Anti-FOLR1 immunoconjugate dosing regimen

Abstract

The present invention provides methods of administering immunoconjugates that bind to FOLR1. The methods comprise administering an anti-FOLR1 immunoconjugate to a human in need thereof, such as a cancer patient, in a therapeutically effective dosing regimen that results in minimal adverse effects.

Description

[0001] This application is a divisional application of the invention patent application with application number 201480027385.9, application date May 13, 2014, applicant is Imunogene Company, and invention name is "Anti-FOLR1 immunoconjugate administration regimen". Field of the Invention

[0002] The field of the invention generally relates to methods of administering anti-FOLR1 immunoconjugates to treat diseases such as cancer. The methods provide dosing regimens that minimize undesirable side effects. Background of the Invention

[0004] Cancer is one of the leading causes of death in developed countries, with more than one million people diagnosed with cancer and 500,000 deaths each year in the United States alone. Overall, it is estimated that more than one-third of people will develop some form of cancer in their lifetime. There are more than 200 different types of cancer, of which four (breast cancer, lung cancer, colorectal cancer, and prostate cancer) account for more than half of all new cases (Jemal et al., 2003, Cancer J. Clin. 53: 5-26).

[0005] Folate receptor 1 (FOLR1), also known as folate receptor-α or folate binding protein, is an N-glycosylated protein expressed on the plasma membrane. FOLR1 has a high affinity for folic acid and several reduced folic acid derivatives. FOLR1 mediates the delivery of physiological folate, 5-methyltetrahydrofolate, into the cell interior.

[0006] FOLR1 is overexpressed in the vast majority of ovarian cancers and in many uterine cancers, endometrial cancers, pancreatic cancers, renal cancers, lung cancers, and breast cancers, while FOLR1 expression in normal tissues is limited to the apical membrane of epithelial cells in the renal proximal tubules, alveolar pneumocytes of the lungs, bladder, testis, choroid plexus, and thyroid gland (Weitman SD et al., Cancer Res 52:3396-3401 (1992); Antony AC, Annu Rev Nutr 16:501-521 (1996); Kalli KR et al. Gynecol Oncol 108:619-626 (2008)). This expression pattern of FOLR1 makes it an ideal target for FOLR1-directed cancer therapy.

[0007] Because ovarian cancer is usually asymptomatic until advanced stages, it is often diagnosed at advanced stages and has a poor prognosis when treated with currently available methods (usually chemotherapeutic drugs followed by surgical de-bulking) (von Gruenigen V et al., Cancer 112:2221-2227 (2008); Ayhan A et al., Am J Obstet Gynecol 196:81e81-86 (2007); Harry VN et al., Obstet Gynecol Surv 64:548-560 (2009)). Therefore, there is a significant unmet medical need for more effective therapeutic agents for ovarian cancer.

[0008] Antibodies are gradually emerging as promising methods for treating such cancers. In addition, immunoconjugates comprising antibodies conjugated to another compound (e.g., cytotoxins) are also being studied as potential therapeutic agents. In particular, immunoconjugates comprising maytansinoids (which are plant-derived antifungal and antitumor agents) have been shown to have some beneficial activities. SMKupchan et al. first reported that three types of bridged ring (ansa) macrolides were isolated from the ethanol extracts of Maytenus ovatus and Maytenus buchananii, which, together with their demonstration of anti-leukemic effects in μg / kg dosage ranges in mouse models, are the subject of U.S. Patent No. 3,896,111. However, maytansinoids have unacceptable toxicity, thereby causing central and peripheral neuropathy and side effects: especially nausea, vomiting, diarrhea, elevated liver function test indicators, and rare weakness and lethargy. This overall toxicity is reduced to a certain extent by conjugating maytansinoids to antibodies, because the toxicity of antibody conjugates to antigen-negative cells is several orders of magnitude lower than that to antigen-positive cells. However, immunoconjugates containing maytansinoids are still associated with unacceptable levels of adverse side effects. For example, animals injected with high doses of anti-FOLR1 immunoconjugates containing maytansinoids show ocular toxicity. The cause of this toxicity (e.g., may be related to Cmax or AUC) is unclear. Therefore, it is still necessary to determine a specific dosing regimen for anti-FOLR1 immunoconjugates that are therapeutically effective in humans but avoid adverse reactions. SUMMARY OF THE INVENTION

[0010] Provided herein are methods of administering an anti-FOLR1 immunoconjugate in a therapeutically effective dosing regimen that minimizes undesirable side effects. Accordingly, described herein are methods for treating a patient suffering from cancer comprising administering to the patient an effective dose of an immunoconjugate that binds to FOLR1, wherein the immunoconjugate is administered at a dose of about 3.0 mg / kg to about 6 mg / kg. The anti-FOLR1 immunoconjugate may comprise a charged linker. In some embodiments, the anti-FOLR1 immunoconjugate comprises the antibody huMov19, the linker sulfo-SPDB, and maytansinoid DM4.

[0011] In some embodiments, the immunoconjugate comprises an antibody or an antigen-binding fragment thereof that competitively inhibits the binding of an antibody having a sequence of SEQ ID NO: 3 and SEQ ID NO: 5 to FOLR1. In some embodiments, the antibody or fragment thereof comprises the CDRs of huMov19 (ie, SEQ ID NO: 6-10 and 12 or SEQ ID NO: 6-9, 11 and 12). In some embodiments, the antibody or fragment does not contain the six CDRs of mouse Mov19 (ie, SEQ ID NO: 6-9, 16 and 12). In some embodiments, the antibody is huMov19. In some embodiments, the immunoconjugate comprises maytansinoid. In some embodiments, maytansinoid is DM4. In some embodiments, the immunoconjugate comprises a sulfo-SPDB linker. In some embodiments, the immunoconjugate is IMGN853 (huMov19-sulfo-SPDB-DM4).

