding of the Fc region to Fc receptors on immune cells with phagocytic or lytic activity or by binding of the Fc region to components of the complement system. Typically, one or more of the effects mediated by Fc-binding cell or complement components results in the inhibition and/or depletion of cells targeted by LIV 1. The Fc region of an antibody can recruit Fc receptor (FcR) -expressing cells and juxtapose them to antibody-coated target cells. Cells expressing surface fcrs against IgG, including Fc γ RIII (CD16), Fc γ RII (CD32), and Fc γ RIII (CD64), may serve as effector cells for the destruction of IgG-coated cells. Such effector cells include monocytes, macrophages, Natural Killer (NK) cells, neutrophils, and eosinophils. Engagement of IgG with Fc γ R activates ADCC or ADCP. ADCC is mediated by CD16+ effector cells through secretion of pore-forming proteins and proteases, whereas phagocytosis is mediated by CD32+ and CD64+ effector cells (see Fundamental Immunology, 4)^thed., Paul ed., Lippincott-Raven, N.Y., 1997, chapters 3, 17 and 30; uchida et al, j.exp.med.199:1659-69, 2004; akewanlop et al, Cancer Res.61:4061-65, 2001; the results of Watanabe et al,Breast CancerRes.Treat.53:199-207，1999)。

in addition to ADCC and ADCP, the Fc region of cell-bound antibodies may activate the classical complement pathway to elicit CDC. When the C1q of the complement system and the Fc region of the antibody are complexed with the antigen, C1q of the complement system will bind to the Fc region of the antibody. Binding of C1q to cell-bound antibodies may initiate a cascade of events involving proteolytic activation of C4 and C2 to produce C3 convertase. Cleavage of C3 to C3b by C3 convertase activates the terminal complement components including C5b, C6, C7, C8 and C9. These proteins together form membrane attack complex pores on antibody coated cells. These pores disrupt the integrity of the cell membrane, killing the target cells (see Immunobiology, 6)^thed., Janeway et al, Garland Science, n.y., 2005, chapter 2).

The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a mechanism for inducing cell death that depends on the interaction between antibody-coated target cells and immune cells with lytic activity (also referred to as effector cells). Such effector cells include natural killer cells, monocytes/macrophages and neutrophils. Effector cells are attached via their antigen binding site to the Fc region of Ig that binds to target cells. The antibody-coated target cells die as a result of effector cell activity. In certain exemplary embodiments, the anti-LIV 1 IgG1 antibodies of the invention mediate equal or increased ADCC relative to the parent antibody and/or relative to the anti-LIV 1 IgG3 antibody.

The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to the process by which antibody-coated cells are fully or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and/or dendritic cells) that bind to the Fc of Ig. In certain exemplary embodiments, the anti-LIV 1 IgG1 antibodies of the invention mediate equal or increased ADCP relative to the parent antibody and/or relative to the anti-LIV 1 IgG3 antibody.

The term "complement dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death: wherein the Fc region of the target-bound antibody activates a series of enzymatic reactions that ultimately form pores in the target cell membrane.

Typically, antigen-antibody complexes (such as those on antibody-coated target cells) bind to and activate complement component C1q, thereby activating the complement cascade, leading to death of the target cell. Activation of complement can also result in deposition of complement components on the surface of target cells, thereby facilitating ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

By "cytotoxic effect" is meant depleting, eliminating and/or killing the target cell. "cytotoxic agent" refers to a compound that has a cytotoxic effect on a cell, thereby mediating the depletion, elimination and/or killing of a target cell. In certain embodiments, the cytotoxic agent is conjugated to or administered in combination with an antibody. Suitable cytotoxic agents are further described herein.

"cytostatic" refers to the inhibition of cell proliferation. "cytostatic agent" refers to a compound that has a cytostatic effect on cells, thereby mediating inhibition of growth and/or expansion of specific cell types and/or cell subsets. Suitable cytostatics are further described herein.

The term "patient" or "subject" includes human and other mammalian subjects receiving any prophylactic or therapeutic treatment, such as non-human primates, rabbits, rats, mice, and the like, as well as transgenic species thereof.

In the context of treating a disorder expressing LIV1 by administering an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) as described herein, the term "effective amount" refers to an amount of such antibody or antigen-binding fragment thereof sufficient to inhibit the development of or alleviate one or more symptoms of a LIV 1-associated disorder (e.g., a cancer expressing LIV 1). An effective amount of the antibody is administered in an "effective regime". The term "effective regime" refers to the combination of the amount and frequency of dosage of antibody administered sufficient to effect prophylactic or therapeutic treatment of the disease (e.g., prophylactic or therapeutic treatment of a cancer expressing LIV 1).

The term "pharmaceutically acceptable" means approved or approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "pharmaceutically compatible ingredient" refers to a pharmaceutically acceptable diluent, adjuvant, excipient, or vehicle formulated with an anti-LIV 1 antibody (e.g., LIV 1-ADC).

The phrase "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1' -methylenebis- (2-hydroxy-3-naphthoate). pharmaceutically acceptable salts can further comprise additional molecules such as acetate, succinate or other counterions In addition, pharmaceutically acceptable salts can have multiple charged atoms in their structure. Where multiple dotted atoms are part of the pharmaceutically acceptable salt, then there may be multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

Unless otherwise apparent from the context, when a value is expressed as "about" X or "approximately" X, the specified value of X should be understood to be accurate to. + -. 10%.

In the context of the present invention, solvates are those forms of the compounds of the present invention which form complexes in the solid or liquid state by coordination with solvent molecules. Hydrates are a particular form of solvates in which coordination occurs with water. In certain exemplary embodiments, in the context of the present invention, the solvate is a hydrate.

anti-LIV 1 antibodies and antigen binding fragments

The present invention provides isolated, recombinant, and/or synthetic anti-LIV 1 human, primate, rodent, mammalian, chimeric, humanized, and/or CDR-grafted antibodies and antigen-binding fragments thereof (e.g., LIV1-ADC), as well as compositions and nucleic acid molecules comprising at least one polynucleotide encoding at least a portion of an anti-LIV 1 antibody molecule. The invention further includes, but is not limited to, methods of making and using such nucleic acids and antibodies, including diagnostic and therapeutic compositions, methods and devices. In a certain exemplary embodiment, a humanized anti-LIV 1 IgG1 antibody is provided. In other exemplary embodiments, humanized anti-LIV 1 IgG1 antibody-drug conjugates are provided.

