Humanized antibodies to LIV-1 and use of same to treat cancer

Abstract

The invention provides humanized antibodies that specifically bind to LIV-1. The antibodies are useful for treatment and diagnoses of various cancers as well as detecting LIV-1.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

• • wherein:
  • -A- is a stretcher unit;
  • a is 0 or 1;
  • each —W— is independently an amino acid unit;
  • w is independently an integer ranging from 0 to 12;
  • —Y— is a spacer unit; and
  • y is 0, 1 or 2.

Representative stretcher units are depicted within the square brackets of Formulas (Ia) and (Ib: see infra), wherein A-, —W—, —Y—, -D, w and y are as defined above and R¹ is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₈-C₁₀ alkylene, —C₃-C₈ heterocyclo-, —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene, —(CH₂CH₂O)₂—, and —(CH₂CH₂O)₂—CH₂—; and r is an integer ranging from 1-10. Ab is antibody.

##STR00001##

The drug loading is represented by p, the number of drug-linker molecules per antibody. Depending on the context, p can represent the average number of drug-linker molecules per antibody, also referred to the average drug loading, P ranges from 1 to 20 and is preferably from 1 to 8. In some preferred embodiments, when p represents the average drug loading, p ranges from about 2 to about 5. In some embodiments, p is about 2, about 3, about 4, or about 5. The average number of drugs per antibody in a preparation may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.

The Amino Acid unit (—W—), if present, links the Stretcher unit (-A-) to the Spacer unit (—Y—) if the Spacer unit is present, and links the Stretcher unit to the cytotoxic or cytostatic agent (Drug unit; D) if the spacer unit is absent.

If present, —W_w— is preferably a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.

The Spacer unit (—Y—), when present, links an Amino Acid unit to the Drug unit. Spacer units are of two general types: self-immolative and non self-immolative. A non self-immolative spacer unit is one in which part or all of the Spacer unit remains bound to the Drug unit after enzymatic cleavage of an amino acid unit from the anti-LIV-1 antibody-linker-drug conjugate or the drug-linker compound. Examples of a non self-immolative Spacer unit include a (glycine-glycine) spacer unit and a glycine spacer unit. When an anti-LIV-1 antibody-linker-drug conjugate containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage via a tumor-cell associated-protease, a cancer-cell-associated protease or a lymphocyte-associated protease, a glycine-glycine-drug moiety or a glycine-drug moiety is cleaved from Ab-A_a-W_w,—. To liberate the drug, an independent hydrolysis reaction should take place within the target cell to cleave the glycine-drug unit bond.

Alternatively, an anti-LIV-1 antibody drug conjugate containing a self-immolative spacer unit can release the drug (D) without the need for a separate hydrolysis step. In some of these embodiments, —Y— is a p-aminobenzyl alcohol (PAB) unit that is linked to —W_w— via the nitrogen atom of the PAB group, and connected directly to -D via a carbonate, carbamate or ether group. Other examples of self-immolative spacers include aromatic compounds that are electronically equivalent to the PAB group such as 2-aminoimidazol-5-methanol derivatives (see Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237 for examples) and ortho or para-aminobenzylacetals. Spacers can be used that undergo facile cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs that are substituted at the α-position of glycine (Kingsbury, et al., 1984, J. Med. Chem. 27:1447) are also examples of self-immolative spacer strategies that can be applied to the anti-LIV-1 antibody-linker-drug conjugates. Alternatively, the spacer unit is a branched bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional drugs.

Useful classes of cytotoxic agents to conjugate to anti-LIV-1 antibodies include, for example, antitubulin agents. DNA minor groove binding agents, DNA replication inhibitors, chemotherapy sensitizers, or the like. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, duocarmycins, etoposides, maytansinoids and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, doxorubicin, morpholino-doxorubicin and cyano-morpholino-doxorubicin.

The cytotoxic agent can be a chemotherapeutic such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. The agent can also be a CC-1065 analogue, calicheamicin, maytansine, an analogue of dolastatin 10, rhizoxin, or palytoxin.

The cytotoxic agent can also be an auristatin. The auristatin can be an auristatin E derivative is, e.g., an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to produce AFB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of various auristatins are described in, for example, US 2005-0238649 and US2006-0074008.

The cytotoxic agent can be a DNA minor groove binding agent. (See, e.g., U.S. Pat. No. 6,130,237.) For example, the minor groove binding agent can be a CBI compound or an enediyne (e.g., calicheamicin).

The cytotoxic or cytostatic agent can be an anti-rubulin agent. Examples of anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). (Exemplary auristatins are shown below in formulae III-XIII. Other suitable antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermolide, and elemtherobin.

##STR00002## ##STR00003##

The cytotoxic agent can be a maytanisinoid, another group of anti-tubulin agents. For example, the maytansinoid can be maytansine or a maytansine containing drug linker such as DM-1 or DM-4 (ImmunoGen, Inc.; see also Chari et al., 1992. Cancer Res. 52:127-131).

Exemplary antibody drug conjugates include vcMMAE and mcMMAF antibody drug conjugates as follows wherein p and Ab are as previously described herein:

##STR00004##
or a pharmaceutically acceptable salt thereof.
VI. Other Antibodies to LIV-1

As well as humanized forms of the BR2-14a and BR2-22a antibodies discussed above, other antibodies binding to an extracellular domain of LIV-1 can be used in some of the methods of the invention, particularly the treatment of triple negative breast cancers. A collection of mouse antibodies to LIV-1 is described in US20080175839. These antibodies include 1.1F10, 1.7A4, BR2-10b, BR2-11a, BR2-13a, BR2-14a, BR2-15a, BR2-16a, BR2-17a, BR2-18a, BR2-19a, BR2-20a, BR2-21a, BR2-22a, BR2-23a, BR2-24a, and BR2-25a, of which BR2-19a produced by the hybridoma ATCC Accession No. PTA-5706 or BR2-23a produced by the hybridoma ATCC Accession No. PTA-5707 in addition to BR2-14a and BR2-22a are preferred. Humanized, chimeric or veneered forms of these antibodies can be made by conventional methods summarized below.

Other antibodies to LIV-1 can be made de novo by immunizing with LIV-1 or one or more extracellular domains thereof. The production of other non-human monoclonal antibodies, e.g., murine, guinea pig, primate, rabbit or rat, against an immunogen can be performed by as described by Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis or by recombinant expression.

Humanized, chimeric or veneered forms of non-human antibodies can be made. General methodology for producing humanized antibodies is described by Queen, U.S. Pat. Nos. 5,530,101 and 5,586,089; 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). A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence. A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions.

Human antibodies against LIV-1 can be provided by a variety of techniques described below. Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al., WO93/12227 (1993); U.S. Pat. Nos. 5,886,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 138, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) and phage display methods (see, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat. Nos. 5,877,218, 5,871,907. 5,858,657, 5,837,242, 5,733,743 and 5,565,332.

Any of the antibodies can be selected to have the same or overlapping epitope specificity as an exemplary antibody, such as the BR2-14a antibody, by a competitive binding assay or otherwise.

VII. Therapeutic Applications

The humanized antibodies of the invention, alone or as LIV-1 antibody drug conjugates thereof, can be used to treat cancer. Some such cancers show detectable levels of LIV-1measured at either the protein (e.g., by immunoassay using one of the exemplified antibodies ) or mRNA level. Some such cancers show elevated levels of LIV-1 relative to noncancerous tissue of the same type, preferably from the same patient. An exemplary level of LIV-1 on cancer cells amenable to treatments is 5000-150000 LIV-1 molecules per cell, although higher or lower levels can be treated. Optionally, a level of LIV-1 in a cancer is measured before performing treatment.

Examples of cancers associated with LIV-1 expression and amenable to treatment include breast cancer, prostate cancer, ovarian cancer, endometrial cancer, cervical, liver, gastric, kidney, and squamous cell carcinomas (e.g., bladder, head, neck and lung), skin cancers, e.g., melanoma, small lung cell carcinoma or lung carcinoid. The treatment can be applied to patients having primary or metastatic tumors of these kinds. The treatment can also be applied to patients who are refractory to conventional treatments (e.g., hormones, tamoxifen, herceptin), or who have replaced following a response to such treatments. The methods can also be used on triple negative breast cancers. A triple negative breast cancer is a term of art for a cancer lacking detectable estrogen and progesterone receptors and lacking overexpression of HER2/neu when stained with an antibody to any of these receptors, such as described in the examples. Staining can be performed relative to an irrelevant control antibody and lack of expression shown from a background level of straining the same or similar to that of the control within experimental error. Likewise lack of overexpression is shown by staining at the same or similar level within experimental error of noncancerous breast tissue, preferably obtained from the same patient. Alternatively or additionally, triple native breast cancers are characterized by lack of responsiveness to hormones interacting with these receptors, aggressive behavior and a distinct pattern of metastasis.

hLIV14 antibodies can be used to treat cancers that express LIV-1. In one embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing breast cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing prostate cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing melanoma. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing ovarian cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing endometrial cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing cervical cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing liver cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing gastric cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing kidney cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing squamous cell carcinomas (e.g., bladder, head, neck and lung cancer). In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing breast cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing skin cancer. In another embodiment, an hLIV14 antibody is used treat a subject with a LIV-1-expressing small lung cell carcinoma or lung carcinoid. hLIV22 antibodies can be used to treat cancers that express LIV-1. In one embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing breast cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing prostate cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing melanoma. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing ovarian cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing endometrial cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing cervical cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing liver cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing gastric cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing kidney cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing squamous cell carcinomas (e.g., bladder, head, neck, and lung cancer). In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing breast cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing skin cancer. In another embodiment, an hLIV22 antibody is used treat a subject with a LIV-1-expressing small lung cell carcinoma or lung carcinoid. This application provides the first disclosure that LIV-1 protein is expressed on the surface of the melanoma cells. Thus, antibodies that bind to LIV-1 can be used to treat patients that are afflicted with melanomas that express LIV-1. Such antibodies include antibodies disclosed herein, e.g., hLIV14 and hLIV22, but are not limited to the antibodies disclosed herein.

Humanized antibodies, alone or as conjugates thereof, are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of cancer. If a patient is already suffering from cancer, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the cancer relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficiency can be observed in an individual patient relative to historical controls or past experiences in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.

Exemplary dosages for a monoclonal antibody are 0.1 mg/kg to 50 mg/kg of the patient's body weight, more typically 1 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 15 mg/kg, 1 mg/kg to 12 mg/kg, or 1 mg/kg to 10 mg/kgl, or 2 mg/kg to 30 mg/kg, 2 mg/kg to 20 mg/kg, 2 mg/kg to 15 mg/kg, 2 mg/kg to 12 mg/kg, or 2 mg/kg to 10 mg/kg, or 3 mg/kg to 30 mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 12 mg/kg, or 3 mg/kg to 10 mg/kb. Exemplary dosages for a monoclonal antibody or antibody drug conjugates thereof are 1 mg/kg to 7.5 mg/kg, or 2 mg/kg to 7.5 mg/kg or 3 mg/kg to 7.5 mg/kg of the subject's body weight, or 0.1-20, or 0.5 mg/kg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg) or 10-1500 or 200-1500 mg as a fixed dosage. In some methods, the patient is administered a dose of at least 1.5 mg/kg, at least 2 mg/kg or at least 3 mg/kb, administered once every three weeks or greater. The dosage depends on the frequency of administration, condition of the patient and response to prior treatment, if any, whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration can also be localized directly into a tumor. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration can be, for example, by infusion over a period such as 30-90 min or by a single bolus injection.