[0012] In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered at a dose of about 3.0 mg / kg. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered at a dose of about 3.3 mg / kg. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered at a dose of about 4.0 mg / kg. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered at a dose of about 5 mg / kg. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered at a dose of about 5.5 mg / kg. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered at a dose of about 6 mg / kg.

[0013] According to the methods described herein, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) can be administered approximately once every 4 weeks. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered approximately once every 3 weeks. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered approximately once every 2 weeks. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered approximately once every 1 week. In some embodiments, the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is administered approximately twice a week.

[0014] In some embodiments, the anti-FOLR1 binding agent (eg, huMov19-sulfo-SPDB-DM4) is administered by intravenous infusion approximately once every 21 days.

[0015] Administration according to the methods described herein can produce an AUC of about 10,000-18,000 h·μg / mL, about 10,000-17,500 h·μg / mL, about 10,000-17,000 h·μg / mL, or about 10,000-16,000 h·μg / mL. (0-inf) In some embodiments, AUC (0-inf) In some embodiments, AUC (0-inf) In some embodiments, AUC (0-inf) The obtained in Example 1 and Figure 1 The AUC shown in (0-inf) .

[0016] Administration according to the methods described herein can produce an AUC of about 7,500-12,500 h·μg / mL, about 7,500-12,000 h·μg / mL, about 7,500-10,000 h·μg / mL, or about 8,000-10,000 h·μg / mL. (0-168) In some embodiments, AUC (0-168) In some embodiments, AUC (0-168) In some embodiments, AUC (0-168) The obtained in Example 1 and Figure 1 The AUC shown in (0-168) .

[0017] Administration according to the methods described herein can produce a Cmax of about 50-250 μg / mL, about 50-200 μg / mL, about 50-175 μg / mL, about 50-150 μg / mL, about 50-125 μg / mL, about 75-250 μg / mL, about 75-200 μg / mL, about 75-175 μg / mL, about 75-150 μg / mL, or about 75-125 μg / mL. In some embodiments, the Cmax is about 100 μg / mL to about 150 μg / mL. In some embodiments, the Cmax is about 100 μg / mL to about 120 μg / mL. In some embodiments, the Cmax is about 108 μg / mL. In some embodiments, the Cmax is that obtained in Example 1 and Figure 1 Cmax shown in .

[0018] According to the methods described herein, the clearance rate of the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) may be less than 1.0 mL / h / kg. In some embodiments, the clearance rate of the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is less than 0.6 mL / h / kg. In some embodiments, the clearance rate of the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is about 0.2 mL / h / kg to about 0.6 mL / h / kg. In some embodiments, the clearance rate of the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is about 0.3 mL / h / kg to about 0.4 mL / h / kg. In some embodiments, the clearance rate of the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is about 0.3 mL / h / kg. In some embodiments, the clearance rate of the anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is about 0.4 mL / h / kg. In some embodiments, the clearance rate is obtained in Example 1 and Figure 1 The clearance rates shown in .

[0019] According to the methods described herein, the half-life of an anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) can be at least about 4 days. In some embodiments, the half-life of an anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) is about 3 to about 5 days, or about 4 to about 4.5 days. In some embodiments, the half-life is about 4.4 days. In some embodiments, the half-life is obtained in Example 1 and in Figure 1 The half-life is shown in .

[0020] According to the methods described herein, the steady-state apparent volume of distribution (Vss) of an anti-FOLR1 binding agent (e.g., huMov19-sulfo-SPDB-DM4) can be about 25 to about 100 mL / kg, about 25 to about 75 mL / kg, about 30 to about 75 mL / kg, or about 35 to about 70 mL / kg. In some embodiments, Vss is about 55 mL / kg to about 65 mL / kg. In some embodiments, Vss is about 61 mL / kg. In some embodiments, Vss is obtained in Example 1 and in Figure 1 Vss shown in .

[0021] In some embodiments, the anti-FOLR1 binding agent (eg, huMov19-sulfo-SPDB-DM4) is administered intravenously.

[0022] The methods described herein can be used to treat cancer. In some embodiments, the cancer is selected from ovarian cancer, brain cancer, breast cancer, uterine cancer, endometrial cancer, pancreatic cancer, kidney cancer (e.g., renal cell carcinoma) and lung cancer (e.g., non-small cell lung cancer or bronchioloalveolar carcinoma (BAC)). In some embodiments, the cancer is ovarian cancer or lung cancer. In some embodiments, the cancer is epithelial ovarian cancer.

[0023] In some embodiments, the cancer expresses a FOLR1 polypeptide or nucleic acid. In some embodiments, the cancer has an elevated expression level of a FOLR1 polypeptide, as measured by immunohistochemistry (IHC). For example, in some embodiments, the cancer is a cancer that expresses a FOLR1 polypeptide at a level of 2 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is a cancer that expresses a FOLR1 polypeptide at a level of 2 homogeneous or higher (determined by IHC). In some embodiments, the cancer is a cancer that expresses a FOLR1 polypeptide at a level of 3 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is a cancer that expresses a FOLR1 polypeptide at a level of 3 homogeneous or higher (determined by IHC). In some embodiments, the cancer is a lung cancer that expresses a FOLR1 polypeptide at a level of 2 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is a lung cancer that expresses a FOLR1 polypeptide at a level of 3 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is an epithelial ovarian cancer (e.g., platinum-resistant or recurrent or refractory) that expresses a FOLR1 polypeptide at a level of 3 heterogeneous or higher.

[0024] In some embodiments, the method further comprises administering a steroid to the patient. The steroid may be administered as a pre-treatment, ie, prior to administering the anti-FOLR1 binding agent. The steroid may be dexamethasone.