Unless otherwise indicated, anti-LIV 1 antibody drug conjugates (i.e., LIV 1-ADCs) include antibodies specific for human LIV-1 protein conjugated to a cytotoxic agent.

SGN-LIV1A is an anti-LIV-1 humanized antibody (also known as hLIV22) conjugated to monomethyl reooxetine e (mmae) through a protease cleavable linker (i.e., a valine-citrulline linker). Upon binding to LIV-1 expressing cells, SGN-LIV1A is internalized and releases MMAE, thereby disrupting tubulin and inducing apoptosis.

SGN-LIV1A is a humanized form of the mouse BR2-22a antibody, described in U.S. Pat. No. 9,228,026. Within experimental error, the SGN-LIV1A antibody was essentially identical to BR2-22a and contained seven back mutations. Methods of making the SGN-LIV1A antibody are also disclosed in U.S. patent No. 9,228,026, which is incorporated by reference herein in its entirety for all purposes.

The amino acid sequence of the heavy chain variable region of SGN-LIV1A is provided herein as SEQ ID NO: 1. The amino acid sequence of the light chain variable region of SGN-LIV1A is provided herein as SEQ ID NO: 2. U.S. patent No. 9,228,026 and U.S. patent publication No. 2005/0238649 further describe the synthesis and conjugation of the drug linker vcMMAE (as shown below; also referred to as 1006), which is incorporated herein by reference in its entirety for all purposes.

TABLE 1 HCVR of SGN-LIV1A (SEQ ID NO:1)

TABLE 2 LCVR of SGN-LIV1A (SEQ ID NO:2)

According to certain exemplary embodiments, the LIV1-ADC comprises monomethyl Riocistatin E (MMAE) (Pubchem CID:53297465):

according to certain exemplary embodiments, the LIV1-ADC comprises vcMMAE conjugated thereto. vcMMAE is a drug-linker conjugate with potent anti-tumor activity for ADCs comprising the antimitotic agent MMAE linked by the lysosomally cleavable dipeptide valine-citrulline (vc):

U.S. patent No. 9,228,026 discloses a method for conjugating vcMMAE to hLIV 22.

vcMMAE-antibody conjugates (e.g., LIV1-ADC) according to certain exemplary embodiments are shown below.

According to certain exemplary embodiments, vcMMAE-antibody conjugates (e.g., LIV1-ADC) are provided as shown above, wherein Ab may comprise an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., hLIV22), and wherein p may be any integer from about 1 to about 8. In some embodiments, vcMMAE-antibody conjugates (e.g., LIV1-ADC) are provided as shown above, wherein Ab may comprise an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., hLIV22), and wherein p is 1, meaning that the ratio of vcMMAE to antibody or antigen-binding fragment thereof is 1. In some embodiments, vcMMAE-antibody conjugates (e.g., LIV1-ADC) are provided as shown above, wherein Ab may comprise an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., hLIV22), and wherein p is 2,3, 4, 5,6, 7, 8, 9, or 10, indicating a ratio of vcMMAE to antibody or antigen-binding fragment thereof (also referred to as a "drug-to-antibody ratio" or "DAR") of 2,3, 4, 5,6, 7, 8, 9, or 10, respectively. Thus, in some embodiments, there is provided a vcMMAE-antibody conjugate (e.g., LIV1-ADC) as shown above, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is 1, 2,3, 4, 5,6, 7, 8, 9, or 10. In certain exemplary embodiments, vcMMAE-antibody conjugates (e.g., LIV1-ADC) are provided as shown above, wherein Ab may comprise an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., hLIV22), and wherein p is 4, meaning that the ratio of vcMMAE to antibody or antigen-binding fragment thereof is 4. Thus, in certain exemplary embodiments, there is provided a vcMMAE-antibody conjugate (e.g., LIV1-ADC) as shown above, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is 4.

SGN-LIV1A can be administered to a subject at a level that inhibits growth of breast cancer cells while being tolerated by the subject.

In certain exemplary embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) comprises CDRs from a HCVR as set forth in SEQ ID NO:1 and/or CDRs from a LCVR as set forth in SEQ ID NO: 2. In certain exemplary embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) comprises an HCVR as set forth in SEQ ID NO:1 and/or an LCVR as set forth in SEQ ID NO: 2. In other embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) comprises the HCVR/LCVR pair SEQ ID NO:1/SEQ ID NO: 2. In other embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) comprises a HCVR having at least about 80% homology or identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to SEQ ID No. 1 and/or comprises a LCVR having at least about 80% homology or identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to SEQ ID No. 2.

The anti-LIV 1 antibodies and antigen-binding fragments thereof (e.g., LIV1-ADC) described herein can be represented in modified form. For example, additional amino acid regions, particularly charged amino acids, can be added to the N-terminus of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) to improve stability and persistence of the host cell during purification or during subsequent handling and storage. Additionally, peptide moieties can be added to the anti-LIV 1 antibodies or antigen-binding fragments thereof of the invention (e.g., LIV1-ADC) to facilitate purification. These regions may be removed prior to final preparation of the antibody molecule or at least one fragment thereof. Many standard laboratory manuals describe such methods, such as Sambrook, supra; ausubel, et al, ed., Current Protocols In Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001).

anti-LIV 1 antibodies or antigen-binding fragments thereof described herein (e.g., LIV1-ADC) typically bind LIV-1 with an equilibrium binding constant of about ≦ 1 μ M, e.g., about ≦ 100nM, about ≦ 10nM, or about ≦ 1nM, as measured using a standard binding assay, e.g., a Biacore-based binding assay.

Antibody molecules of the invention can be characterized relative to a reference anti-LIV-1 antibody, e.g., BR2-22 a. The antibody BR2-22a is described in U.S. Pat. No. 8,591,863 and is commercially available from the American Type Culture Collection.

Antibody-drug conjugates

In certain embodiments, the anti-LIV 1 antibodies of the invention can be combined with a conjugate (ADC). An exemplary anti-LIV 1-ADC antibody is SGN-LIV 1A. A particular ADC may comprise a cytotoxic agent (e.g., a chemotherapeutic agent), a prodrug converting enzyme, a radioisotope or compound or toxin (these moieties are collectively referred to as therapeutic agents). For example, the ADC may be conjugated to a cytotoxic agent, e.g., a chemotherapeutic agent or toxin (e.g., a cytostatic or cytocidal agent such as abrin (abrin), ricin a, pseudomonas exotoxin, or diphtheria toxin). Examples of useful classes of cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic agents include, for example, reocidins, haplotypesDendrimers, calicheamicins (calicheamicins), duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids, e.g., DM1, DM2, DM3, DM4), taxanes, benzodiazepines, and pharmaceutically acceptable salts thereofClass (e.g. pyrrolo [1,4 ]]BenzodiazepinesIndolebenzodiazepines, indolophenyldiazepinesAnd oxazolidinebenzodiazepinesClass (I) including pyrrolo [1,4 ]]BenzodiazepinesDimer, indolophenyldinitrogenDimer and oxazolidinebenzodiazepineDimer-like) and vinca alkaloids.