The frequency of administration depends on the half-life of the antibody or conjugate in the circulation, the condition of the patient and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or progression of the cancer being treated. An exemplary frequency for intravenous administration is between twice a week and quarterly over a continuous course of treatment, although more or less frequent dosing is also possible. Other exemplary frequencies for intravenous administration are between weekly or three out of every four weeks over a continuous course of treatment, although more or less frequency dosing is also possible. For subcutaneous administration, an exemplary dosing frequency is daily to monthly, although more or less frequent dosing is also possible.

The number of dosages administered depends on the nature of the cancer (e.g., whether presenting acute or chronic symptoms) and the response of the disorder to the treatment. For acute disorders or acute exacerbations of a chronic disorder between 1 and 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided form, is sufficient for an acute disorder or acute exacerbation of a chronic disorder. Treatment can be repeated for recurrence of an acute disorder or acute exacerbation. For chronic disorders, an antibody can be administered at regular intervals, e.g., weekly, fortnightly, monthly, quarterly, every six months for at least 1, 5 or 10 years, or the life of the patient.

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical composition can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution. Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The concentration of antibody in a liquid formulation can be e.g., 1-100 mg/mL such as 10 mg/ml.

Treatment with antibodies of the invention can be combined with chemotherapy, radiation, stem cell treatment, surgery other treatments effective against the disorder being treated. Useful classes of other agents that can be administered with humanized antibodies to LIV-1 include, for example, antibodies to other receptors expressed on cancerous cells, antitubulin agents (e.g., auristatins), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

Treatment with the humanized anti-LIV-1 antibody, optionally in combination with any of the other agents or regimes described above along or as an antibody drug conjugate, can increase the median progression-free survival or overall survival time of patients with tumors (e.g., breast, prostate, melanoma), especially when relapsed or refractory, by at least 30% or 40% but preferably 50%, 60% to 70% or even 100% or longer, compared to the same treatment (e.g., chemotherapy) but without an anti-LIV-1 antibody alone or as a conjugate. In addition or alternatively, treatment (e.g., standard chemotherapy) including the anti-LIV-1 antibody alone or as a conjugate can increase the complete response rate, partial response rate, or objective response rate (complete+partial) of patients with tumors by at least 30% or 40% but preferably 50%, 60% to 70% or even 100% compared to the same treatment (e.g., chemotherapy) but without the anti-LIV-1 antibody.

Typically, in a clinical trial (e.g., a phase II, phase II/III or phase III trial), the aforementioned increased in medium progression-free survival and/or response rate of the patients treated with standard therapy plus the humanized anti-LIV-1 antibody, relative to the control group of patients receiving standard therapy alone (or plus placebo), are statistically significant, for example at the p=0.05 or 0.01 or even 0.001 level. The complete and partial response rates are determined by objective criteria commonly used in clinical trials for cancer, e.g., as listed or accepted by the National Cancer Institute and/or Food and Drug Administration.

VIII. Other Applications

The anti-LIV-1 humanized antibodies can be used for detecting LIV-1 in the context of clinical diagnosis or treatment or in research. Expression of LIV-1 on a cancer provides an indication that the cancer is amenable to treatment with the antibodies of the present invention. The antibodies can also be sold as research reagents for laboratory research in detecting cells bearing LIV-1 and their response to various stimuli. In such uses, monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotypes, and can be provided in the form of kit with all the necessary reagents to perform the assay for LIV-1. The antibodies described herein, BR2-14a, BR2-22a and humanized versions thereof, e.g., hLIV14 and hLIV22, can be used to detect LIV-1 protein expression and determine whether a cancer is amenable to treatment with LIV-1 ADCs. As an example, BR2-14a, BR2-22a and humanized versions thereof, e.g., hLIV14 and hLIV 22 can be used to detect LIV-1 expression on breast cancer cells, melanoma cells, cervical cancer cells, or prostate cancer cells. The antibodies can also be used to purify LIV-1, e.g., by affinity chromatography.

IX. Cynomolgus Monkey LIV-1

The invention further provides an amino acid sequence for LIV-1 (CY LIV-1) from cynomolgus monkeys at SEQ ID NO:85 with or without a signal peptide, which occupies approximately residues 1-28 of SEQ ID NO:85, as well as nucleic acids that encode that amino acid sequences. Variants differing by up to 1, 2, 3, 4, or 5 substitutions, deletions or insertions are also included provided CY variants do not include a natural human LIV-1 sequence. Analogous to human LIV-1, reference to CY-LIV-1 means at least one extracellular domain of the protein and usually the complete protein other than a cleavable signal peptide (amino acids 1-28). The invention further provides antibodies that specifically bind to SEQ ID NO:85 with or without specifically binding to human LIV-1 (i.e., binding to human LIV-1 at level of negative control irrelevant antibody). The invention further provides antibodies that preferentially bind CY-LIV-1 over human LIV-1 and vice versa. Preferential binding means an association higher beyond experimental error and preferably at least 2, 3 or 4 fold higher. The invention further provides antibodies that show the same binding profile to human and CY-LIV-1 within experimental error as any of the exemplified antibodies described below. The invention further provides methods of analyzing binding of an antibody to CY LIV-1. Such methods involve contacting an antibody with CY LIV-1, determining whether the antibody specifically binds to CY LIV-1 and optionally determining a measure of binding strength, such as an association constant.

All patent filings, website, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The efective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention can be used in combination with any other unless specifically indicated otherwise. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

EXAMPLES

I. Humanization of BR2-14a

Materials

Cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC), the National Cancer Institute (NCI) or the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany (DMSZ). Cell culture reagents were obtained from Invitrogen Corp. (Carlsbad, Calif.) or other suppliers.

Methodologies:

Saturation Binding Assays

1×10⁵ antigen expressing cells (either MCF7 cells (ATCC) expressing human LIV-1, a transfected CHO cell line expressing human LIV-1 or a transferred CHO cell line expressing cyno LIV-1) were aliquoted per well of a 96-well v-bottom plates. AlexaFluor-647 labeled murine LIV-1 mAb, e.g., BR2-14a, was added in concentrations ranging from 0.66 pM to 690 nM and incubated on ice for 30 minutes. Cells were pelleted and washed 3× with PBS/BSA. The cells were the pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence was analyzed by flow cytometry, using percent of saturated fluorescent signal to determine percent bound and to subsequently calculate apparent Kd.

Competition Binding Assays

1×10⁵ CHO cells expressing recombinant human LIV-1 in PBS/BSA were aliquoted on ice. The cells were incubated for 1 hours with 5 nM AlexaFluor-647 (AF) labeled parental murine LIV-1 mAb and increasing concentrations (from 0.038 nM to 600 nM) of unlabeled humanized LIV-1 mAb, combinations of humanized light chains LA-LF and humanized heavy chains HA-HE. Cells were pelleted and washed 3 times with PBS/BSA. The cells were pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence was analyzed by flow cytometry, using percent of saturated fluorescent signal to determine percent labeled murine LIV-1 mAb bound and to subsequently extrapolate the EC50 by fitting the data to a sigmoidal dose-response curve with variable slope.

1×10⁵ MCF7 cells expressing LIV-1 in PBS/BSA were aliquoted in each well of a 96-well v-bottom plates on ice. The cells were incubated for 1 hour with 5 nM AlexaFluor-647 labeled murine LIV-1 mAb and increasing concentrations (from 0.38 nM to 600 nM) of unlabeled humanized LIV-1 mAb, combinations of humanized light chains LA-LF and humanized heavy chains HA-HE. Cells were pelleted and washed 3 times with PBS. The cells were pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence was analyzed by flow cytometry, using percent of saturated fluorescent signal to determine percent labeled murine LIV-1 mAb bound and to subsequently extrapolate the BC50 by fitting the data to a sigmoidal dose-response curve with variable slope.

1×10⁵ CHO cells expressing recombinant cyno LIV-1 in PBS were aliquoted in each well of a 96-well v-bottom plates on ice. The cells were incubated for 1 hour with 5 nM AlexaFluor-647 labeled murine LIV-1 mAb and increasing concentrations (from 0.038 nM to 600 nM) of unlabeled humanized LIV-1 mAb, combinations of humanized light chains LA-LF and humanized heavy chains HA-HE. Cells were pelleted and washed 3 times with PBS. The cells were pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence was analyzed by flow cytometry, using percent of saturated fluorescent signal to determine percent labeled murine LIV-1 mAb bound and to subsequently extrapolate the EC50 by fitting the data to a sigmoidal dose-response curve with variable slope.

Quantitative Flow Cytometric Analysis

Quantification of LIV-1 copy number on the cell surfaces was determined using murine LIV-1 mAb as primary antibody and the DAKO QiFiKit flow cytometric indirect assay as described by the manufacturer (DAKO A/S, Glostrup, Denmark) and evaluated with a Becton Dickinson FACS®can flow cytometer.

Cytotoxicity Assay

Tumor cells were incubated with LIV-1 antibody drug conjugates for 96-144 hours at 37° C. A non-binding (H00) ADC was used as a negative control. Cell viability was measured by resazurin (Sigma) at the final concentration of 50 μM. Cells were incubated for four to six hours at 37°. Fluorescent signal was measured on a Fusion HT fluorescent plate reader (Perkin concentration of compound needed to yield a 50% reduction in viability compound to vehicle-treated cells (control=100%).

Production of antibody Drug Conjugates

Antibody drug conjugates of the LIV-1 antibodies were prepared as described in US20050238649. The drug linkers vcMMAE (also referred to as 1006) and mcMMAF (referred to as 1269) are both described in US20050238649. Preparation of cysteine mutants of IgG1 antibodies is generally described in US20100158919. US20050238649 and US20100158919 are herein incorporated by reference for all purposes.

Production of Non-fucosylated Anti-LIV-1 mAb

A CHO DG44 cell line producing the humanized IgG1 anti-LIV-1 monoclonal antibody, HBLB mAb (hLIV-14), was cultured at 3.0×10⁵ cells per mL in 30 mL of CHO culture media at 37°, 5% CO₂ and shaking at 100 RPM in a 125 mL shake flask. Media was supplemented with insulin like growth factor (IGF), penicillin, streptomycin and 65 μM 2-fluorofucose peracetate (SGD-2084) (see US20090317869). Cultures were fed on day 3 with 2% volume of feed media. On day four, the culture was split L4 into fresh culture media. Cultures were fed with a 6% volume of production feed media on days 5, 7, 9 and 10. Conditioned media was collected on day 13 by passing the culture through 0.2 μm filter. Antibody purification was performed by applying the conditioned media to a protein A column pre-equilibrated with 1× phosphate buffered saline (PBS), pH 7.4.