[0025] The methods described herein may result in a reduction in tumor size. The methods described herein may result in a reduction in CA125 levels in ovarian cancer patients. In one example, the CA125 level in a sample obtained from an ovarian cancer patient is measured before treatment, and then measured once or more after treatment, and the reduction in CA125 levels over time indicates the efficacy. The methods described herein may result in an extension of the time between cancer treatments. The methods described herein may result in an extension of progression-free survival (PFS). The methods described herein may result in an extension of disease-free survival (DFS). The methods described herein may result in an extension of overall survival (OS). The methods described herein may result in an increase in complete response (CR). The methods described herein may result in an increase in partial response (PR). The methods described herein may result in an increase in stable disease (SD). The methods described herein may result in a reduction in progressive disease (PD). The methods described herein may result in a reduction in time to progression (TTP).

[0026] The methods described herein may also result in reduced adverse effects.

[0027] In particular, the dosing regimens provided herein achieve an optimal balance between efficacy (e.g., PR) and reduced toxicity, as described, for example, in Examples 1 and 2 and Figure 1 as shown in . BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Pharmacokinetic data resulting from administration of IMGN853 (0.15 mg / kg to 7.0 mg / kg) as described in Example 1 are provided. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides novel dosing regimens for FOLR1 -binding immunoconjugates.

[0032] I. Definitions

[0033] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0034] As used herein, the terms "human folate receptor 1," "FOLR1," or "folate receptor alpha (FR-α)" refer to any native human FOLR1 unless otherwise indicated. Thus, all of these terms may refer to a protein or nucleic acid sequence as indicated herein. The term "FOLR1" encompasses "full-length," unprocessed FOLR1, as well as any form of FOLR1 produced by processing within a cell. The term also encompasses naturally occurring FOLR1 variants, such as splice variants, allelic variants, and isoforms. The FOLR1 polypeptides described herein can be isolated from a variety of sources, such as from a human tissue type or from another source, or prepared by recombinant or synthetic methods. Examples of FOLR1 sequences include, but are not limited to, NCBI reference numbers P15328, NP_001092242.1, AAX29268.1, AAX37119.1, NP_057937.1, and NP_057936.1.

[0035] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target through at least one antigen recognition site in the variable region of the immunoglobulin molecule, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated by at least two complete antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigenic determinant of an antibody, and any other modified immunoglobulin molecules comprising an antigen recognition site, as long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the species of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked antibodies or conjugated to other molecules, such as toxins, radioisotopes, etc.

[0036] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds, such as FOLR1. In some embodiments, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The biological activity may be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0037] The term "anti-FOLR1 antibody" or "antibody that binds to FOLR1" refers to an antibody that is capable of binding to FOLR1 with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting FOLR1. The extent of binding of the anti-FOLR1 antibody to unrelated, non-FOLR1 proteins may be less than about 10% of the binding of the antibody to FOLR1, as determined, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to FOLR1 has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.

[0038] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0039] "Monoclonal antibody" refers to a homogeneous group of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies against different antigenic determinants. The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising an antigen recognition site. In addition, "monoclonal antibody" refers to such antibodies prepared by any of a variety of means, including but not limited to by hybridomas, phage screening, recombinant expression, and transgenic animals.

[0040] The term "humanized antibody" refers to a non-human (e.g., mouse) antibody form that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof that contains minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin in which residues from a complementary determining region (CDR) are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and ability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some cases, the Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues from a non-human species antibody having the desired specificity, affinity, and ability. Humanized antibodies can be further modified by the replacement of other residues in the Fv framework region and / or in the replaced non-human residues, to improve or optimize antibody specificity, affinity and ability. Generally speaking, humanized antibodies will comprise substantially all of at least one usually two or three of the following variable domains, which contain all or substantially all of the CDR regions corresponding to non-human immunoglobulins, and all or substantially all of the FR regions of the consensus sequences of human immunoglobulins. Humanized antibodies can also comprise at least a portion of immunoglobulin constant regions or domains (Fc) that are typically human immunoglobulins. Examples of methods for generating humanized antibodies are described in U.S. Patent No. 5,225,539. In some embodiments, "humanized antibodies" are surface remodeled antibodies.

[0041] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable region of both the heavy and light chains consists of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also called hypervariable regions). The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. At least two techniques are used to determine CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0042] The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0043] The amino acid position numbering as in Kabat refers to the numbering system for heavy chain variable domains or light chain variable domains mapped for antibodies, as found in: Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after the 52nd residue of H2 (residue 52a according to Kabat) and an inserted residue after the 82nd residue of the heavy chain FR (e.g., residues 82a, 82b, and 82c according to Kabat). For a given antibody, the Kabat numbering of the residues can be determined by comparing the antibody sequence homology region with the "standard" Kabat numbering sequence. In contrast, Chothia refers to the position of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop differ between H32 and H34 depending on the length of the loop when numbered using the Kabat numbering convention (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at position 32; if only 35A is present, the loop ends at position 33; if both 35A and 35B are present, the loop ends at position 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0044]

[0045] The term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, prepared using any technique known in the art. This definition of a human antibody includes complete or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide, such as, for example, antibodies comprising a mouse light chain and a human heavy chain polypeptide.

[0046] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0047] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, the epitope can be formed by either continuous amino acids or non-continuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed by continuous amino acids are usually retained upon protein denaturation, while epitopes formed by tertiary folding are usually lost upon protein denaturation. An epitope usually includes at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0048] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound for a longer time. A variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific exemplary embodiments are described below.

[0049] "Or better" when used herein to refer to binding affinity refers to stronger binding between a molecule and its binding partner. "Or better" when used herein to refer to stronger binding is represented by a smaller Kd value. For example, for an antibody with an antigen affinity of "0.6 nM or better", the antigen affinity of the antibody is <0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM, etc., or any value less than 0.6 nM.