The ADC may be conjugated to a prodrug converting enzyme. The prodrug converting enzyme may be recombinantly fused to the antibody, or chemically conjugated thereto, using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin G amidase, beta-lactamase, beta-glucosidase, nitroreductase and carboxypeptidase a.

The art of conjugating therapeutic agents to proteins, particularly to antibodies, is well known (see, e.g., Alley et al, Current Opinion in Chemical Biology 201014: 1-9; Senter, Cancer J., 2008, 14(3): 154-) -169). A therapeutic agent can be conjugated in a manner such that its activity is reduced unless it cleaves the antibody (e.g., by hydrolysis, by proteolytic degradation, or by a cleaving agent). In some aspects, the therapeutic agent is attached to the antibody by a cleavable linker that is sensitive to cleavage in the intracellular environment of the cancer cell expressing LIV-1 but is substantially insensitive to the extracellular environment, such that the conjugate is cleaved when the antibody is internalized by the cancer cell expressing LIV-1 (e.g., in an endosome, or in a lysosomal environment or in a niche environment, e.g., by pH-sensitivity or protease-sensitivity). In some embodiments, the therapeutic agent may also be attached to the antibody through a non-cleavable linker.

In certain exemplary embodiments, the ADC may include a linker region between the cytotoxic or cytostatic agent and the antibody. As previously described, generally, the linker can be cleaved under intracellular conditions such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., within lysosomes or endosomes or pits). For example, the linker may be a peptidyl linker that is cleaved by intracellular peptidases or proteases, including lysosomal or endosomal proteases. Lysing agents can include cathepsins B and D, as well as plasmin (see, e.g., Dubowchik and Walker, pharm. therapeutics 83:67-123, 1999). Most typically a peptidyl linker that is cleavable by an enzyme present in a cell expressing LIV-1. For example, a peptidyl linker that is highly expressed in cancer tissues and cleaved by the thiol-dependent protease cathepsin-B (e.g., a linker comprising Phe-Leu or Val-Cit peptides) may be used.

At certain pH values, the cleavable linker is pH sensitive, i.e. sensitive to hydrolysis. Typically, the pH sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-acrylamides, orthoesters, acetals, ketals, etc.) may be used (see, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, pharm. therapeutics 83:67-123, 1999; Neville et al, biol. chem.264:14653-14661, 1989). Such linkers are relatively stable under neutral pH conditions (e.g., conditions in the blood), but are unstable at pH 5.5 or below 5.0 (the approximate pH of lysosomes).

Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), SPDB (N-succinimidyl-3- (2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene), SPDB, and SMPT (see, e.g., Thorpe et al, Cancer Res.47: 5924. 5931, 1987; Wawrzynczak et al, In immunoconjunates: Antibody Conjugates In Radioimage and Therapy of Cancer (C.W.Vogel ed., Oxford U.Press, 1987. see also U.S. Pat. No. 4,880,935).

The linker can be a malonate linker (Johnson et al, Anticancer Res.15:1387-93, 1995), a maleimidobenzoyl linker (Lau et al, Bioorg-Med-chem.3:1299-1304, 1995) or a 3' -N-amide analog (Lau et al, Bioorg-Med-chem.3:1305-12, 1995).

The linker may also be a non-cleavable linker, such as a maleimide-alkylene or maleimide-aryl linker directly attached to the therapeutic agent and released by proteolytic degradation of the antibody.

Typically, the linker is substantially insensitive to the extracellular environment, which means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linker in an ADC sample is cleaved when the ADC is present in the extracellular environment (e.g., plasma). It can be determined whether the linker is substantially insensitive to the extracellular environment, for example, by: the method can be performed by incubating (a) the ADC ("ADC sample") and (b) an equimolar amount of the unconjugated antibody or therapeutic agent ("control sample") independently with plasma for a predetermined period of time (e.g., 2, 4,8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample to the amount of unconjugated antibody or therapeutic agent present in the control sample, e.g., as measured by high performance liquid chromatography.

For example, when conjugated with a therapeutic agent (i.e., in the context of a linker-therapeutic agent portion of an ADC or ADC derivative as described herein), the linker may also promote cellular internalization. Alternatively, the linker may promote cellular internalization when conjugated to both the therapeutic agent and the antibody (i.e., in the context of an ADC as described herein).

The anti-LIV-1 antibody may be conjugated to the linker through a heteroatom of the antibody. These heteroatoms may be present on the antibody in their native state or may be incorporated into the anti-LIV-1 antibody. In some aspects, the anti-LIV-1 antibody will be conjugated to the linker through the sulfur atom of the cysteine residue. Methods of conjugating linkers and drug linkers to antibodies are known in the art.

Exemplary antibody-drug conjugates include reoxidine-based antibody-drug conjugates, meaning that the drug component is a reoxidine drug. Reoxidine binds to tubulin, has been shown to interfere with microtubule dynamics and nuclear and cellular division, and has anti-cancer activity. Typically, reoxidine-based antibody-drug conjugates comprise a linker between the reoxidine drug and the anti-LIV-1 antibody. For example, the linker can be a cleavable linker (e.g., a peptidyl linker) or a non-cleavable linker (e.g., a linker that is released by degradation of the antibody). Reooxetine includes MMAF and MMAE. The synthesis and structure of exemplary reocidins are described in U.S. patent nos. 7,659,241, 7,498,298, 7,968,687, and U.S. patent publication nos. 2009/0111756 and 2009/0018086, which are incorporated herein by reference in their entirety for all purposes.

In certain embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof can be combined with an Antibody Drug Conjugate (ADC), and the drug moiety ratio of each antibody can be about 1 to about 8. In a certain embodiment, an anti-LIV 1 antibody or antigen-binding fragment thereof can be combined with an ADC, and the drug moiety ratio of each antibody can be about 2 to about 5. In some embodiments, the drug moiety ratio of each antibody is 1, 2,3, 4, 5,6, 7, 8, 9, or 10. In certain exemplary embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof can be combined with an ADC and the drug moiety ratio of each antibody is 4. Methods of determining the drug moiety ratio of the antibody or antigen-binding fragment thereof for each ADC are known to those skilled in the art.