After washing column with 20 column volumes of 1×PBS, antibodies were eluted with 5 column volumes of Immunopure IgG elution buffer (Pierce Biotechnology, Rockford, Ill.). A 10% volume of 1M Tris pH 8.0 was added to eluted fraction. Sample was dialyzed overnight into 1×PBS.

ADCC activity was measured using the standard ³¹Cr-release assay. Briefly, the MCP-7 target tumor cells were labeled with 100 μCi Na⁵¹CrO₄, washed, and pre-incubated with test antibodies prior to addition of effector (natural killer, NK) cells. NK (Cd16⁺ CD56⁺) cells were prepared from non-adherent peripheral blood mononuclear cells (PBMCs) obtained from normal FcγRIIIA 158V/V donors (Lifeblood, Memphis, Tenn.) with immunomagnetic beads (EasyStep, StemCell Technologies, Vancouver, BC, Canada). Viable NK cells were added to target cells at an effector to target cell ratio of 10:1. A human IgG1κ (Ancell, Bayport, Minn.) was used as negative control in this assay. After 4 hours of incubation, supernatants were collected and dried overnight on Luma plates. Gamma radiation emitted from lysed MCF-7 cells was then detected using the TopCount Microplate Scintillation and Luminescence Counter (Perkin Elmer, Waltham, Mass.). ADCC activity is reported as % specific lysis.

In Vivo Activity Study

Nude (m/m) mice (7-8 animals/group) were implanted with tumor cells grown in culture. MCF-7 from NCl (5×10⁶ cells in 25% matrigel), PC3 from ATCC (2.5×10⁶ cells in 25% matrigel), and PC3 from DSMZ (5×10⁵ in 25% matrigel). For in vivo growth of MCF-7 cells, female mice also received estrogen supplementation by implanting a slow-release estrogen pellet (90 day release). Dosing with either chimeric or humanized LIV-1 ADC or nonbinding control ADC (3 mg/kg) started when tumors reached 100 mm³ (q4d×4 intra-peritoneal injections). Tumor volumes were monitored using calipers and animals were euthanized when tumor volume reached ˜800 mm³ Median tumor volume plots were continued for each group until one or more animals were euthanized. All animal procedures were performed under a protocol approved by the Institutional Animal Care and Use Committee in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

LIV-1 Immunohistochemical (IHC) Staining Method

Tumor microarrays (TMAs) and individual tumor samples were obtained from commercial sources. Tissue microarrays from normal or tumor formalin fixed and paraffin embedded (FFPE) tissues were purchased either from US Biomax Inc. or Cybrdi. A frozen array was purchased from BioChain. Single sections were purchased from NDRI, Asterand, Tissue Solution or CHTN. A set of 25 paraffin-embedded samples of metastatic hormone refractory prostate cancer (corresponding bone and soft tissue metastatic sites) was provided by Dr. R. Vessella, University of Washington, Genitourinary Cancer Department. All samples were processed on Bond-Max™ auto-stainer (Leica).

IHC Staining of FFPE Tissues:

FPPE slides or TMAs sectioned on glass slides were deparaffinized using Bond™ Dewax solution (Leica, cat #AR9222) at 72° C. and rehydrated. Antigen retrieval was performed using EDTA based Bond™ Epitope Retrieval Solution 2 (Leica, cat #AR9640) for 20 min at 95-100° C. before incubation with the primary murine LIV-1 mAb (1-2 μg/ml for 30-45 minutes at 25° C.). Isotype-matched murine IgG1 (Sigma; cat #M5284) was used as negative control for background staining. For automated IHC staining we used either a Refine DAB kit or an alkaline phosphatase based detection kit: Bond™ Polymer AP Red Detection kit (Leica, cat #DS9305). Slides were incubated with murine monoclonal primary antibodies against murine LIV-1 mAb for 45 min at 1 μg/ml with a preliminary 30 min protein block (DAKO cat #X0909). After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by a pathologist and images were taken using a Zeiss Axiovert 200M microscope (Car Zeiss, Inc., Thornwood, N.Y.).

IHC of Frozen Tissues:

5 μm sections of frozen/OCT samples were acetone fixed for 10 min., air dried for 30 min, and pretreated 20 min with 1× Morphosave at room temperature. The slides were loaded into Bond-Max™ auto-stainer (Leica) and stained for 45 min with primary antibody with preliminary 30 min protein block (DAKO cat# X0909). Mouse IgG1 (BD Pharmingen cat #550878) was used as negative control. For detection we used DAB-based Bond Polymer Refine kit (Leica, cat #DS9800). After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by pathologist.

Results

1. Binding of Mouse Antibody

The K_D for the murine LIV-1 monoclonal antibody BR2-14a antibody (US2004141983) was determined for human LIV-1 expressed as an endogenous protein in a human breast cancer cell line or as a recombinant protein in a CHO cell line. The K_D for the murine LIV-1 antibody BR2-14a was also determined for cyano LIV-1 expressed as a recombinant protein in a CHO cell line. MCF7 is a human breast cancer cell line. 293F is a human embryonic kidney cell line. Table 1 shows that the antibody had about 5-fold lower dissociation constant for non-recombinant LIV-1 expressed from a human cell line than recombinant LV-1, wherein human (hLIV-1) or from cynomolgus monkeys (cyLIV-1).

TABLE 1
	Cell line	Antigen	Kd (nM)
	MCF-7 (ATCC)	hLIV-1	2.4
	293F (hLIV-1)	hLIV-1	2.7
	CHO (hLIV-1)	hLIV-1	12.5
	CHO (cyLIV-1)	cLIV-1	14.0

2. Design and Testing of Humanized Antibodies

The starting point or donor antibody for humanization in this Example is the mouse antibody BR2-14a produced by the hybridoma having ATCC Accession No. PTA-5705A and descried in US2004141983. Suitable human acceptor sequences are genomic sequences provided by VH1-02 and JH5 for the heavy chain and by VK2-30 and Jk4 for the light chain. The human acceptor sequences show 68 and 85 percentage identity to the donor sequences in the variable region frameworks. The light chain CDRs of the human acceptor sequences are of the same canonical type as the CDRs of the donor sequences. In contrast, the heavy chain CDRs of the human acceptor sequences differed in their canonical type (the germline was 1-3 versus 1-2 for the murine donor).

Alignment of the donor sequences identified eleven positions in the heavy chain (H27, H28, H29, H30, H48, H66, H67, H71, H76, H93 and H94) and five positions in the light chain (L36, L37, L45, L46 and L39) at which the human acceptor sequence differed from the donor sequence and that may affect antibody binding as a result of contacting antigen directly, affecting conformation of CDRs or affecting packing between heavy and light chains. Five humanized heavy chains and six humanized light chains were made incorporating back mutations at different permutations of these positions (FIG. 1 (sequence alignment) and Table 2).

TABLE 2
Backmutations
	VH exon
	acceptor
VH variant	sequence	donor framework residues
hVHA	VH1-02	none
hVHB	VH1-02	H29, H30, H76
hVHC	VH1-02	H66, H67, H71
hVHD	VH1-02	H27, H93, H94
hVHE	VH1-02	H27, H28, H29, H30, H48, H76,
		H66, H67, H71, H93, H94
	VL exon
	acceptor
VL variant	sequence	donor framework residues
hVKA	VK2-30	none
hVKB	VK2-30	L36
hVKC	VK2-30	L37
hVKD	VK2-30	L45
hVKE	VK2-30	L46
hVKF	VK2-30	L36, L37, L39, L45, L46

Humanized antibodies were then expressed representing every permutation of these chains (30 possibilities) of the humanized heavy and light chains. The binding curves for recombinant human LIV-1 expressed from CHO cells are shown in FIG. 2. The EC50's are summarized in the Table 3 below.

TABLE 3

EC50s for humanized LIV-1 mAb antibodies, derived
from BR2-14a, on human LIV-1 expressed in CHO cells
Ab   EC50 (μg/mL)

HALA DNB
HALB 37.8
HALC 25.5
HALD 4.9
HALE DNB
HALF 8.8
HBLA 19.9
HBLB 0.3
HBLC 44.0
HBLD 17.4
HBLE DNB
HBLF 0.7
HCLA DNB
HCLB 1.8
HCLC DNB
HCLD 66.6
HCLE DNB
HCLF 1.3
HDLA DNB
HDLB 2.3
HDLC DNB
HDLD 67.9
HDLE DNB
HDLF 1.4
HELA 12.5
HELB 173.3
HELC DNB
HELD 24.2
HELE 0.3
HELF 1.5

DNB means “did not bind”

These data indicate considerable variation of EC50 between the 30 humanized antibodies tested with HBLB and HELE showing at least two fold better binding that the next humanized antibody HBLF and larger margins over most of the humanized antibodies. The binding curves of FIG. 2 show that both HBLB and HELE had stronger binding than the original mouse antibody.

The HBLB antibody was selected as the best of the humanized antibodies because it has (together with HELE) the strongest binding but has fewer backmutations versus HELE, there being four back mutations in HBLB and twelve in HELE.

The EC50s for the humanized LIV-1 mAb which bound human LIV-1 expressed on CHO cells were determined for human LIV-1 expressed as a native protein in an MCF7 cell line (FIG. 3). Again, LIV-1 mAb HBLB and HELE were determined to be the tightest binders.

The Kd for HBLB to human LIV-1 on the MCF7 cell line was determined from the average of several saturation binding curves as 1.5 nM whereas that for the mouse antibody is 2.9 nM. In other words, the HBLB antibody has about twice the affinity for native human LIV-1 as the mouse antibody. The saturation binding curve shown in FIG. 4 is a representative example.

Two forms of the HBLB were compared for binding to human LIV-1 recombinantly expressed from CHO cells. One form was expressed with wildtype human IgG1 and kappa constant regions. The other form was the same except for an S239C mutation (EU numbering) in the IgG1 heavy chain (referred to as LIV-14d or HBLB S239C), which reduces binding of the antibody to Fc gamma receptors. The binding curves and EC50's of these antibodies compared with the mouse donor antibody are shown in FIG. 5. The EC50's of both forms of HBLB were similar to one another (within the error of the study), and both were stronger than the mouse antibody.

The EC50s for the humanized LIV-1 mAb HBLB and HBLB S239C were also determined for cyno LIV-1 expressed as a recombinant protein in a CHO cell line. Both antibodies bound with equal affinity (better than murine LIV-1 mAb).

Expression Data for LIV-1

Murine LIV-1 mAbs (at least 2 for concordance) were used for immunohistochemical analysis of various tumor types using formalin-fixed paraffin embedded tissues.