[0050] By "specific binding" is generally meant that an antibody binds to an epitope through its antigen binding domain and that the binding requires a certain complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope more easily through its antigen binding domain than it would to a random, unrelated epitope. The term "specificity" is used herein to verify the relative affinity of an antibody for binding to an epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for a related epitope "D".

[0051] By "preferentially binds" is meant that an antibody specifically binds to an epitope more readily than it will bind to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" a given epitope will bind to that epitope more readily than it will to a related epitope, even though such an antibody may cross-react with the related epitope.

[0052] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope when the antibody preferentially binds to the epitope so as to block binding of the reference antibody to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. An antibody is said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0053] As used herein, the phrase "substantially similar" or "substantially identical" refers to a sufficiently high degree of similarity between two numerical values ​​(usually one associated with an antibody of the invention and the other associated with a reference / comparison antibody) such that one skilled in the art will recognize that the difference between the two values ​​is almost biologically and / or statistically significant in the context of the biological property measured by the value (e.g., Kd value). The difference between the two values ​​may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% depending on the value of the reference / comparison antibody.

[0054] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition refers to a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form that does not exist in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to an extent that they are no longer in a form found in nature. In some embodiments, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0055] As used herein, "substantially pure" refers to a material that is at least 50% pure (ie, free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0056] As used herein, the term "immunoconjugate" or "conjugate" refers to a compound or derivative thereof linked to a cell-binding agent (i.e., an anti-FOLR1 antibody or fragment thereof) and is defined by the following general formula: CLA, wherein C = cytotoxin, L = linker and A = anti-FOLR1 antibody or antibody fragment. An immunoconjugate may also be defined by the reversed general formula ALC.

[0057] The term "IMGN853" refers to the immunoconjugate described herein containing the huMov19 antibody, a sulfo-SPDB linker, and DM4 maytansine.

[0058] A "linker" is any chemical moiety capable of linking a compound, typically a drug, such as maytansinoids, to a cell binding agent, such as an anti-FOLR1 antibody or fragment thereof, in a stable covalent manner. The linker may be susceptible to or substantially resistant to the following cleavages, under conditions where the compound or antibody remains active: acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers may also include charged linkers and hydrophilic forms thereof as described herein and known in the art.

[0059] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, malignancies, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver malignancies, and various types of head and neck cancer. The cancer may be a cancer that expresses FOLR1.

[0060] "Tumor" and "neoplasm" refer to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (noncancerous) or malignant (cancerous), including precancerous lesions.

[0061] The terms "cancer cell", "tumor cell" and grammatical equivalents refer to the total cell population derived from a tumor or precancerous lesion, including both non-tumorigenic cells (which constitute the majority of the tumor cell population) and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring only to those tumor cells that lack the ability to renew and differentiate, to distinguish those tumor cells from cancer stem cells.

[0062] The term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. In general, the terms "subject" and "patient" are used interchangeably herein when referring to human subjects.

[0063] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) administration and consecutive administration in any order.

[0064] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient to exert its effect, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. The formulation may be sterile.

[0065] An "effective amount" of an antibody or immunoconjugate as disclosed herein is an amount sufficient to achieve a specifically specified purpose. An "effective amount" can be determined empirically and in a routine manner in relation to the specified purpose.

[0066] The term "therapeutically effective amount" refers to the amount of an antibody or other drug that effectively "treats" a disease or condition in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down to a certain extent and stop in a certain embodiment) the infiltration of cancer cells to surrounding organs; inhibit (i.e., slow down to a certain extent and stop in a certain embodiment) tumor metastasis; inhibit tumor growth to a certain extent; alleviate one or more symptoms associated with cancer to a certain extent; and / or lead to a favorable response, such as extended progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR), or in some cases, stable disease (SD), progressive disease (PD) reduction, reduced progression time (TTP), CA125 reduction or any combination thereof for ovarian cancer.

[0067] See the definition of "treatment" herein. To the extent that the drug can prevent cancer cell growth and / or kill existing cancer cells, the drug can be cytostatic and / or cytotoxic. In certain embodiments, determination of elevated FOLR1 levels allows for administration of reduced amounts of a therapeutic agent targeting FOLR1 to achieve the same therapeutic effect as seen with a higher dose. A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the dosage and for the period of time necessary. Typically, but not necessarily, the prophylactic effective amount will be less than the therapeutically effective amount because the prophylactic dose is used in subjects prior to disease or at an earlier stage of disease.

[0068] The term "favorably respond" generally refers to causing a beneficial state in a subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on a subject. Positive therapeutic effects in cancer can be measured in a variety of ways (see WA Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess the efficacy of anticancer therapeutics. With respect to tumor growth inhibition, according to NCI standards, T / C ≤ 42% is the lowest level of antitumor activity. T / C < 10% is considered a high level of antitumor activity, where T / C (%) = median tumor volume of the treatment group / median tumor volume of the control group x 100. A favorable response can be assessed, for example, by prolonged progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR) or in some cases stable disease (SD), reduction in progressive disease (PD), reduced time to progression (TTP), reduction in CA125 for ovarian cancer, or any combination thereof.

[0069] PFS, DFS and OS can be measured by criteria established by the National Cancer Institute and the US Food and Drug Administration, which approves new drugs. See Johnson et al., (2003) J. Clin. Oncol. 21(7): 1404-1411.

[0070] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is generally measured using the Kaplan-Meier method and the Response Evaluation Criteria for Solid Tumors (RECIST) 1.1 criteria. Generally speaking, progression-free survival refers to a situation in which a patient remains alive without cancer worsening.

[0071] "Time to tumor progression" (TTP) is defined as the time from enrollment to disease progression. TTP is generally measured using RECIST 1.1 criteria.