Therapeutic applications

The present invention provides methods of treating a disorder associated with cells expressing LIV-1, e.g., cancer (e.g., breast cancer, such as locally advanced breast cancer or metastatic breast cancer). Accordingly, the present invention provides methods of treating a subject (e.g., a subject having breast cancer) using the anti-LIV 1 antibodies and antigen-binding fragments thereof (e.g., LIV1-ADC) described herein. The methods comprise administering to a subject in need thereof an effective amount of an anti-LIV 1 antibody or a composition comprising an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV 1-ADC).

As used herein, the terms "subject" and "patient" refer to an organism treated by the methods of the invention. These organisms preferably include, but are not limited to, mammals (e.g., mice, apes, horses, cows, pigs, dogs, cats, etc.), and more preferably include humans. As used herein, the terms "treat", "treating" and "treating" include any effect that can ameliorate a condition, disease, disorder, etc., or reduce a symptom thereof, e.g., reduce, modulate, reduce or eliminate, e.g., reduce the number of cancer cells, reduce the size of a tumor, reduce the rate at which cancer cells infiltrate surrounding organs, or reduce the rate of tumor metastasis or tumor growth.

The positive therapeutic effect of cancer can be measured in a number of ways (see, w.a. weber, j.null. med.50:1S-10S (2009); Eisenhauer et al, supra). In certain exemplary embodiments, the response to an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is assessed using RECIST 1.1 criteria. In some embodiments, the treatment achieved by the therapeutically effective amount is any one of Partial Response (PR), Complete Response (CR), Progression Free Survival (PFS), Disease Free Survival (DFS), Objective Response (OR), OR Overall Survival (OS). The dosage regimen of the therapies described herein that are effective to treat a breast cancer patient may vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the therapy to elicit an anti-cancer response in the subject. Despite the therapeutic methods, medicaments and uses of the inventionEmbodiments of use may not be effective in achieving a positive therapeutic effect in each subject, but should be achieved in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student's t-test, chi²Test, U-test according to Mann-Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpsra test and Wilcoxon test.

As used herein, "RECIST 1.1Response Criteria" ("RECIST 1.1Response criterion") means the definition shown in Eisenhauer et al, e.a. et al, eur.j Cancer 45: 228-.

A "tumor," as applied to a subject diagnosed with or suspected of having a cancer (e.g., breast cancer), refers to a malignant or potentially malignant tumor or tissue mass of any size. A solid tumor is an abnormal growth of tissue or a tissue mass, which typically does not contain cysts or fluid areas. Different types of solid tumors are named as the cell types that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemia (hematological Cancer) does not typically form solid tumors (national Cancer institute, Cancer terminology Dictionary).

"tumor burden," also known as "tumor burden," refers to the total amount of tumor mass distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of the tumor, including lymph nodes and bone marrow, throughout the body. Tumor burden can be determined by a variety of methods known in the art, for example, measuring the size of a tumor using calipers after the tumor is removed from a subject or in vivo using imaging techniques (e.g., ultrasound, bone scan, Computed Tomography (CT), or Magnetic Resonance Imaging (MRI) scan).

The term "tumor size" refers to the overall size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, for example, measuring the size of the tumor using calipers or in vivo using imaging techniques (e.g., bone scan, ultrasound, CT, or MRI scan) after the tumor is removed from the subject.

As used herein, the term "effective amount" refers to an amount of a compound (e.g., an anti-LIV 1 antibody or antigen-binding fragment thereof) sufficient to produce a beneficial or desired result. An effective amount of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) can be administered, or dosed in one or more doses, and is not intended to be limited to a particular formulation or route of administration. Typically, a therapeutically effective amount of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) at a maximum dose of about 200mg ranges from 0.5mg/kg to 2.8 mg/kg. The dosage administered may vary according to known factors, such as the pharmacodynamic properties of the particular agent and its mode and route of administration; age, health, and weight of the recipient; the type and extent of the disease or indication to be treated, the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. In order to quickly reach the desired blood or tissue level, the initial dose may be increased above the upper limit. Alternatively, the initial dose may be less than the optimal dose and the daily dose may be increased gradually over the course of treatment. The frequency of administration may vary depending on factors such as the route of administration, the dose, the serum half-life of the antibody and the disease being treated. Exemplary dosing frequencies are once daily, once weekly, once every two weeks, and once every three weeks. The formulation of monoclonal antibody-based drugs is within the ordinary skill in the art. In some embodiments, the monoclonal antibody is lyophilized and then reconstituted in buffered saline at the time of administration.

In some embodiments, an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is administered to a patient who has not obtained a sustained response after a prior treatment (e.g., after failure or ineffective treatment of a systemic anti-cancer therapy with a non-anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC)), i.e., has undergone a cancer therapy.

In some embodiments, a medicament comprising an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) as described above can be provided in the form of a liquid formulation or prepared by reconstituting a lyophilized powder with sterile water for injection prior to use.

In certain embodiments, throughout the course of treatment, a dosing regimen will comprise administering an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) at a dose of about 2.5mg/kg of subject's body weight every about 21 days (± 2 days). In certain embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is used at a dose of less than about 200mg every three weeks.

In certain embodiments, throughout the course of treatment, a dosing regimen will comprise administering an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) at a dose of about 2.5mg/kg of subject's body weight every about 21 days (± 2 days). In certain embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is used at a dose of less than or equal to about 250mg every three weeks. In certain embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is used at a dose of less than or equal to 250mg every three weeks. In certain embodiments, the subject is further administered Granulocyte Colony Stimulating Factor (GCSF). In certain embodiments, the subject is further administered GCSF if the anti-LIV 1 antibody or antigen binding fragment thereof (e.g., LIV1-ADC) is used at a dose of greater than or equal to about 200mg and less than or equal to about 250mg every three weeks. In certain embodiments, the subject is further administered GCSF if the anti-LIV 1 antibody or antigen binding fragment thereof (e.g., LIV1-ADC) is used at a dose of greater than or equal to 200mg and less than or equal to 250mg every three weeks. In certain embodiments, the GCSF is administered prophylactically. In certain embodiments, the GCSF is recombinant human GCSF. In a certain embodiment, the GCSF is filgrastim (filgrastim,). In certain embodiments, the GCSF is PEG-filgrastimIn certain embodiments, the GCSF is a peptide of legrastim (lenograstim,). At a certain pointIn some embodiments, GCSF is tbo-filgrastim

In certain embodiments, a medicament comprising an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is to be administered parenterally (e.g., Intravenous (IV) infusion) to a subject.