TABLE 4
Summary of the expression data for LIV-1 in tumor samples
Origin	Type	LIV-1+	# cases	%
Breast	Primary & meta-			28-46
	static (TMA)
	Primary tumors	12	12	100
	Metastatic tumors	17	19	89
	Post-hormone	19	22	86
	treatment
	Triple Negative	13	20	65
Prostate	Metastatic hormone	15	25	60
	refractory: bone
	mets
	soft tissue mets	21	25	84
Ovarian	Primary (TMA)	9	72	13
	Metastatic (TMA)	4	11	36
	Post-chemo treated	5	17	29
Endometrial		7	56	12
Squamous cell	Primary tumors	8	114	7
carcinoma (uterine
and multiple organs)
Pancreatic	Primary tumors	9	95	9
Lung	Primary tumors	3	192	2
	(TMA)

We observed lower LIV-1 IHC positivity in studies done using tissue microarrays compared to large tissue sections. The difference in expression is highly significant suggesting analysis of LIV-1 expression in larger tissue sections is preferred. There was good concordance of expression using at least 2 different anti-LIV-1 mAbs. FIGS. 6 and 7 show a high level of LIV-1 expression in post-hormone (tamoxifen or aromatase inhibitors) treated breast and prostate tumors providing a strong rationale to target these tumors using a LIV-1 ADC. FIG. 8 shows detectable LIV-1 expression in triple negative (ER-, PgR-, Her2-) breast cancer tissues. The LIV-1 level of expression in triple negative breast cancer by immunohistochemistry staining was comparable to the level in the PC3 animal model, where we demonstrated anti-tumor activity of LIV-1 ADC. Triple negative breast cancers were therefore a potential target population, particularly triple negative breast cancers which have been found to express LIV-1.

In Vitro Anti-tumor Activity of hLIV-14 mAb as ADC and Effector Function Enhanced mAb (SEA)

Anti-tumor activity of LIV-1 ADCs in vitro was measured using both cytotoxicity assays (FIG. 9) and antibody dependent cell cytotoxicity (ADCC) (FIGS. 10 and 11). First, we performed a survey of LIV-1 expression in various cell lines by quantitative FACS analysis. The breast cancer cell line MCF-7 from ATCC had the highest level of LIV-1 binding sites/cell, as compared to the MCF-7 cell line from other sources (data not shown). We used this cell line for both assays in vitro. Referring to FIG. 9, various hLIV-14 ADCs (the HBLB antibody conjugated with vcMMAE (referred to as 1006) or mcMMAF (referred to as 1269) (both small molecules and/or linkers described in US20050238649)) were highly effective in killing MCF-7 cells, as compared with the nonbinding and murine control conjugates (mIgG-1006, mIgG-1269, hIgG-1006, and hIgG-1269). In addition cysteine mutant LIV-14d ADCs, having an average of two drug linkers per antibody were also highly effective in killing MCF-7 cells as measured by the cytotoxic assay. Referring to FIGS. 10 and 11, in ADCC assays the activity of the fucosylated/wild-type (WT) mAb and ADC's were compared with the effector-function enhanced versions (non-fucosylated mAbs and ADCs, referred to as SEA). The results demonstrated that effector function enhanced LIV-1 mAbs and ADCs have good ADCC activity against MCF-7 cells, as computed to non-effector function enhanced mAbs or ADCs (compare, for example, FIG. 10 hLIV-1 SEA and vcMMAE with hLIV-1 vcMMAE). Referring again to FIG. 9, an effector function enhanced LIV-1 ADC (indicated as SEA) also had a similar level of cytotoxic activity as wildtype (non-fucosylated) ADCs (compare hLIV-1 SEA 1006 (vcMMAE) with hLIV-1 1006 (vcMMAE)). Thus cytotoxicity can be affected by both effector function and conjugate action.

In Vivo Anti-tumor Activity of hLIV-14 ADC

Using breast cancer (MCF-7) and prostate cancer (PC-3) models, we determined the anti-tumor activity of LIV-1 ADCs (chimeric and humanized (HBLB) mAbs with an average of 4 drugs per antibody) in vivo (FIGS. 12-15). LIV-1 ADCs conjugated to vcMMAE showed significant tumor delay compared to untreated and control ADCs. At least one complete regression (CR) was observed in all the studies using LIV-1-vcMMAE at 3 mg/kg with a number of animals having tumors that were static or grew slowly compared to controls. Referring to FIG. 12, a chimeric form of the parental murine antibody conjugated to vcMMAE resulted in complete regressions in 3 out of 7 mice. Referring to FIG. 13, the same chimeric ADC produced a complete regression in 1 out of 8 mice. Referring to FIG. 14, a humanized ADC (HBLB) conjugated to vcMMAE (hLIV-14-mvMMAE)(4)) produced a complete regression in 1 out of 8 times. In addition, a cysteine mutant form of the HBLB antibody, a vcMMAE drug linker conjugated to each heavy chain at position 239, producing at conjugate with an average drug load of 2 drug linkers per antibody; designated hLIV-14d-vcMMAE(2)) exhibited similar activity as the 4-loaded form. Referring to FIG. 15, the humanized ADC (HBLB) conjugated to vcMMAE (hLIV-14-vcMMAE(4)) produced a complete regression in 1 out of 8 mice in a prostate carcinoma model. In contrast, the activity of the two loaded cysteine mutants was not as pronounced in this model (compare hLIV-14-vcMMAe(4) with hLIV-14d-vcMMAE(2), and hLIV-14-mcMMAF(4) with hLIV-14d-mcMMAF(2). In summary, these studies demonstrate that LIV-1 ADC can stop or delay growth of LIV-1 expressing cancers including breast and prostate.

II. Humanization of BR2-22a

BR2-22a, sometimes also referred to as an mAb2, in a mouse monoclonal antibody of isotype IgG1Kappa.

Methodologies:

Unless otherwise stated below, methods described for humanization and testing of BR2-14a are also applicable to BR2-22.

Saturation Binding Assays

1×10⁵ antigen expressing cells (either MCF7 cells expressing human LIV-1, 293F cells, a transfected CHO cell line expressing human LIV-1 or a transfected CHO cell line expressing cyno LIV-1) were aliquoted per wall of 96-well v-bottom plates. AtexaFlour-647 labeled murine BR2-22a was added in concentrations ranging from 0.66 pM to 690 nM and incubated on ice for 30 minutes. Cells were pelleted and washed 3× with PBS/BSA. The cells were then pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence was analyzed by flow cytometry, using percent of saturated fluorescent signal to determine percent bound and to subsequently calculate apparent Kd.

Competition Binding Assays

1×10⁵ CHO cells expressing recombinant LIV-1 in PBS were aliquoted into each cell well of a 96-well v-bottom plates on ice. The cells were incubated for 1 hour with 5 nM AlexaFluor-647 (AF) labeled parental BR2-22a and increasing concentrations (from 0.038 nM to 600 mM) of unlabeled humanized BR2-22a antibody in all combinations of humanized light Chains LA-LG and humanized heavy chains HA-HG. Cells were pelleted and washed 3 times with PBS. The cells were then pelleted and resuspended in 125 μL of PBS/BSA. Fluorescence was analyzed by flow cytometry, using percent of saturated fluorescent signal to determine percent labeled humanized BR2-22a antibody bound and to subsequently extrapolate the EC50 by fitting the data to a sigmoidal dose-response curve with variable slope.

In Vitro Activity Study

Nude (nn/nn) mice (7-8 animals/group) were implanted with tumor cells grown in culture MCF-7 (NCI) at 5×10⁶ in 25% matrigel, PC3 from ATCC (2.5×10⁶ cells in 25% matrigel), and PC3 from DSMZ (5×10⁵ in 25% matrigel). For in vivo growth of MCF-7 cells, female mice also received estrogen supplementation by implanting a slow-release estrogen pellet (90 day release). Dosing with either chimeric or humanized LIV-1 ADC or nonbinding control ADC (3 mg/kg) started when tumors reached 100 mm³ (q4d×4 intraperitoneal injections). Tumor volumes were monitored using calipers and animals were euthanized when tumor volume reached ˜800 mm³ Median tumor volume plots were continued for each group until one or more animals were euthanized. All animal procedures were performed under a protocol approved by the Institutional Animal Care and Use Committee in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

Summary of Results and Discussion

Saturation Binding

BR2-22a shows 94% identity to BR2-14a in the mature heavy chain variable region and 91% identity in the mature light chain variable region. The KD for the murine LivI of BR2-22a (Table 5) was determined for human LIV-1 expressed as an endogenous protein in a human breast cancer cell line, in 293F cells or as a recombinant protein in a CHO cell line. The KD for BR2-22a was also determined for cyno LIV-1 expressed as a recombinant protein in a CHO cell line.

TABLE 5
Affinity measurements of BR2-22a for human
(hLIV-1) and cyno LIV-1 (cyLIV-1).
	Cell line	Antigen	Kd (nM)
	MCF7 (ATCC)	hLIV-1	1.1
	293F (hLIV-1)	hLIV-1	0.5
	Cho hLIV-1	hLIV-1	1.5
	Cho cyLIV-1	cLIV-1	4.2

Humanization Study

The BR2-22a antibody was humanized using a VH1-02 JH5 germline acceptor sequence for the heavy chain and a VK2-30 JK4 acceptor sequence for the light chain. These acceptor sequences were chosen based on their having the highest sequence identity to the mature variable region frameworks of BR2-22A heavy and light chains. Initially five variant heavy chains were constructed. Each included the three Kabat CDRs from the heavy chain of BR2-22a, the chains differing in having from zero (VA) to 11 (VE) backmutations. Initially six variant light chains were constructed. Each included the three Kabat CDRs from the light chain of BR2-22a and from zero (LA) to four backmutations (LF). These backmutations were chosen as a result of modeling the BR2-22A antibody to identify positions with potential to interact with antigen directly, affect CDR conformation or affect the interface between heavy and light chains and based on prior experience in humanizing BR2-14a because of the high sequence identity between BR2-14a and BR2-22a. In fact, the same eleven positions in the heavy chain and same four positions in the light chain were considered for backmutation in both BR2-14a and BR2-22a (1.39 was not considered in BR2-22a because the mouse residue is the same as the human residue). The back mutations present in each variant of humanized BR2-22a are shown in Tables 6 and 7 below.

TABLE 6
		VH exon
		acceptor
	VH variant	sequence	donor framework residues
	hVHA	VH1-02	None
	hVHB	VH1-02	H29, H30, H76
	hVHC	VH1-02	H66, H67, H71
	hVHD	VH1-02	H27, H93, H94
	hVHE	VH1-02	H27, H28, H29, H30, H48, H66, H67,
			H71, H76, H93, H94
	hVHF	VH1-02	H27, H29, H30, H94
	hVHG	VH1-02	H27, H29, H30, H76, H94

TABLE 7
		VL exon
		acceptor
	VL variant	sequence	donor framework residues
	hVKA	VK2-30	None
	hVKB	VK2-30	L36
	hVKC	VK2-30	L37
	hVKD	VK2-30	L45
	hVKE	VK2-30	L46
	hVKF	VK2-30	L36, L37, L45, L46
	hVKG	VK2-30	L36, L46

The full sequence of the mature variable region of each variant is shown in FIGS. 16A and 16B.