[0072] A "complete response" or "complete remission" or "CR" indicates that in response to treatment, all signs of the tumor or cancer disappeared. This does not always mean that the cancer has been cured.

[0073] A "partial response" or "PR" refers to a decrease in the size or volume of one or more tumors or lesions, or a reduction in the extent of cancer in the body, in response to treatment.

[0074] "Stable disease" refers to disease that is not progressing or recurring. In stable disease, there is neither sufficient tumor shrinkage to verify a partial response nor sufficient tumor enlargement to verify progressive disease.

[0075] "Progressive disease" refers to the appearance of a new lesion or tumor and / or clear progression of existing non-target lesions. Progressive disease may also refer to tumor growth of more than 20% since the start of treatment, either due to enlargement of the mass or spread of the tumor.

[0076] "Disease-free survival" (DFS) refers to the length of time during and after treatment that a patient remains disease-free.

[0077] "Overall survival" (OS) refers to the time from patient enrollment to death or the last known date alive. OS includes an extension of life expectancy compared to trial-naive or untreated individuals or patients. Overall survival refers to a situation in which a patient remains alive for a defined period of time, such as one year, five years, etc., e.g., from the time of diagnosis or treatment.

[0078] "The reduction of CA125 level" can be assessed according to the Gynecologic Cancer Intergroup (GCIG) guidelines. For example, CA125 level can be measured before treatment to establish baseline CA125 level. CA125 level can be measured once or multiple times during or after treatment, and the reduction of CA125 level over time compared to baseline level is regarded as the reduction of CA125 level.

[0079] Terms such as "treating", "treatment", "treat", or "alleviating", "toalleviate" refer to therapeutic measures to cure, alleviate, lessen the symptoms of, and / or prevent the progression of, a diagnosed pathological condition or disorder. Thus, those in need of treatment include those who have been diagnosed with or are suspected of having the disorder. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the invention if the patient exhibits one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; inhibition of or absence of cancer cell infiltration into surrounding organs (including, for example, spread of cancer into soft tissue and bone); inhibition of or absence of tumor metastasis; inhibition of or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improved quality of life; reduced tumorigenicity, tumorigenic frequency, or tumorigenic capacity of the tumor; a decrease in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells to a non-tumorigenic state; prolonged progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), reduction in progressive disease (PD), reduced time to progression (TTP), a decrease in CA125 for ovarian cancer, or any combination thereof.

[0080] Prophylactic or preventative measures refer to therapeutic measures that prevent and / or slow the development of a targeted pathological condition or disorder. Thus, those in need of prophylactic or preventative measures include those susceptible to the disorder and those in which the disorder is to be prevented.

[0081] The term "pre-treat" or "pre-treatment" refers to a therapeutic measure performed prior to the administration of an anti-FOLR1 therapeutic agent. For example, as described in more detail herein, a prophylactic agent, such as a steroid, may be administered within about one week, about five days, about three days, about two days, or about one day or 24 hours prior to the administration of an anti-FOLR1 therapeutic agent. A prophylactic agent may also be administered prior to the anti-FOLR1 therapeutic agent on the day of the anti-FOLR1 therapeutic agent.

[0082] A "chemotherapeutic agent" is a compound useful for treating cancer regardless of mechanism of action. Chemotherapeutic agents include, for example, CD20 antagonists such as rituximab and cyclophosphamide, doxorubicin, vincristine, prednisone, fludarabine, etoposide, methotrexate, lenalidomide, chlorambucil, betahistine, and / or modified forms of such chemotherapeutic agents.

[0083] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that since the polypeptides of the present invention are based on antibodies, in certain embodiments, the polypeptides may exist as single chains or associated chains.

[0084] The term "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence (introducing gaps if necessary) without considering any conservative amino acid substitutions that are part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain amino acid or nucleotide sequence alignments are known in the art. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87: 2264-2268, as improved in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90: 5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25: 3389-3402). In certain embodiments, Gapped BLAST may be used as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266: 460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are other publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software (e.g., using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 90 and a length weight of 1, 2, 3, 4, 5 or 6). In certain alternative embodiments, the GAP program in the GCG software package incorporating the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using a Blossum62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, or 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)).For example, percent identity can be determined using the ALIGN program (version 2.0) and using a gap length penalty of 12 and a gap penalty of 4 with a residue table. The appropriate parameters for the maximum comparison adopted by a specific comparison software can be determined by those skilled in the art. In certain embodiments, the default parameters of the comparison software are used. In certain embodiments, the percent identity "X" of the first amino acid sequence and the second sequence amino acid is calculated as 100x (Y / Z), wherein Y is the number of amino acid residues that are evaluated as identical matches in the comparison of the first and second sequences (such as compared by visual estimation or specific sequence comparison software), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be greater than the percent identity of the second sequence to the first sequence.

[0085] As a non-limiting example, the Bestfit program (Wisconsin Sequence Analysis Software Package, Unix Version 8, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) can be used in certain embodiments to determine whether any particular polynucleotide has a certain percentage sequence identity (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98% or 99% identical) to a reference sequence. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482 489 (1981) to find the best segment of homology between two sequences. When Bestfit or any other sequence alignment program is used according to the present invention to determine whether a particular sequence is, for example, 95% identical to a reference sequence, the parameters are set so that the percentage of identity is calculated over the entire length of the reference nucleotide sequence, and so that gaps are allowed in a homology of up to 5% of the total number of nucleotides in the reference sequence.

[0086] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, that is, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. The identity can exist over a sequence region of at least about 10, about 20, about 40-60, or any integer number of residues therebetween, and can be over a region longer than 60-80 residues, for example, at least about 90-100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared (such as the coding region of the nucleotide sequence).