In particular embodiments of the invention, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is administered to the subject in liquid pharmaceutical form at a dose selected from about 0.5mg/kg body weight once every three weeks (three weeks) or once every 21 days (once every 21 days), about 1.0mg/kg body weight once every three weeks or once every 21 days, about 1.5mg/kg body weight once every 21 days, about 2.0mg/kg body weight once every three weeks or once every 21 days, about 2.5mg/kg body weight once every 21 days, or about 2.8mg/kg body weight once every three weeks or once every 21 days, and for example, the maximum equivalent of any of these doses is less than about 200mg once every three weeks or once every 21 days.

In a particular embodiment of the invention, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is administered to the subject in liquid pharmaceutical form at a dose selected from about 0.5mg/kg body weight once every three weeks (three weeks) or once every 21 days (once every 21 days), about 1.0mg/kg body weight once every three weeks or once every 21 days, about 1.5mg/kg body weight once every three weeks or once every 21 days, about 2.0mg/kg body weight once every three weeks or once every 21 days, about 2.5mg/kg body weight once every 21 days, or once every three weeks or once every 21 days, and for example the maximum equivalent of any of these doses is less than or equal to about 250mg once every three weeks or once every 21 days. In certain embodiments, the subject is further administered GCSF. In certain embodiments, the subject is further administered GCSF if the dose is greater than or equal to about 200mg and less than or equal to about 250mg once every three weeks or once every 21 days. In certain embodiments, the subject is further administered GCSF. In certain embodiments, if the dose is greater than or equal to 200mg and less than or equal to 250mg once every three weeks or once every 21 days, then one more dose is administered to the subjectStep (2) GCSF is administered. In certain embodiments, the GCSF is administered prophylactically. In certain embodiments, the GCSF is recombinant human GCSF. In certain embodiments, the GCSF is filgrastimIn certain embodiments, the GCSF is PEG-filgrastimIn a certain embodiment, the GCSF is legrostatIn certain embodiments, GCSF is tbo-filgrastim

In some embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is provided at a dose of about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 191mg, about 192mg, about 193mg, about 194mg, about 195mg, about 196mg, about 197mg, about 198mg, about 199mg, or about 200 mg. In certain exemplary embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is provided at a dose of less than about 200mg, e.g., at a dose of about 200mg, at a dose of about 199mg, about 198mg, about 197mg, about 196mg, about 195mg, about 190mg, about 185mg, about 180mg, about 175mg, about 170mg, about 165mg, about 160mg, about 155mg, about 150mg, about 145mg, about 140mg, about 135mg, about 130mg, about 125mg, about 120mg, about 115mg, about 110mg, about 105mg, or about 100 mg.

In some embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is provided at a dose of about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 245mg, or about 250 mg. In certain exemplary embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is provided at a dose of less than or equal to about 250mg, e.g., at a dose of about 250mg, at a dose of about 245mg, at about 240mg, at about 235mg, at about 230mg, at about 225mg, at about 220mg, at about 215mg, at about 210mg, at about 205mg, at about 200mg, at about 195mg, at about 190mg, at about 185mg, at about 180mg, at about 175mg, at about 170mg, at about 165mg, at about 160mg, at about 155mg, at about 150mg, at about 145mg, at about 140mg, at about 135mg, at about 130mg, at about 125mg, at about 120mg, at about 115mg, at about 110mg, at about 105mg, or at about 100 mg.

In certain exemplary embodiments, the present invention provides a method for treating cancer in a cell, tissue, organ, animal or patient. In a particular embodiment, the present invention provides a method for treating breast cancer in a human.

Certain breast cancers exhibit detectable levels of LIV-1 measured at protein (e.g., by immunoassay using an exemplary antibody) or mRNA levels. In certain embodiments, the breast cancer exhibits elevated levels of LIV-1 relative to non-cancerous tissue or cells of the same type, e.g., other breast cells or breast tissue of the same patient. In other embodiments, the breast cancer exhibits similar levels of LIV-1 relative to the same type of non-cancerous breast tissue or breast cells, e.g., from the same patient.

Although breast cancers associated with higher or lower levels can be treated, exemplary levels of LIV-1 protein on breast cancer cells suitable for treatment are 5,000-150,000 LIV-1 proteins per cell. Optionally, the level of LIV-1 (e.g., LIV-1 protein level) in the breast cancer from the subject is measured prior to treatment.

Exemplary breast cancers are those that express LIV-1 in cells that express the cancer (i.e., cancers that express LIV 1). In certain exemplary embodiments, the breast cancer is selected from the group consisting of carcinoma, sarcoma, phyllode, paget's disease, and angiosarcoma. Breast cancer can be in situ (e.g., Ductal Carcinoma In Situ (DCIS), Lobular Carcinoma In Situ (LCIS), etc.) or invasive/invasive (e.g., Invasive Ductal Carcinoma (IDC), Invasive Lobular Carcinoma (ILC), and Invasive Breast Cancer (IBC), etc.).

Breast cancer may have the following characteristics: estrogen receptor positive (ER +); progesterone receptor positive (PR +); hormone receptor negative (HR-); HER2 gene overexpression (HER2 +); HER2 gene wild type or under-expressed (HER 2-); group 1 (Chamber A), i.e., ER +/PR +/HER 2-; group 2 (Chamber B), i.e., ER +/PR-/HER2 +; group 3(HER2+), i.e., ER-/PR-/HER2 +; and group 4 (substrate-like or Triple Negative (TN)), i.e., ER-/PR-/HER 2-.

Breast cancer can be further classified as grade 1, 2 or 3. Breast cancer of grade 1 or well differentiated (score 3, 4 or 5) contains slower growing cells and looks like normal breast tissue than high grade breast cancer. Grade 2 or moderately differentiated (fraction 6, 7) breast cancer cells grow at rates between grade 1-3 and look like cells between grade 1-3. Grade 3 or low differentiated (fraction 8, 9) breast cancer cells appear very different from normal cells, usually growing and spreading faster than grade 1 or 2 cells.

In certain exemplary embodiments, the breast cancer is incurable, unresectable locally advanced or metastatic breast cancer (LA/MBC). In certain embodiments, the breast cancer is Triple Negative (TN) (ER-/PR-/HER2-) breast cancer, ER-and/or PR +/HER 2-breast cancer, and LA/MBC breast cancer. In certain exemplary embodiments, the breast cancer is HER2+ and LA/MBC. In certain exemplary embodiments, the breast cancer is TN and LA/MBC. In certain exemplary embodiments, the breast cancer is selected from TN breast cancer, metastatic breast cancer, and metastatic TN breast cancer.