All permutations of these five heavy chains and six light chains were then tested in a competition assay compared with BR2-22a (see FIG. 17). Surprisingly, in view of the experience with the BR2-14a antibody in which improved binding relative to the mouse antibody was obtained with only four backmutations and further backmutations did not necessarily improve binding affinity, the only combination of humanized chains that showed binding affinity approximating that of BR2-22a was HELF with 15 backmutations. The other permutations showed poor or no significant binding to LIV-1. The EC50s of the different permutations are shown in Table 8 below.

TABLE 8

EC50s for humanized BR2-22a antibodies
Ab   EC50 (μg/mL)

HALA DNB
HALB DNB
HALC DNB
HALD DNB
HALE DNB
HALF 33.2
HBLA DNB
HBLB 4.9
HBLC DNB
HBLD DNB
HBLE DNB
HBLF 6.5
HCLA DNB
HCLB >100
HCLC DNB
HCLD DNB
HCLE DNB
HCLF >100
HDLA DNB
HDLB DNB
HDLC DNB
HDLD DNB
HDLE DNB
HDLF 14.4
HELA 68.2
HELB >100
HELC 65.7
HELD >100
HELE 25.1
HELF 0.3
HELG 0.2
HFLF 0.8
HFLG 0.8
HGLF 0.4
HGLG 0.5

DNB means did not bind

Although HELF shows satisfactory binding, the antibody contains a total of 15 backmutations, a number larger than ideal with respect to potential immunogenicity. Therefore, the HE and LF chains were systematically varied to test the effect of removing individual backmutations. FIG. 18 shows the variants tested. LF-1 through LF-4 differ from LF in each lacking a different backmutation prevent in LF. Similarly, HE-I through HE-I1 lack one of the backmutations present in HE. FIG. 19 compares LF-1 through LF-4 (each paired with HE). FIG. 19 shows that LF-2 and LF-3 lose substantial binding affinity relative to LF (indicated as HELF historic control in the graph), whereas LF-1 and LF-4 do not. It is concluded that backmutations L36 and L46 contribute substantially to retention to binding affinity, while backmutations at positions L37 and L45 can be dispensed without significant effect on binding. FIG. 20 shows similar binding curves for the HE variants. FIG. 20 shows that HF-11 lost most of its binding indicating that backmutation at position H94 has the greatest effect on binding affinity of the backmutations tested. Loss of backmutations at positions H27, H29 and H30 also caused significant loss of affinity. The role of H30 can be rationalized by the mouse residue being the result of somatic mutation. Loss of a back mutation at position H76 caused some loss of affinity. The other back mutations at positions H28, H48, H66, H67, H71 and H93 could be dispensed with little or no effect on binding affinity.

In light of these experiments, heavy chains HF and HG were constructed as was light chain LG. HF included backmutations at H27, H29, H30 and H94 and HG included these mutations and a backmutation at H76. LG contains backmutations at L36 and L46. Several permutations of HF, HG, LE and LF were tested for competition binding as shown in FIG. 21 and all showed binding within a factor of three of that of mouse BR2-22a.

In light of this experiment, HGLG was selected for further experiments as representing the best combination of binding affinity and fewest backmutations. This antibody is hereafter referred to as hLIV22. The saturation binding affinity of hLIV22 for human and cyano LIV-1 expressed from CHO cells is shown in FIG. 22 compared with that of hLIV14. FIG. 22 shows that hLIV22 has about four fold higher affinity (inverse of dissociation constant) for human LIV-1 that does hLIV14. Furthermore, the affinity of hLIV22 for human LIV-1 is the same within experimental error as its affinity for cynomolgus LIV-1, whereas hLIV14 shows twice the affinity for human LIV-1 as for cynomolgus LIV-1. The affinity of hLIV22 for human LIV-1 is the same within experimental error as that of the parent mouse antibody, BR2-22a.

In Vitro Anti-tumor Activity of hLIV22 ADCs

Anti-tumor activity of hLIV22 ADC in vitro was measured using cytotoxicity assays. First, we performed a survey of LIV-1 expression in various cell lines by quantitative FACS analysis. The breast cancer cell line MCF-7 from ATCC had the highest level of LIV-1 binding sites/cell, as compared to the MCF-7 cell line from other sources (data not shown). We used this cell line for in vitro assays. We observed that various hLIV22 ADCs (configured with vcMMAE (referred to as 1006) or mcMMAF (referred to as 1269) (both small molecules described in US 2005-0238469)) were highly effective in killing MCF-7 cells as measured by the in vitro cytotoxic assay. FIGS. 23 and 24 compare hLIV22-conjugated to 1006 or 1269 with a nonbinding control antibody conjugated to 1006 or 1269.

In Vitro Anti-tumor Activity of LIV-1 ADC

Using prostate cancer (PC-3) and breast cancer (MCF-7) models as shown in FIGS. 25 and 26, we determined the anti-tumor activity of hLIV22 ADCs (with an average of 4 drugs per antibody) in vivo. hLIV22 ADCs conjugated to vcMMAE showed significant tumor delay compared to untreated and control ADCs. There were multiple complete regressions was observed in the MCF-7 study using hLIV22-vcMMAE at 3 mg/kg. Additionally, in all studies there were a number of animals that had tumors that were static or grew slowly compared to controls. These studies demonstrate that hLIV 22 ADC can stop or delay growth of LIV-1 expressing cancers, including breast and prostate. FIG. 27 compares the activity of hLIV22 and hLIV14 ADCs in the MCF-7 model. Although both antibodies were effective, hLIV22 was slightly more effective. hLIV22 ADCs were also tested in a model of cervical cancer. A HeLA cell xenograft model was used for the assay. After tumors grew to an appropriate six, hLIV22 conjugated to vcMMAE was administered to animals at 3 mg/kg and 1 mg/kg. A control antibody conjugate was administered at 3 mg/kg. Complete and partial regression were observed in animals that received 3 mg/kg hLIV22 vcMMAE conjugate. (Data not shown.) Thus, LIV-1 antibodies and antibody drug conjugates can be used to treat LIV-1 expressing cervical cancers.

III. Treatment of Skin Cancer Using Anti-LIV-1 Antibodies Expression of LIV-1 Protein on Melanoma Tumor Samples

Melanoma samples from patients were assessed for LIV-1 expression, using IHC staining. FFPE slides were de-paraffinized using Bond™ Dewax solution (Leica, cat #AR9222), at 72° C. Antigen retrieval was performed using EDTA based Bond™ Epitope Retrieval Solution 2 (Leica, cat #AR9640) for 20 min at 100° C. For IHC staining we used alkaline phosphatase based detection kit. Bond™ Polymer Refine Red Detection kit (Leica, cat #DS9390). Slides were incubated with murine monoclonal primary antibodies against LIV-1 (BR2-14a) for 45 min at 1 μg/ml with preliminary 30 min protein block (DAKO cat #X0909). Mouse IgG (Sigma, cat #M5284) was used as negative control. After chromogen development, sections were counterstained with hematoxylin and coverslipped. Slides were evaluated and scored by pathologist.

Results are shown in FIG. 28. Seventy-two percent of the tested melanoma patient samples (21/29) were positive for LIV-1 expression. This indicates that LIV-1 inhibitors, e.g., anti-LIV-1antibodies, can be used to treat melanoma cancers.

In Vivo Anti-melanoma Activity of LIV-1 ADC

Nude (nu/nu) mice (7-8 animals/group) are implanted with 10×10⁶ SK-MEL-5 cells (a melanoma tumor-derived cell line) grown in culture. Tumors are allowed to grow in vivo until they are 100 mm³, as measured using a caliper. Humanized LIV-1 ADCs, e.g., hLIV14 or hLIV22, are administered at 3 mg/kg. Drug conjugates are, e.g., vcMMAE or mcMMAF. Control ADC's are also administered to control animals at 3 mg/kg. ADC's are given as q4d×4 intraperitoneal injections. Tumor volumes are monitored using calipers and animals are euthanized when tumor volume reaches −800 mm³. Administration of hLIV14 ADC or hLIV22 ADC greatly reduced tumor growth in animals as compared to those animals that received control ADC's.

Sequence listing


<LIV-1 mAb light chain leader; PRT/1; mus
musculus>
SEQ ID NO: 1
MKLPVRLLVLMFWIPVSTS

<LIV-1 mAb heavy chain leader; PRT/1; mus
musculus>
SEQ ID NO: 2
MKCSWVIFFLMAVVLGINS

<replacement heavy chain leader sequence;
PRT/1; mus musculus>
SEQ ID NO: 3
MAWVWTLLFLMAAAQSAQA

<Light chain constant region; PRT/1; homo
sapiens>
SEQ ID NO: 4
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
KSFNRGEC

<CH1—CH3; PRT/1; homo sapiens>
SEQ ID NO: 5
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<heavy chain CH1—CH3 (no c-term K); PRT/1; homo
sapiens>
SEQ ID NO: 6
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

<S239C heavy chain CH1—CH3; PRT/1; homo sapiens>
SEQ ID NO: 7
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<S239C heavy chain CH1—CH3 (no c-term K); PRT/1;
homo sapiens>
SEQ ID NO: 8
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

<hLIV-1 mAb HA; PRT/1; artificial>
SEQ ID NO: 9
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYAPTFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
HDAHYGTWFAYWGQGTLVTVSS

<hLIV-1 mAb HB; PRT/1; artificial>
SEQ ID NO: 10
QVQLVQSGAEVKKPGASVKVSCKASGYTIEDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYAPTFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAR
HDAHYGTWFAYWGQGTLVTVSS

<hLIV-1 mAb HC; PRT/1; artificial>
SEQ ID NO: 11
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYAPTFQGKATMTADTSISTAYMELSRLRSDDTAVYYCAR
HDAHYGTWFAYWGQGTLVTVSS

<hLIV-1 mAb HD; PRT/1; artificial>
SEQ ID NO: 12
QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYAPTFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCNV
HDAHYGTWFAYWGQGTLVTVSS

<hLIV-1 mAb HE; PRT/1; artificial>
SEQ ID NO: 13
QVQLVQSGAEVKKPGASVKVSCKASGFNIEDYYMHWVRQAPGQGLEWIG
WIDPENGDTEYAPTFQGKATMTADTSINTAYMELSRLRSDDTAVYYCNV
HDAHYGTWFAYWGQGTLVTVSS

<hLIV-1 mAb LA; PRT/1; artificial>
SEQ ID NO: 14
DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFQQRPGQSP
RRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLIV-1 mAb LB; PRT/1; artificial>
SEQ ID NO: 15
DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWYQQRPGQSP
RRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLIV-1 mAb LC; PRT/1; artificial>
SEQ ID NO: 16
DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFLQRPGQSP
RRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLIV-1 mAb LD; PRT/1; artificial>
SEQ ID NO: 17
DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFQQRPGQSP
KRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLIV-1 mAb LE; PRT/1; artificial>
SEQ ID NO: 18
DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWFQQRPGQSP
RLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLIV-1 mAb LF; PRT/1; artificial>
SEQ ID NO: 19
DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWYLQKPGQSP
KLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