[0087] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, a substitution of tyrosine with phenylalanine is a conservative substitution. In some embodiments, conservative substitutions in the polypeptide and antibody sequences of the invention do not eliminate binding of the polypeptide or antibody containing the amino acid sequence to the antigen (i.e., FOLR1 to which the polypeptide or antibody binds). Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0088] As used in the disclosure and claims, the singular forms "a", "an", "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0089] It should be understood that wherever herein an embodiment is described with the language "comprising," other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided.

[0090] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone), and C (alone).

[0091] II. FOLR1 Binders

[0092] The methods described herein provide methods of administering a sequence that specifically binds to FOLR1 ("FOLR1 binder"). In certain embodiments, the FOLR1 binder is an antibody, an immunoconjugate, or a polypeptide. The amino acid sequence and nucleotide sequence of human FOLR1 are known in the art and are also given herein, as represented by SEQ ID NO: 1 and SEQ ID NO: 2. Thus, in some embodiments, the FOLR1 binder can bind to the epitope of SEQ ID NO: 1.

[0093] Examples of therapeutically effective anti-FOLR1 antibodies can be found in U.S. Application Publication No. US 2012 / 0009181, which is incorporated herein by reference. An example of a therapeutically effective anti-FOLR1 antibody is huMov19 (M9346A). The polypeptides of SEQ ID NOs: 3-5 comprise the variable domain of the heavy chain of huMov19 (M9346A) and the variable domain light chain version 1.00 and variable domain light chain version 1.60 of huMov19, respectively. In certain embodiments, the huMov19 (M9346A) antibody is encoded by a plasmid deposited on April 7, 2010 with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110 under the terms of the Budapest Treaty and having ATCC deposit numbers PTA-10772 and PTA-10773 or 10774. Examples of FOLR1 immunoconjugates useful in the therapeutic methods of the invention are provided below.

[0094] In some embodiments, the FOLR1 binding agent is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the humanized antibody or fragment is a resurfaced antibody or antigen-binding fragment thereof. In other embodiments, the FOLR1 binding agent is a fully human antibody or antigen-binding fragment thereof.

[0095] In certain embodiments, a FOLR1 binding agent has one or more of the following effects: inducing stable disease, inhibiting tumor cell proliferation, reducing tumorigenicity by reducing the frequency of cancer stem cells in the tumor, inhibiting tumor growth, increasing survival, triggering cell death of tumor cells, differentiating tumorigenic cells to a non-tumorigenic state, or preventing tumor cell metastasis.

[0096] In certain embodiments, a FOLR1 binding agent is an antibody having antibody-dependent cellular cytotoxicity (ADCC) activity.

[0097] In some embodiments, the FOLR1 binding agent is capable of reducing tumor volume. The ability of the FOLR1 binding agent to reduce tumor volume can be assessed, for example, by measuring the %T / C value, which is the median tumor volume of the treated subjects divided by the median tumor volume of the control subjects. In certain embodiments, an immunoconjugate or other agent that specifically binds to human FOLR1 triggers cell death by a cytotoxic agent. For example, in certain embodiments, an antibody to a human FOLR1 antibody is conjugated to maytansine, which is activated by protein internalization in tumor cells expressing FOLR1. In certain embodiments, the FOLR1 binding agent is capable of inhibiting tumor growth. In certain embodiments, the FOLR1 binding agent is capable of inhibiting tumor growth in vivo (e.g., in a xenograft mouse model and / or in a person with cancer).

[0098] The FOLR1 binding molecule can be an antibody or antigen-binding fragment that specifically binds to FOLR1, comprising the CDRs of huMov19 (M9346A), and each CDR has up to four (i.e., 0, 1, 2, 3 or 4) conservative amino acid substitutions, for example, wherein the antibody or fragment does not contain the six CDRs of murine Mov19 (i.e., SEQ ID NOs: 6-9, 16 and 12). The polypeptide can comprise one of the variable light chains or variable heavy chains described herein. The antibodies and polypeptides can also comprise both variable light chains and variable heavy chains.

[0099] In some embodiments, the FOLR1 binding molecule is an antibody or antigen binding fragment comprising a sequence of SEQ ID NOs: 6-10 and a sequence of SEQ ID NO: 12. In some embodiments, the FOLR1 binding molecule is an antibody or antigen binding fragment comprising a sequence of SEQ ID NOs: 6-9 and a sequence of SEQ ID NOs: 11 and 12.

[0100] Also provided are polypeptides comprising a polypeptide having at least about 90% sequence identity to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. In certain embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO:3, and / or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NO:4 or SEQ ID NO:5. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO:3; and / or (b) a polypeptide having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. In certain embodiments, the polypeptide is an antibody and / or polypeptide that specifically binds to FOLR1. In certain embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to FOLR1. In certain embodiments, a polypeptide having a certain percentage of sequence identity to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 differs from SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 only by conservative amino acid substitutions.

[0101] The polypeptide may comprise one of the light chains or heavy chains described herein. Antibodies and polypeptides may also comprise both light and heavy chains.

[0102] Monoclonal antibodies can be prepared using hybridoma methods, such as those described in Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, mice, hamsters or other appropriate host animals are immunized as described above to induce the production of antibodies that specifically bind to the immunizing antigen by lymphocytes. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are separated and fused with suitable myeloma cell lines using, for example, polyethylene glycol to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. Then, hybridomas that produce monoclonal antibodies specifically against selected antigens can be propagated in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in animals as ascites tumors, where anti-antigenicity is determined by immunoprecipitation, immunoblotting or by in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)). The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described above for the polyclonal antibodies.