Throughout the specification, when compositions and kits are described as having, including, or containing specific components, or when processes and methods are described as having, including, or containing specific steps, it is contemplated that compositions and kits of the present invention additionally exist that consist essentially of, or consist of, the recited components, and that processes and methods according to the present invention exist that consist essentially of, or consist of, the recited process steps and method steps.

Pharmaceutical compositions and formulations

For therapeutic use, an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is conjugated to a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to buffers, carriers, and excipients that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio. The carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like which are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

Accordingly, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) compositions of the invention can comprise at least one of any suitable excipient, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable excipients are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, such as, but not limited to, the examples and methods described in Gennaro, Ed., Remington's pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers may be routinely selected for the mode of administration, solubility and/or stability of the antibody molecule, fragment or variant compositions well known in the art or as described herein.

Suitable pharmaceutical excipients and/or additives for use in The antibody molecule composition according to The invention are known in The art, for example as listed in "Remington: The Science & Practice of Pharmacy," 19th ed., Williams & Williams, (1995) and in "Physician's Desk Reference," 52nd ed., medical Economics, Montvale, N.J. (1998).

Pharmaceutical compositions containing an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) as disclosed herein can be presented in dosage unit form and can be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are Intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal and rectal administration. The preferred route of administration of the monoclonal antibody is IV infusion. Useful formulations may be prepared by methods known in the pharmaceutical art. See, for example, Remington's Pharmaceutical Sciences (1990) supra. Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers such as acetate, citrate or phosphate; and tonicity adjusting agents such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM(BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be protected from microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

The pharmaceutical formulation is preferably sterile. Sterilization may be accomplished by any suitable method, such as filtration through sterile filtration membranes. When the composition is lyophilized, filter sterilization may be performed before or after lyophilization and reconstitution.

The compositions of the present invention may take a variety of forms. Including, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and liposomes. The particular form depends on the intended mode of administration and therapeutic application. In exemplary embodiments, the compositions provided are in the form of injectable or infusible solutions. Exemplary administration is parenteral (e.g., intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular). In exemplary embodiments, the formulation is administered by intravenous infusion or injection. In another preferred embodiment, the formulation is administered by intramuscular or subcutaneous injection.

The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, subcutaneous, intraarterial, intrathecal, intracapsular, intraorbital, intravitreal, intracardiac, intradermal, intraperitoneal, transtracheal, inhalation, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

An exemplary dose of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is about 0.5mg/kg of subject body weight, about 1.0mg/kg of subject body weight, about 1.5mg/kg of subject body weight, about 2.0mg/kg of subject body weight, about 2.5mg/kg of subject body weight, or about 2.8mg/kg of subject body weight. In particular embodiments, an exemplary dose of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is about 2.5mg/kg of subject's body weight. In another particular embodiment, the maximum exemplary dose of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is about 200 mg/cycle. In another particular embodiment, the maximum exemplary dose of an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is about 250 mg/cycle.

In certain exemplary embodiments, a dose of about 2.5mg/kg is administered to a subject once every three weeks at a maximum dose of about 200 mg. In certain exemplary embodiments, an intravenous dose of 2.5mg/kg is administered to a subject once every three weeks at a maximum dose of about 200 mg.

In certain exemplary embodiments, a dose of about 2.5mg/kg is administered to a subject once every three weeks at a maximum dose of about 250 mg. In certain exemplary embodiments, an intravenous dose of about 2.5mg/kg is administered to a subject once every three weeks at a maximum dose of about 250 mg. In certain exemplary embodiments, the subject is further administered GCSF. In certain exemplary embodiments, the subject is further administered GCSF if the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is used at a dose of greater than or equal to about 200mg and less than or equal to about 250mg once every three weeks. In certain exemplary embodimentsIn a regimen, if the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is used at a dose of greater than or equal to 200mg and less than or equal to 250mg once every three weeks, the subject is further administered GCSF. In certain embodiments, the GCSF is administered prophylactically. In certain embodiments, the GCSF is recombinant human GCSF. In certain embodiments, the GCSF is filgrastimIn certain embodiments, the GCSF is PEG-filgrastimIn certain embodiments, the GCSF is legrostatinIn certain embodiments, GCSF is tbo-filgrastim

The present invention provides a kit comprising packaging materials and at least one vial containing a solution of at least one anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) and a defined buffer and/or preservative, optionally in an aqueous diluent. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. Such concentrations depend on the preservative selected and are readily determined by the skilled artisan.

A variety of delivery systems can be used to administer an anti-LIV 1 antibody or antigen-binding fragment thereof to a subject. In certain exemplary embodiments, the anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) is administered by intravenous infusion.

Any of the above formulations may be stored in liquid or frozen form and may optionally be subjected to a preservation process. In some embodiments, the above formulations are lyophilized, i.e., they are subjected to lyophilization. In some embodiments, the above-described formulations are subjected to a preservation process (e.g., lyophilization) and then reconstituted with a suitable liquid (e.g., water). By lyophilized is meant that the composition has been freeze-dried under vacuum. Lyophilization is typically accomplished by freezing a particular formulation to separate the solute from the solvent. The solvent is then removed by sublimation (i.e., primary drying) and by desorption (i.e., secondary drying).

The formulations of the present invention may be used with the methods described herein or with other methods of treating disease. The anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV1-ADC) formulation can be further diluted prior to administration to a subject. In some embodiments, the formulation will be diluted with saline and contained in an IV bag or syringe prior to administration to a subject. Thus, in some embodiments, a method for treating a cancer expressing LIV-1 in a subject will comprise administering to the subject in need thereof per week a pharmaceutical composition comprising an anti-LIV 1 antibody or antigen-binding fragment thereof (e.g., LIV 1-ADC).

It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, they will be more clearly understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to be limiting. All patents, patent applications, and references described herein are incorporated by reference in their entirety for all purposes.