DNA sequences:
<LIV-1 mAb heavy chain leader; DNA; mus musculus>
SEQ ID NO: 20
atgaaatgcagctgggtcatcttcttcctgatggcagtggttctaggaa
tcaattca

<LIV-1 mAb light chain leader; DNA; mus musculus>
SEQ ID NO: 21
atgaagttgcctgttaggctgttggtgctgatgttctggattcctgttt
ctaccagt

>replacement heavy chain leader sequence; DNA;
mus musculus>
SEQ ID NO: 22
atggcttgggtgtggaccttgctattcctgatggcagctgcccaaagtg
cccaagca

>light chain constant region; DNA; mus musculus>
SEQ ID NO: 23
acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagt
tgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcc
cagagaggccaaagtacagtggaaggtggataacgccctccaatcgggt
aactcccaggagagtgtcacagagcaggacagcaaggacagcacctaca
gcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa
agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcaca
aagagcttcaacaggggagagtgt

<CH1—CH3; DNA; homo sapiens>
SEQ ID NO: 24
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggtaaa

<CH1—CH3 (w/o c-term K); DNA; homo sapiens>
SEQ ID NO: 25
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggt

<S239C CH1—CH3; DNA; artificial>
SEQ ID NO: 26
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggtaaa

<S239C CH1—CH3 (w/o c-term K); DNA; artificial>
SEQ ID NO: 27
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggt

<hLIV-1 mAb HA; DNA; artificial>
SEQ ID NO: 28
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacaccttcacagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgagaatggtgatactgaatatgcccccaccttccagg
gcagggtcaccatgaccagggacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
catgatgctcactatgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLIV-1 mAb HB; DNA; artificial>
SEQ ID NO: 29
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacaccattgaagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgagaatggtgatactgaatatgcccccaccttccagg
gcagggtcaccatgaccagggacacctccatcaacacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
catgatgctcactatgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLIV-1 mAb; DNA; artificial>
SEQ ID NO: 30
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacaccttcacagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgagaatggtgatactgaatatgcccccaccttccagg
gcaaggccactatgactgcagacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
catgatgctcactatgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLIV-1 mAb HD; DNA; artificial>
SEQ ID NO: 31
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggattcaccttcacagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgagaatggtgatactgaatatgcccccaccttccagg
gcagggtcaccatgaccagggacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
catgatgctcactatgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLIV-1 mAb HE; DNA; artificial>
SEQ ID NO: 32
Caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggattcaacattgaagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggattgga
tggattgatcctgagaatggtgatactgaatatgcccccaccttccagg
gcaaggccactatgactgcagacacctccatcaacacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtaatgtc
catgatgctcactatgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLIV-1 mAb LA; DNA; artificial>
SEQ ID NO: 33
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagcattataaggaatga
tggaaacacctatttggaatggtttcagcagaggccaggccaatctcca
aggaggctaatttatagagtttccaacaggttttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<hLIV-1 mAb LB; DNA; artificial>
SEQ ID NO: 34
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagcattataaggaatga
tggaaacacctatttggaatggtaccagcagaggccaggccaatctcca
aggaggctaatttatagagtttccaacaggttttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<hLIV-1 mAb LC; DNA; artificial>
SEQ ID NO: 35
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagcattataaggaatga
tggaaacacctatttggaatggtttctgcagaggccaggccaatctcca
aggaggctaatttatagagtttccaacaggttttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<hLIV-1 mAb LD; DNA; artificial>
SEQ ID NO: 36
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagcattataaggaatga
tggaaacacctatttggaatggtttcagcagaggccaggccaatctcca
aagaggctaatttatagagtttccaacaggttttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<hLIV-1 mAb LE; DNA; artificial>
SEQ ID NO: 37
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggaa
gccctgcctccatctcctgcagatctagtcagagcattataaggaatga
tggaaacacctatttggaatggtttcagcagaggccaggccaatctcca
aggctcctaatttatagagtttccaacaggttttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<hLIV-1 mAb LF; DNA; artificial>
SEQ ID NO: 38
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagcattataaggaatga
tggaaacacctatttggaatggtacctgcagaaaccaggccaatctcca
aagctcctaatttatagagtttccaacaggttttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<Liv1 mAb2 light chain leader; PRT/1; mus
musculus>
SEQ ID NO: 39
MKLPVRLLVLMFWIPVATSS

<Liv1 mAb2 heavy chain leader; PRT/1; mus
musculus>
SEQ ID NO: 40
MKCSWVIFFLMAVVIGINS

<replacement heavy chain leader sequence; PRT/1;
mus musculus>
SEQ ID NO: 41
MAWVWTLLFLMAAAQSAQA

<Light chain constant region; PRT/1; homo
sapiens>
SEQ ID NO: 42
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
KSFNRGEC

<CH1—CH3; PRT/1; homo sapiens>
SEQ ID NO: 43
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

<heavy chain CH1—CH3 (no c-term K); PRT/1; homo
sapiens>
SEQ ID NO: 44
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

<S239C heavy chain CH1—CH3; PRT/1; homo sapiens>
SEQ ID NO: 45
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<S239C heavy chain CH1—CH3 (no c-term K); PRT/1;
homo sapiens>
SEQ ID NO: 46
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPCVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

<hLiv1 mAb2 HA; PRT/1; artificial>
SEQ ID NO: 47
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYGPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 HB; PRT/1; artificial>
SEQ ID NO: 48
QVQLVQSGAEVKKPGASVKVSCKASGYTIEDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAR
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 HC; PRT/1; artificial>
SEQ ID NO: 49
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYGPKFQGKATMTADTSISTAYMELSRLRSDDTAVYYCAR
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 HD; PRT/1; artificial>
SEQ ID NO: 50
QVQLVQSGAEVKKPGASVKVSCKASGLTFTDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYGPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCTV
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 HE; PRT/1; artificial>
SEQ ID NO: 51
QVQLVQSGAEVKKPGASVKVSCKASGLNIEDYYMHWVRQAPGQGLEWIG
WIDPENGDTEYGPKFQGKATMTADTSINTAYMELSRLRSDDTAVYYCTV
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 HF; PRT/1; artificial>
SEQ ID NO: 52
QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYGPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAV
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 HG; PRT/1; artificial>
SEQ ID NO: 53
QVQLVQSGAEVKKPGASVKVSCKASGLTIEDYYMHWVRQAPGQGLEWMG
WIDPENGDTEYGPKFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCAV
HNAHYGTWFAYWGQGTLVTVSS

<hLiv1 mAb2 LA; PRT/1; artificial>
SEQ ID NO: 54
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFQQRPGQSP
RRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLiv1 mAb2 LB; PRT/1; artificial>
SEQ ID NO: 55
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSP
RRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLiv1 mAb2 LC; PRT/1; artificial>
SEQ ID NO: 56
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFLQRPGQSP
RRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLiv1 mAb2 LD; PRT/1; artificial>
SEQ ID NO: 57
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFQQRPGQSP
KRLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLiv1 mAb2 LE; PRT/1; artificial>
SEQ ID NO: 58
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWFQQRPGQSP
RPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLiv1 mAb2 LF; PRT/1; artificial>
SEQ ID NO: 59
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYLQRPGQSP
KPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

<hLiv1 mAb2 LG; PRT/1; artificial>
SEQ ID NO: 60
DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSSGNTYLEWYQQRPGQSP
RPLIYKISTRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH
VPYTFGGGTKVEIKR

DNA sequences:
<Liv1 mAb2 heavy chain leader; DNA; mus musculus>
SEQ ID NO: 61
atgaaatgcagctgggtcatcttcttcctgatggcagtggttataggaa
tcaattca

<Liv1 mAb2 light chain leader; DNA; mus musculus>
SEQ ID NO: 62
atgaagttgcctgttaggctgttggtgctgatgttctggattcctgcta
ccagcagt

<replacement heavy chain leader sequence; DNA;
mus musculus>
SEQ ID NO: 63
atggcttgggtgtggaccttgctattcctgatggcagctgcccaaagtg
cccaagca

<light chain constant region; DNA; homo sapiens>
SEQ ID NO: 64
acgacggtggctgcaccatctgtcttcatcttcccgccatctgatgagc
agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcta
tcccagagaggccaaagtacagtggaaggtggataacgccctccaatcg
ggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacct
acagcctcagcagcaccctgacgctgagcaaagcagactacgagaaaca
caaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtc
acaaagagcttcaacaggggagagtgttag

<CH1-CH3; DNA; homo sapiens>
SEQ ID NO: 65
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacatcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggtaaa

<CH1—CH3 (w/o c-term K); DNA; homo sapiens>
SEQ ID NO: 66
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggt

<S239C CH1—CH3; DNA; artificial>
SEQ ID NO: 67
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacagaagagcctctccctgtc
tccgggtaaa

<S239C CH1—CH3 (w/o c-term K); DNA; artificial>
SEQ ID NO: 68
gctagcaccaagggcccatctgtcttccccctggcaccctcctccaaga
gcacctctgggggcacagctgccctgggctgcctggtcaaggactactt
ccctgaacctgtgacagtgtcctggaactcaggcgccctgaccagcggc
gtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacat
ctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagtt
gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcac
ctgaactcctggggggaccgtgtgtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtg
gacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg
gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactgg
ctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccag
cccccatcgagaaaaccatctccaaagccaaagggcagccccgagaacc
acaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg
tggagtgggagagcaatgggcagccggagaacaactacaagaccacgcc
tcccgtgctggactccgacggctccttcttcctctacagcaagctcacc
gtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtga
tgcatgaggctctgcacaaccactacacacaggagagcctctccctgtc
tccgggt

<hLiv1 mAb2 HA; DNA; artificial>
SEQ ID NO: 69
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacaccttcacagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcagggtcaccatgaccagggacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLiv1 mAb2 HB; DNA; artificial>
SEQ ID NO: 70
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacaccattgaagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcagggtcaccatgaccagggacacctccatcaacacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLiv1 mAb2 HC; DNA; artificial>
SEQ ID NO: 71
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggatacaccttcacagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcaaggccaccatgaccgcagacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccaga
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<Liv1 mAb2 HD; DNA; artificial>
SEQ ID NO: 72
Caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggactcaccttcacagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcagggtcaccatgaccagggacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtactgtc
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLiv1 mAb2 HE; DNA; artificial>
SEQ ID NO: 73
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggactcaacattgaagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggattgga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcaaggccaccatgaccgcagacacctccatcaacacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtactgtc
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLiv1 mAb2 HF; DNA; artificial>
SEQ ID NO: 74
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggactcaccattgaagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcagggtcaccatgaccagggacacctccatcagcacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccgtc
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLiv1 mAb2 HG; DNA; artificial>
SEQ ID NO: 75
caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcct
cagtgaaggtctcctgcaaggcttctggactcaccattgaagactacta
tatgcactgggtgaggcaggcccctggacaagggcttgagtggatggga
tggattgatcctgaaaatggtgatactgaatatggcccgaagttccagg
gcagggtcaccatgaccagggacacctccatcaacacagcctacatgga
gctgagcaggctgagatctgatgacacagctgtgtattactgtgccgtc
cataatgctcactacgggacctggtttgcttactggggccaaggaaccc
tggtcacagtctcctca