[0103] Alternatively, monoclonal antibodies can also be prepared using recombinant DNA methods as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding antibody heavy and light chains, and then their sequences are determined using conventional procedures. The isolated polynucleotides encoding heavy and light chains are then cloned into a suitable expression vector, which, when transfected into a host cell such as an E. coli cell, ape COS cell, Chinese hamster ovary (CHO) cell, or myeloma cell that would not otherwise produce immunoglobulins, produces monoclonal antibodies by the host cell. Alternatively, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

[0104] Recombinant DNA technology can be used to further modify the polynucleotide encoding the monoclonal antibody in a variety of different ways to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be replaced with 1) those regions of, for example, a human antibody to generate a chimeric antibody, or 2) a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant region is truncated or removed to generate the desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0105] In some embodiments, the monoclonal antibody against human FOLR1 is a humanized antibody. In some embodiments, the humanized antibody is a resurfaced antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to human subjects. Humanized antibodies can be produced using a variety of techniques known in the art. In certain alternative embodiments, the antibody against FOLR1 is a human antibody.

[0106] Human antibodies can be directly prepared using a variety of techniques known in the art. Immortalized human B lymphocytes isolated from individuals immunized in vitro or producing antibodies against a target antigen can be generated (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147 (1): 86-95; and U.S. Pat. No. 5,750,373). In addition, human antibodies can be selected from phage libraries that express human antibodies as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314; Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; and Marks et al., 1991, J. Mol. Biol., 222:581. Techniques for generating and using antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484, and 7,264,963; and Rothe et al., 2007, J. Mol. Bio., doi: 10.1016 / j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783, incorporated by reference in its entirety) are known in the art and can be used to generate high affinity human antibodies.

[0107] Humanized antibodies can also be prepared in transgenic mice containing human immunoglobulin loci that are capable of producing a full spectrum of human antibodies upon immunization in the absence of endogenous immunoglobulin production. This approach is described in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016.

[0108] The present invention also encompasses bispecific antibodies that specifically recognize FOLR1. Bispecific antibodies are antibodies that are capable of specifically recognizing and binding at least two different epitopes. The different epitopes can be within the same molecule (e.g., the same FOLR1), or on different molecules such that, for example, the antibody can specifically recognize and bind FOLR1 and, for example, 1) an effector molecule on a leukocyte, such as a T cell receptor (e.g., CD3) or an Fc receptor (e.g., CD64, CD32, or CD16) or 2) a cytotoxic agent as described in detail below.

[0109] The polypeptide of the present invention may be a recombinant polypeptide comprising an anti-human FOLR1 antibody or a fragment thereof, a natural polypeptide or a synthetic polypeptide.

[0110] Polypeptides and analogs can be further modified to contain additional chemical moieties that are not normally part of a protein. Those derivatized moieties can improve solubility, biological half-life, or protein absorption. The moieties can also reduce or eliminate any desired side effects of the protein, etc. An overview of these moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th edition, Mack Publishing Co., Easton, PA (2000).

[0111] The isolated polypeptide described herein can be produced by any suitable method known in the art. Such methods cover from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in suitable transformed hosts. In some embodiments, recombinant technology is used to construct DNA sequences by separating or synthesizing DNA sequences encoding wild-type proteins of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide its functional analogs. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81: 5662-5066 (1984) and U.S. Patent No. 4,588,585.

[0112] In some embodiments, the DNA sequence of the polypeptide of interest will be constructed by a chemical synthesis method using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the following aspects: the amino acid sequence of the desired polypeptide, and the codons that are selected in the host cell that will produce the recombinant polypeptide of interest. Standard methods can be used to synthesize the separation polynucleotide sequence of the separation polypeptide of interest of the coding. For example, the complete amino acid sequence can be used to construct the gene of reverse translation. In addition, the DNA oligomer containing the nucleotide sequence of the specific separation polypeptide of the coding can be synthesized. For example, a plurality of small oligonucleotides of the multiple parts of the desired polypeptide of the coding can be synthesized and then connected. Each oligonucleotide usually contains 5' or 3' overhangs, for complementary assembly.

[0113] Once assembled (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest can be inserted into an expression vector and operably linked to expression control sequences suitable for expressing the protein in the desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping and expression of the biologically active polypeptide in a suitable host. It is well known in the art that in order to obtain high expression levels of the transfected gene in the host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0114] In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding an anti-human FOLR1 antibody or a fragment thereof. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an anti-FOLR1 antibody or a fragment thereof, which is operably linked to a suitable transcriptional or translational regulatory element derived from a mammalian, microbial, viral or insect gene. A transcription unit generally comprises an assembly of the following parts: (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer; (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and (3) appropriate transcriptional and translational initiation and termination sequences, as will be described in detail below. Such regulatory elements may include an operator sequence that controls transcription. In addition, it may also include replication ability in a host, which is usually conferred by an origin of replication, and a selection gene that facilitates the identification of transformants. DNA regions are operably linked when they are functionally related to each other. For example, the DNA of the signal peptide (secretion leader sequence) is operably linked to the DNA of the polypeptide in the following cases: the DNA of the signal peptide is expressed as a precursor involved in the secretion of the polypeptide; the promoter is operably linked to the coding sequence in the following cases: the promoter controls the transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence in the following cases: the positioning of the ribosome binding site allows translation. Structural elements intended for use in yeast expression systems include leader sequences that enable extracellular secretion of translated proteins by host cells. Alternatively, if the recombinant protein is expressed without a leader sequence or a transport sequence, it may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0115] The selection of expression control sequences and expression vectors will depend on the choice of the host. A variety of expression host / vector combinations can be used. Expression vectors that can be used for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors that can be used for bacterial hosts include known bacterial plasmids, such as plasmids derived from Escherichia coli, including pCR 1, pBR322, pMB9, and derivatives thereof; plasmids with a wider host range, such as M13 and filamentous single-stranded DNA phages.