Examples

Example 1: phase 1 study of antibody-drug conjugate SGN-LIV1A in patients with severe pre-treated triple-negative metastatic breast cancer

Method of producing a composite material

This ongoing phase 1 study evaluated the safety, tolerability, pharmacokinetics and antitumor activity of SGN-LIV1A (IV once every three weeks) in women with LIV-1 positive, unresectable, locally advanced or metastatic breast cancer (LA/MBC) (NCT 01969651). Patients with measurable disease and LA/MBC ≧ 2 previous cytotoxicity regimens are eligible. Patients with neuropathy of grade 2 or more were excluded. Responses were assessed according to RECIST v 1.1; patients with Stable Disease (SD) or better can continue treatment until disease progression or intolerable toxicity. After dose escalation was completed in hormone receptor positive/HER 2-negative (HR +/HER 2-) and Triple Negative (TN) patients, an expanded cohort was opened to further assess the safety and anti-tumor activity of single therapies in TN patients. Tumor biopsies were evaluated for LIV1 expression.

Results

To date, 69 patients (18HR +/HER2-, 51TN) received SGN-LIV1A with a median of 3 cycles (range 1-12) at a dose of 0.5 to 2.8 mg/kg. The median age of the patients was 56 years. Patients received a median of three LA/MBC previous cytotoxic treatment regimens. 58 patients had visceral disease and 37 patients had bone metastases. No dose-limiting toxicity (DLT) occurred in the 19 DLTs evaluated patients. The maximum tolerated dose does not exceed 2.8 mg/kg. The extended cohort openings for TN patients were 2.0 and 2.5 mg/kg.

Treatment emergent Adverse Events (AE) reported in > 25% of patients were fatigue (59%), nausea (51%), peripheral neuropathy (44%), alopecia (36%), appetite decline (33%), constipation (30%), abdominal pain (25%), diarrhea (25%) and neutropenia (25%). Most AEs were grade 1/2. AE grade > 3 include neutropenia (25%) and anemia (15%). Febrile neutropenia occurred in 2 patients with a total dose of over 200 mg/cycle, including one with treatment-related death due to sepsis. No other treatment-related deaths occurred in the study. Treatment was discontinued in 7 patients due to AE.

In dose escalation, activity was observed in 17 (EE) HR +/HER2 patients whose efficacy was evaluated, with a disease control rate (DCR ═ CR + PR + SD) of 59% (10SD), including one patient with SD ≧ 24 weeks. In 44 EE TN patients (dose escalation plus extension cohort), the Objective Response Rate (ORR) was 32% (14PR), the confirmed PR rate was 21%, the DCR was 64% (14PR, 14SD), and the clinical benefit rate (CBR ═ CR + PR + SD ≧ 24 weeks) was 36% (16 patients). For TN patients, median PFS was 11.3 weeks (95% CI: 6.1, 17.1). 10 patients were still receiving treatment.

Of the 631 MBC tumor samples of all clinical subtypes evaluated against LIV-1, 91% were positive and 75% had moderate to high expression (H-score ≧ 100).

After dose escalation was completed, multiple extended cohorts of SGN-LIV1A monotherapy (part a) were opened at recommended dose levels to recruit up to 15 patients with a particular breast cancer subtype to further determine safety and anti-tumor activity. After analysis of the safety and activity of the 2.0mg/kg and 2.5mg/kg dose cohorts, the recommended phase 2 dose was determined to be 2.5mg/kg (maximum dose of 200 mg/cycle).

Review of the safety data available to date found that the incidence of grade 3 or higher neutropenia AE (57%) was higher at the part a 2.5mg/kg dose level than in the overall monotherapy study population (39%). In addition, 1 of the 2.5mg/kg groups had neutropenia with sepsis-induced death. As a result, it was decided to evaluate the 2mg/kg dose level in the extended cohort. Patients enrolled at or after this decision date began receiving a starting dose of 2mg/kg, while subsequent doses were reduced to 2mg/kg in patients who previously received 2.5mg/kg SGN-LIV1A in earlier cycles.

At the end of the data, the mTNBC 2.0mg/kg extended cohort entry was nearly complete and 10 patients were still receiving treatment. A safety comparison between 2.0mg/kg (N ═ 26) and 2.5mg/kg (dose ≦ 200mg) (N ═ 18) shows no febrile neutropenic events or SAEs associated with neutropenia. In contrast, at a dose of 2.5mg/kg, neutropenia of >200mg (N ═ 11) is more common, with 5 of 11 patients developing SAEs associated with neutropenia. In 2 of 11 patients, febrile neutropenia occurred, including 1 treatment-related death due to sepsis. No other treatment-related deaths occurred in the study.

Example 2: phase 1 study of antibody-drug conjugate SGN-LIV1A in patients with severe pre-treated triple-negative metastatic breast cancer

Method of producing a composite material

This study was a continuation of the SGN-LIV1A monotherapy (part a) dose extension cohort study of the phase 1 study described in example 1, which included data from 22 additional doses (IV once three weeks) of SGN-LIV 1A. The patient was as described in example 1. These additional administrations were performed to assess the safety of a total maximum dose of 250 mg/cycle. The patient was dosed at 2.5mg/kg, and each of the 22 additional doses was greater than or equal to 200 mg/cycle due to the patient weighing greater than or equal to 80 kg.

Results

Of 22 additional administrations greater than or equal to 200 mg/cycle up to a maximum dose of 250 mg/cycle, 7 SGN-LIV1A were co-administered with Granulocyte Colony Stimulating Factor (GCSF) and 15 SGN-LIV1A were not co-administered with it. Of the 15 SGN-LIV1A doses not co-administered with GCSF, 5 resulted in the development of neutropenia (33.3%). However, administration of 7 SGN-LIV1A co-administered with GCSF did not result in the development of neutropenia. These results indicate that the incidence of neutropenia can be significantly reduced by the use of GCSF in patients receiving doses greater than or equal to 200mg doses/cycle up to a maximum dose of 250 mg/cycle.

Sequence listing

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Kostic, Ana

Corwin, Elizabeth

Drachman, Jonathan

Haughney, Peter

Zhao, Baiteng

Garfin, Phillip

Palanca-Wessels, Corinna

Abidoye, Oyewale O.

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Claims

1. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

administering to the subject a therapeutically effective dose of an antibody or antigen-binding fragment thereof that specifically binds human LIV-1,

wherein the antibody or antigen-binding fragment thereof is administered at a dose of less than about 200mg per treatment cycle, and

wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having at least 95% identity to SEQ ID NO. 1 and a Light Chain Variable Region (LCVR) having at least 95% identity to SEQ ID NO. 2.

2. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

wherein the antibody or antigen-binding fragment thereof is administered at a dose of less than or equal to about 250mg per treatment cycle,

wherein the antibody or antigen-binding fragment thereof comprises a Heavy Chain Variable Region (HCVR) having at least 95% identity to SEQ ID NO. 1 and a Light Chain Variable Region (LCVR) having at least 95% identity to SEQ ID NO. 2, and

wherein if the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg per treatment cycle, the method further comprises administering to the subject Granulocyte Colony Stimulating Factor (GCSF).