<hLiv1 mAb2 LA; DNA; artificial>
SEQ ID NO: 76
gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtct
ccactctccctgcctgtcacccttggacagcctgcctccatctcctgca
gatctagtcagagccttttacacagtagtggaaacacctatttagaatg
gtttcagcagaggccaggccaatctccaaggaggctaatttataaaatt
tccacccgattttctggggtcccagacagattctctggcagtgggtcag
gcactgatttcacactgaaaatcagcagggtggaggctgaggatgttgg
ggtttattactgctttcaaggttcacatgttccctacacctttggagga
gggaccaaggtggagatcaaacgtacg

<hLiv1 mAb2 LB; DNA; artificial>
SEQ ID NO: 77
gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtct
ccactctccctgcctgtcacccttggacagcctgcctccatctcctgca
gatctagtcagagccttttacacagtagtggaaacacctatttagaatg
gtaccagcagaggccaggccaatctccaaggaggctaatttataaaatt
tccacccgattttctggggtcccagacagattctctggcagtgggtcag
gcactgatttcacactgaaaatcagcagggtggaggctgaggatgttgg
ggtttattactgctttcaaggttcacatgttccctacacctttggagga
gggaccaaggtggagatcaaacgtacg

<hLiv1 mAb2 LC; DNA; artificial>
SEQ ID NO: 78
gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtct
ccactctccctgcctgtcacccttggacagcctgcctccatctcctgca
gatctagtcagagccttttacacagtagtggaaacacctatttagaatg
gtttctgcagaggccaggccaatctccaaggaggctaatttataaaatt
tccacccgattttctggggtcccagacagattctctggcagtgggtcag
gcactgatttcacactgaaaatcagcagggtggaggctgaggatgttgg
ggtttattactgctttcaaggttcacatgttccctacacctttggagga
gggaccaaggtggagatcaaacgtacg

<hLiv1 mAb2 LD; DNA; artificial>
SEQ ID NO: 79
gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtct
ccactctccctgcctgtcacccttggacagcctgcctccatctcctgca
gatctagtcagagccttttacacagtagtggaaacacctatttagaatg
gtttcagcagaggccaggccaatctccaaagaggctaatttataaaatt
tccacccgattttctggggtcccagacagattctctggcagtgggtcag
gcactgatttcacactgaaaatcagcagggtggaggctgaggatgttgg
ggtttattactgctttcaaggttcacatgttccctacacctttggagga
gggaccaaggtggagatcaaacgtacg

<hLiv1 mAb2 LE; DNA; artificial>
SEQ ID NO: 80
gatgttctggattcctgctaccagcagtgatgttgtgatgactcagtct
ccactctccctgcctgtcacccttggacagcctgcctccatctcctgca
gatctagtcagagccttttacacagtagtggaaacacctatttagaatg
gtttcagcagaggccaggccaatctccaaggcccctaatttataaaatt
tccacccgattttctggggtcccagacagattctctggcagtgggtcag
gcactgatttcacactgaaaatcagcagggtggaggctgaggatgttgg
ggtttattactgctttcaaggttcacatgttccctacacctttggagga
gggaccaaggtggagatcaaacgtacg

<Liv1 mAb2 LF; DNA; artificial>
SEQ ID NO: 81
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagccttttacacagtag
tggaaacacctatttagaatggtacctgcagaggccaggccaatctcca
aagcccctaatttataaaatttccacccgattttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<hLiv1 BR2-22a LG; DNA; artificial>
SEQ ID NO: 82
gatgttgtgatgactcagtctccactctccctgcctgtcacccttggac
agcctgcctccatctcctgcagatctagtcagagccttttacacagtag
tggaaacacctatttagaatggtaccagcagaggccaggccaatctcca
aggcccctaatttataaaatttccacccgattttctggggtcccagaca
gattctctggcagtgggtcaggcactgatttcacactgaaaatcagcag
ggtggaggctgaggatgttggggtttattactgctttcaaggttcacat
gttccctacacctttggaggagggaccaaggtggagatcaaacgt

<Q13433; protein
SEQ ID NO: 83
MARKLSVILI LTFALSVTNP LHELKAAAFP QTTEKISPNW
ESGINVDLAI STRQYHLQQL FYRYGENNSL SVEGFRKLLQ
NIGIDKIKRI HIHHDHDHHS DHEHHSDHER HSDHEHHSEH
EHHSDHDHHS HHNHAASGKN KRKALCPDHD SDSSGKDPRN
SQGKGAHRPE HASGRRNVKD SVSASEVTST VYNTVSEGTH
FLETIETPRP GKLFPKDVSS STPPSVTSKS RVSRLAGRKT
NESVSEPRKG FMYSRNTNEN PQECFNASKL LTSHGMGIQV
PLNATEFNYL CPAIINQIDA RSCLIHTSEK KAEIPPKTYS
LQIAWVGGFI AISIISFLSL LGVILVPLMN RVFFKFLLSF
LVALAVGTLS GDAFLHLLPH SHASHHHSHS HEEPAMEMKR
GPLFSHLSSQ NIEESAYFDS TWKGLTALGG LYFMFLVEHV
LTLIKQFKDK KKKNQKKPEN DDDVEIKKQL SKYESQLSTN
EEKVDTDDRT EGYLRADSQE PSHFDSQQPA VLEEEEVMIA
HAHPQEVYNE YVPRGCKNKC HSHFHDTLGQ SDDLIHHHHD
YHHILHHHHH QNHHPHSHSQ RYSREELKDA GVATLAWMVI
MGDGLHNFSD GLAIGAAFTE GLSSGLSTSV AVFCHELPHE
LGDFAVLLKA GMTVKQAVLY NALSAMLAYL GMATGIFIGH
YAENVSMWIF ALTAGLFMYV ALVDMVPEML HNDASDHGCS
RWGYFFLQNA GMLLGFGIML LISIFEHKIV FRINF

<AAA96258.2; protein
SEQ ID No: 84
MARKLSVILI LTFALSVTNP LHELKAAAFP QTTEKISPNW
ESGINVDLAI STRQYHLQQL FYRYGENNSL SVEGFRKLLQ
NIGIDKIKRI HIHHDHDHHS DHEHHSDHER HSDHEHHSDH
EHHSDHNHAA SGKNKRKALC PDHDSDSSGK DPRNSQGKGA
HRPEHASGRR NVKDSVSASE VTSTVYNTVS EGTHFLETIE
TPRPGKLFPK DVSSSTPPSV TSKSRVSRLA GRKTNESVSE
PRKGFMYSRN TNENPQECFN ASKLLTSHGM GIQVPLNATE
FNYLCPAIIN QIDARSCLIH TSEKKAEIPP KTYSLQIAWV
GGFIAISIIS FLSLLGVILV PLMNRVFFKF LLSFLVALAV
GTLSGDAFLH LLPHSHASHH HSHSHEEPAM EMKRGPLFSH
LSSQNIEESA YFDSTWKGLT ALGGLYFMFL VEHVLTLIKQ
FKDKKKKNQK KPENDDDVEI KKQLSKYESQ LSTNEEKVDT
DDRTEGYLRA DSQEPSHFDS QQPAVLEEEE VMIAHAHPQE
VYNEYVPRGC KNKCHSHFHD TLGQSDDLIH HHHDYHHILH
HHHHQNHHPH SHSQRYSREE LKDAGVATLA WMVIMGDGLH
NFSDGLAIGA AFTEGLSSGL STSVAVFCHE LPHELGDFAV
LLKAGMTVKQ AVLYNALSAM LAYLGMATGI FIGHYAENVS
MWIFALTAGL FMYVALVDMV PEMLHNDASD HGCSRWGYFF
LQNAGMLLGF GIMLLISIFE HKIVFRINF

<Cyno LIV-1
SEQ ID NO: 85
MARKLSVILILTFTLSVTNPLHELKSAAAFPQTTEKISPNWESGINVDL
AITTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHD
HHSDHEHHSDHEHHSDHEHHSHRNHAASGKNKRKALCPEHDSDSSGKDP
RNSQGKGAHRPEHANGRRNVKDSVSTSEVTSTVYNTVSEGTHFLETIET
PKLFPKDVSSSTPPSVTEKSLVSRLAGRKTNESMSEPRKGFMYSRNTNE
NPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTS
EKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFL
LSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSH
LSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQ
KKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHF
DSQQPAILEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDD
LIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGIATLAWMVIMG
DGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAG
MTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYV
ALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKI
VFRINF

Claims

1. A humanized antibody, that specifically binds human LIV-1 comprising a mature heavy chain variable region comprising three CDRs of SEQ ID NO:53 and having an amino acid sequence at least 95% identical to SEQ ID NO:53 provided that position H27 is occupied by L, position H29 is occupied by I, H30 by E and H94 by V and a mature light chain variable region comprising three CDRs of SEQ ID NO:60 at least 95% identical to SEQ ID NO:60 provided position L36 is occupied by Y and position L46 by P.

2. The humanized antibody of claim 1, further provided position H76 is occupied by N.

3. The humanized antibody of claim 1 wherein the mature heavy chain variable region is fused to a heavy chain constant region and the mature light chain variable region is fused to a light chain constant region.

4. The humanized antibody of claim 3, wherein the heavy chain constant region is a mutant form of a natural human constant region which has reduced binding to an Fcgamma receptor relative to the natural human constant region.

5. The humanized antibody of claim 3, wherein the heavy chain constant region is of igg1 isotope.

6. The humanized antibody of claim 3, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:44 and the light chain constant region has an amino acid sequence comprising SEQ ID NO:42.

7. The humanized antibody of claim 3, wherein the heavy chain constant region has an amino acid sequence comprising SEQ ID NO:46 (S239C) and the light chain constant region has an amino acid sequence comprising SEQ ID NO:42.

8. The humanized antibody of claim 1, wherein the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO:52 or 53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO: 59 or 60.

9. The humanized antibody of claim 1, wherein the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO:53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO:60.

10. The humanized antibody of claim 1, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.

11. The humanized antibody of claim 1, having an association constant for human or cynomolgus monkey LIV-1 of 0.5 to 2×10⁹ M⁻¹.

12. A nucleic acid encoding a the mature heavy chain variable region and/or a the mature light chain variable region as defined by claim 1.

13. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of a the humanized antibody of claim 1.

14. The method of claim 13, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.

15. A pharmaceutical composition comprising a the humanized antibody of claim 1.

16. The method of claim 13, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.

17. The method of claim 13, wherein the isotype of the antibody is human igg1.

18. A vector comprising the nucleic acid of claim 12.

19. The vector of claim 18, wherein the vector is an expression vector.

20. A host cell comprising the nucleic acid of claim 12.

21. The host cell of claim 20, wherein the host cell is a Chinese hamster ovary (CHO) cell.

22. A method of producing an anti-LIV-1 antibody comprising culturing the host cell of claim 20 under a condition suitable for production of the anti-LIV-1 antibody.