3. The method of claim 2, wherein the GCSF is administered prophylactically, if at all.

4. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

administering to the subject a Granulocyte Colony Stimulating Factor (GCSF),

wherein the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg and less than or equal to about 250mg per treatment cycle,

5. The method of claim 4, wherein the GCSF is administered prophylactically.

6. The method of any one of claims 1-5, wherein the LIV-1 associated cancer is breast cancer.

7. The method of claim 6, wherein the breast cancer is triple negative breast cancer.

8. The method of claim 6, wherein the breast cancer is metastatic breast cancer.

9. The method of claim 6, wherein the breast cancer is triple negative, metastatic breast cancer.

10. The method of claim 6, wherein the breast cancer is hormone receptor positive, metastatic breast cancer.

11. The method of any one of claims 1-10, wherein the treatment cycle is about every three weeks (once every three weeks).

12. The method of any one of claims 1-11, wherein the dose is about 2.5mg/kg of the subject's body weight.

13. The method of any one of claims 1-12, wherein the antibody or antigen-binding fragment thereof is conjugated to monomethyl reooxetine e (mmae):

14. the method of any one of claims 1-13, wherein the antibody or antigen-binding fragment thereof is conjugated to valine-citrulline-monomethyl reoscitine e (vcmmae):

15. the method of claim 14, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is from about 1 to about 8.

16. The method of claim 15, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is about 4.

17. The method of any one of claims 1-16, wherein the HCVR has at least 97% sequence identity to SEQ ID No. 1 and the LCVR has at least 97% sequence identity to SEQ ID No. 2.

18. The method of any one of claims 1-17, wherein the HCVR has at least 99% sequence identity to SEQ ID No. 1 and the LCVR has at least 99% sequence identity to SEQ ID No. 2.

19. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

wherein the antibody or antigen-binding fragment thereof is administered at a dose of less than about 200mg per treatment cycle,

wherein the antibody or antigen-binding fragment thereof comprises a HCVR at least 95% identical to SEQ ID NO. 1 and a LCVR at least 95% identical to SEQ ID NO. 2, and

wherein the antibody or antigen binding fragment thereof is conjugated to vcMMAE:

20. a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

wherein the antibody or antigen-binding fragment thereof comprises a HCVR at least 95% identical to SEQ ID NO. 1 and a LCVR at least 95% identical to SEQ ID NO. 2,

vcMMAE, and

21. The method of claim 20, wherein the GCSF is administered prophylactically, if at all.

22. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

administering to the subject a Granulocyte Colony Stimulating Factor (GCSF),

wherein the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg per treatment cycle and less than or equal to about 250mg per treatment cycle,

23. the method of claim 22, wherein the GCSF is administered prophylactically.

24. The method of any one of claims 19-23, wherein the dose is administered at a concentration of about 2.5mg/kg of body weight of the subject.

25. The method of any one of claims 19-24, wherein each treatment cycle is administered to the subject once every three weeks.

26. The method of any one of claims 19-25, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is from about 1 to about 8.

27. The method of claim 26 wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is about 4.

28. The method of any one of claims 19-27, wherein the LIV-1 associated cancer is breast cancer.

29. The method of claim 28, wherein the breast cancer is triple negative breast cancer.

30. The method of claim 28, wherein the breast cancer is metastatic breast cancer.

31. The method of claim 28, wherein the breast cancer is triple negative, metastatic breast cancer.

32. The method of claim 28, wherein the breast cancer is hormone receptor positive, metastatic breast cancer.

33. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

wherein the antibody or antigen-binding fragment thereof is administered at a dose of less than about 200mg per treatment cycle once every three weeks,

wherein the antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO. 1 and the LCVR of SEQ ID NO. 2, and

34. a method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

wherein the antibody or antigen-binding fragment thereof is administered at a dose of less than or equal to about 250mg per treatment cycle once every three weeks,

wherein the antibody or antigen-binding fragment thereof comprises the HCVR of SEQ ID NO. 1 and the LCVR of SEQ ID NO. 2,

vcMMAE, and

35. The method of claim 34, wherein the GCSF is administered prophylactically, if at all.

36. A method of treating a subject having or at risk of having an LIV-1 associated cancer, comprising:

administering to the subject a Granulocyte Colony Stimulating Factor (GCSF),

wherein the antibody or antigen-binding fragment thereof is administered at a dose of greater than or equal to about 200mg per treatment cycle once every three weeks and less than or equal to about 250mg per treatment cycle once every three weeks,

37. the method of claim 36, wherein the GCSF is administered prophylactically.

38. The method of any one of claims 33-37, wherein the LIV-1 associated cancer is breast cancer.

39. The method of claim 38, wherein the breast cancer is triple negative breast cancer.

40. The method of claim 38, wherein the breast cancer is metastatic breast cancer.

41. The method of claim 38, wherein the breast cancer is triple negative, metastatic breast cancer.

42. The method of claim 38, wherein the breast cancer is hormone receptor positive, metastatic breast cancer.

43. The method of any one of claims 33-42, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is about 4.

44. The method of any one of claims 33-43, wherein the dose is about 2.5mg/kg of the subject's body weight.

45. A method of treating a subject having or at risk of having LIV-1 associated breast cancer, comprising:

administering to the subject an antibody or antigen-binding fragment thereof that specifically binds human LIV-1 at a dose of about 2.5mg/kg of the subject's body weight,

46. a method of treating a subject having or at risk of having LIV-1 associated breast cancer, comprising:

vcMMAE, and

47. The method of claim 46, wherein the GCSF, if administered, is administered prophylactically.

48. A method of treating a subject having or at risk of having LIV-1 associated breast cancer, comprising:

administering to the subject a Granulocyte Colony Stimulating Factor (GCSF),

49. the method of claim 48, wherein the GCSF is administered prophylactically.

50. The method of any one of claims 45-49, wherein the breast cancer is triple negative breast cancer.

51. The method of any one of claims 45-50, wherein the breast cancer is metastatic breast cancer.

52. The method of claim 51, wherein the breast cancer is triple negative, metastatic breast cancer.

53. The method of any one of claims 45-50, wherein the breast cancer is hormone receptor positive, metastatic breast cancer.

54. The method of any one of claims 45-53, wherein the ratio of vcMMAE to antibody or antigen-binding fragment thereof is about 4.

55. The method of any one of claims 1-54, wherein the subject is a human.