23. The method of claim 22, further comprising isolating the anti-LIV-1 antibody produced by the host cell.

24. A method of producing an anti-LIV-1 antibody-drug conjugate comprising culturing the host cell of claim 20 under a condition suitable for production of an anti-LIV-1 antibody; isolating the anti-LIV-1 antibody produced from the host cell; and conjugating the anti-LIV-1 antibody to a cytotoxic or cytostatic agent.

25. The method of claim 24, wherein the cytotoxic or cytostatic agent is an antitubulin agent.

26. The method of claim 24, wherein the cytotoxic or cytostatic agent is an auristatin.

27. The method of claim 26, wherein the auristatin is monomethyl auristatin E.

28. The method of claim 26, wherein the auristatin is monomethyl auristatin F.

29. The method of claim 24, wherein the anti-LIV-1 antibody is conjugated to the cytotoxic or cytostatic agent via a linker.

30. The method of claim 29, wherein the linker is a cleavable peptide linker.

31. The method of claim 30, wherein the cleavable peptide linker has a formula:

-A_a-W_w—Y_y—,

wherein:

-A- is a stretcher unit;

a is 0 or 1;

each —W— is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

−Y— is a spacer unit; and

y is 0, 1 or 2.

32. The method of claim 29, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.

33. The method of claim 29, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.

34. The method of claim 33, wherein the anti-LIV-1 antibody-drug conjugate has the following structure:

35. The method of claim 34, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

36. A nucleic acid encoding the mature heavy chain variable region and/or the mature light chain variable region as defined by claim 9.

37. A vector comprising the nucleic acid of claim 36.

38. The vector of claim 37, wherein the vector is an expression vector.

39. A host cell comprising the nucleic acid of claim 36.

40. The host cell of claim 39, wherein the host cell is a Chinese hamster ovary (CHO) cell.

41. A method of producing an anti-LIV-1 antibody comprising culturing the host cell of claim 39 under a condition suitable for production of the anti-LIV-1 antibody.

42. The method of claim 41, further comprising isolating the anti-LIV-1 antibody produced by the host cell.

43. A method of producing an anti-LIV-1 antibody-drug conjugate comprising culturing the host cell of claim 39 under a condition suitable for production of an anti-LIV-1 antibody; isolating the anti-LIV-1 antibody produced from the host cell; and conjugating the anti-LIV-1 antibody to a cytotoxic or cytostatic agent.

44. The method of claim 43, wherein the cytotoxic or cytostatic agent is an antitubulin agent.

45. The method of claim 43, wherein the cytotoxic or cytostatic agent is an auristatin.

46. The method of claim 45, wherein the auristatin is monomethyl auristatin E.

47. The method of claim 45, wherein the auristatin is monomethyl auristatin F.

48. The method of claim 43, wherein the anti-LIV-1 antibody is conjugated to the cytotoxic or cytostatic agent via a linker.

49. The method of claim 48, wherein the linker is a cleavable peptide linker.

50. The method of claim 49, wherein the cleavable peptide linker has a formula:

-A_a-W_w—Y_y—

wherein:

-A- is a stretcher unit;

a is 0 or 1;

each —W— is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a spacer unit; and

y is 0, 1 or 2.

51. The method of claim 48, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.

52. The method of claim 48, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.

53. The method of claim 52, wherein the anti-LIV-1 antibody-drug conjugate has the following structure:

54. The method of claim 53, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

55. The humanized antibody of claim 6, wherein the mature heavy chain variable region has an amino acid sequence designated SEQ ID NO:53 and the mature light chain variable region has an amino acid sequence designated SEQ ID NO:60.

56. The humanized antibody of claim 10, wherein the cytotoxic or cytostatic agent is an antitubulin agent.

57. The humanized antibody of claim 10, wherein the cytotoxic or cytostatic agent is an auristatin.

58. The humanized antibody of claim 57, wherein the auristatin is monomethyl auristatin E.

59. The humanized antibody of claim 57, wherein the auristatin is monomethyl auristatin F.

60. The humanized antibody of claim 10, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.

61. The humanized antibody of claim 60, wherein the linker is a cleavable peptide linker.

62. The humanized antibody of claim 61, wherein the cleavable peptide linker has a formula:

-A_a-W_w—Y_y—,

wherein:

-A- is a stretcher unit;

a is 0 or 1;

each —W— is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a spacer unit; and

y is 0, 1 or 2.

63. The humanized antibody of claim 60, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.

64. The humanized antibody of claim 60, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.

65. The humanized antibody of claim 64, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure:

66. The humanized antibody of claim 65, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

67. The humanized antibody of claim 6, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.

68. The humanized antibody of claim 67, wherein the cytotoxic or cytostatic agent is an antitubulin agent.

69. The humanized antibody of claim 67, wherein the cytotoxic or cytostatic agent is an auristatin.

70. The humanized antibody of claim 69, wherein the auristatin is monomethyl auristatin E.

71. The humanized antibody of claim 69, wherein the auristatin is monomethyl auristatin F.

72. The humanized antibody of claim 67, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.

73. The humanized antibody of claim 72, wherein the linker is a cleavable peptide linker.

74. The humanized antibody of claim 73, wherein the cleavable peptide linker has a formula:

-A_a-W_w—Y_y—,

wherein:

-A- is a stretcher unit;

α is 0 or 1;

each —W— is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a spacer unit; and

y is 0, 1 or 2.

75. The humanized antibody of claim 72, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.

76. The humanized antibody of claim 72, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.

77. The humanized antibody of claim 76, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure:

78. The humanized antibody of claim 77, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

79. The humanized antibody of claim 9, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.

80. The humanized antibody of claim 79, wherein the cytotoxic or cytostatic agent is an antitubulin agent.

81. The humanized antibody of claim 79, wherein the cytotoxic or cytostatic agent is an auristatin.

82. The humanized antibody of claim 81, wherein the auristatin is monomethyl auristatin E.

83. The humanized antibody of claim 81, wherein the auristatin is monomethyl auristatin F.

84. The humanized antibody of claim 79, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.

85. The humanized antibody of claim 84, wherein the linker is a cleavable peptide linker.

86. The humanized antibody of claim 85, wherein the cleavable peptide linker has a formula:

-A_a-W_w—Y_y—,

wherein:

-A- is a stretcher unit;

a is 0 or 1;

each —W— is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a spacer unit; and

y is 0, 1 or 2.

87. The humanized antibody of claim 84, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.

88. The humanized antibody of claim 84, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.

89. The humanized antibody of claim 88, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure:

90. The humanized antibody of claim 89, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

91. The humanized antibody of claim 55, wherein the antibody is conjugated to a cytotoxic or cytostatic agent.

92. The humanized antibody of claim 91, wherein the cytotoxic or cytostatic agent is an antitubulin agent.

93. The humanized antibody of claim 91, wherein the cytotoxic or cytostatic agent is an auristatin.

94. The humanized antibody of claim 93, wherein the auristatin is monomethyl auristatin E.

95. The humanized antibody of claim 93, wherein the auristatin is monomethyl auristatin F.

96. The humanized antibody of claim 91, further comprising a linker between the humanized antibody and the cytotoxic or cytostatic agent.

97. The humanized antibody of claim 96, wherein the linker is a cleavable peptide linker.

98. The humanized antibody of claim 97, wherein the cleavable peptide linker has a formula:

-A_a-W_W—Y_y—,

wherein:

-A- is a stretcher unit;

a is 0 or 1;

each —W— is independently an amino acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a spacer unit; and

y is 0, 1 or 2.

99. The humanized antibody of claim 96, wherein the linker is attached to sulphydryl residues of the humanized antibody obtained by partial reduction or full reduction of the humanized antibody.

100. The humanized antibody of claim 96, wherein the cytotoxic or cytostatic agent is monomethyl auristatin E.

101. The humanized antibody of claim 100, wherein the linker is attached to monomethyl auristatin E forming an antibody-drug conjugate having the structure:

102. The humanized antibody of claim 101, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

103. A pharmaceutical composition comprising the humanized antibody of claim 6.

104. A pharmaceutical composition comprising the humanized antibody of claim 9.

105. A pharmaceutical composition comprising the humanized antibody of claim 55.

106. A pharmaceutical composition comprising the antibody-drug conjugate of claim 65.

107. A pharmaceutical composition comprising the antibody-drug conjugate of claim 101.

108. The method of claim 14, wherein the breast cancer is triple negative breast cancer.

109. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the humanized antibody of claim 55.

110. The method of claim 109, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.

111. The method of claim 110, wherein the breast cancer is triple negative breast cancer.

112. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the antibody-drug conjugate of claim 65.

113. The method of claim 112, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.

114. The method of claim 113, wherein the breast cancer is triple negative breast cancer.

115. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the antibody-drug conjugate of claim 101.

116. The method of claim 115, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.

117. The method of claim 116, wherein the breast cancer is triple negative breast cancer.

118. A pharmaceutical composition comprising an antibody-drug conjugate, wherein the antibody-drug conjugate comprises a humanized antibody conjugated to monomethyl auristatin E, wherein the humanized antibody specifically binds to human LIV-1 and comprises a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO:53 and a mature light chain variable region comprising the amino acid sequence of SEQ ID NO:60, wherein the mature heavy chain variable region is fused to an igg1 isotype heavy chain constant region comprising the amino acid sequence of SEQ ID NO:44 and the mature light chain variable region is fused to a light chain constant region comprising the amino acid sequence of SEQ ID NO:42, wherein the antibody-drug conjugate further comprises a linker between the humanized antibody and the monomethyl auristatin E, wherein the antibody-drug conjugate has the structure:

119. The pharmaceutical composition of claim 118, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

120. An antibody-drug conjugate comprising a humanized antibody conjugated to monomethyl auristatin E, wherein the humanized antibody specifically binds to human LIV-1 and comprises a mature heavy chain variable region comprising the amino acid sequence of SEQ ID NO:53 and a mature light chain variable region comprising the amino acid sequence of SEQ ID NO:60, wherein the mature heavy chain variable region is fused to an igg1 isotype heavy chain constant region comprising the amino acid sequence of SEQ ID NO:44 and the mature light chain variable region is fused to a light chain constant region comprising the amino acid sequence of SEQ ID NO:42, wherein the antibody-drug conjugate further comprises a linker between the humanized antibody and the monomethyl auristatin E, wherein the antibody-drug conjugate has the structure:

121. The antibody-drug conjugate of claim 120, wherein the average value of p in a population of the antibody-drug conjugate is about 4.

122. A method of treating a patient having a cancer expressing LIV-1, comprising administering to the patient an effective regime of the antibody-drug conjugate of claim 120.

123. The method of claim 122, wherein the cancer is a breast cancer, a prostate cancer, a cervical cancer or a melanoma.

124. The method of claim 123, wherein the breast cancer is triple negative breast cancer.