[0116] Host cells suitable for expressing FOLR1 binding polypeptides or antibodies (or FOLR1 proteins to be used as antigens) include prokaryotes, yeast, insects or higher eukaryotic cells controlled by appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be used. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is hereby incorporated by reference. Additional information on methods for producing proteins (including producing antibodies) can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated by reference in its entirety.

[0117] A variety of mammalian or insect cell culture systems are also advantageously used to express recombinant proteins. Expression of recombinant proteins can be carried out in mammalian cells because such proteins are usually correctly folded, appropriately modified and have complete functions. Examples of suitable mammalian host cell lines include the COS-7 cell line of monkey kidney cells described by Gluzman (Cell 23: 175, 1981), and other cell lines capable of expressing appropriate vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may include non-transcriptional elements, such as replication origins, suitable promoters and enhancers connected to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites and transcription termination sequences. The baculovirus system for producing heterologous proteins in insect cells is reviewed in Luckow and Summers, Bio / Technology 6: 47 (1988).

[0118] The protein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography and size exclusion column chromatography), centrifugation, differential dissolution or by any other standard techniques for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza virus capsid sequence and glutathione S-transferase can be attached to the protein to allow easy purification by passing through a suitable affinity column. The separated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0119] For example, the supernatant of a system that secretes the recombinant protein into the culture medium can be first concentrated using a commercially available protein concentration filter such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an ion exchange resin can be used, such as a matrix or substrate with diethylaminoethyl (DEAE) side groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include a variety of insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reversed phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (e.g., silica gel with methyl side groups or other aliphatic side groups) can be used to further purify the FOLR1 binder. Some or all of the above purification steps can also be used in various combinations to provide a uniform recombinant protein.

[0120] Recombinant proteins produced in bacterial culture can be isolated, for example, by first extracting from the cell pellet and then performing one or more concentration, salting out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for the final purification step. Microbial cells used for recombinant protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption or the use of cell lysing agents.

[0121] Methods known in the art for purifying antibodies and other proteins also include those described, for example, in U.S. Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is hereby incorporated by reference in its entirety.

[0122] III. Immunoconjugates

[0123] Also described herein are methods of administering conjugates comprising anti-FOLR1 antibodies, antibody fragments, and functional equivalents thereof as disclosed herein, linked to or conjugated to a drug or prodrug (also referred to herein as immunoconjugates). Suitable drugs or prodrugs are known in the art. The drug or prodrug may be a cytotoxic agent. The cytotoxic agent used in the cytotoxic conjugates of the present invention may be any compound that causes cell death, or induces cell death, or reduces cell viability in some way, and includes, for example, maytansinoids and maytansinoid analogs. Other suitable cytotoxic agents are, for example, benzodiazepines. class, taxanes, CC-1065 and CC-1065 analogs, duocarmycin and duocarmycin analogs, enediynes (such as calicheamicin), dolesta and dolesta analogs (including auristatins), tomaymycin derivatives, leptomycin derivatives, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholinodoxorubicin.

[0124] Such conjugates can be prepared by using a linker to attach the drug or prodrug to the antibody or functional equivalent. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups.

[0125] The drug or prodrug may be linked to the anti-FOLR1 antibody or fragment thereof, for example, via a disulfide bond. The linker molecule or cross-linker comprises a reactive chemical group that can react with the anti-FOLR1 antibody or fragment thereof. The reactive chemical group that reacts with the cell binding agent may be an N-succinimidyl ester and an N-sulfosuccinimidyl ester. In addition, the linker molecule comprises a reactive chemical group that may be a dithiopyridyl group that can react with the drug to form a disulfide bond. Linker molecules include, for example, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al., Biochem. J., 173:723-737 (1978)), N-succinimidyl 4-(2-pyridyldithio) butyrate (SPDB) (see, e.g., U.S. Pat. No. 4,563,304), N-succinimidyl 4-(2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) (see U.S. Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry No. 341498-08-6), 2-iminothiolane, or acetyl succinic anhydride. For example, an antibody or cell binding agent can be modified by a cross-linking agent, and the antibody or cell binding agent thus derived containing a free or protected thiol group is then reacted with a maytansinoid containing a disulfide bond or thiol to produce a conjugate. The conjugate can be purified by chromatography (including but not limited to HPLC, size exclusion chromatography, adsorption chromatography, ion exchange chromatography, and affinity capture chromatography), dialysis, or tangential flow filtration.

[0126] In another aspect of the invention, an anti-FOLR1 antibody is linked to a cytotoxic drug via a disulfide bond and a polyethylene glycol spacer to enhance the potency, solubility or efficacy of the immunoconjugate. Such cleavable hydrophilic linkers are described in WO2009 / 0134976. Additional benefits of this linker design are the desired high monomer ratio and minimal aggregation of the antibody-drug conjugate. Specifically contemplated in this aspect are the use of a cleavable hydrophilic linker having a polyethylene glycol spacer ((CH2CH2O) n＝1-14 ) disulfide (-SS-) linked cell binding agent and drug conjugates, which are described to have a narrow range of drug loading of 2-8, exhibit relatively efficient biological activity against cancer cells, and have the desired biochemical properties of high conjugation yield and high monomer ratio and minimal protein aggregation.

[0127] Antibody-maytansinoid conjugates with non-cleavable linkers can also be prepared.Such cross-linking agents are described in the art (see US Publication No. 20050169933) and include, but are not limited to, 4-(maleimidomethyl)cyclohexanecarboxylic acid N-succinimidyl ester (SMCC). In some embodiments, the antibody is modified with a cross-linking reagent such as 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or succinimidyl-iodoacetate as described in the literature to introduce 1-10 reactive groups (Yoshitake et al., Eur. J. Biochem., 101: 395-399 (1979); Hashida et al., J. Applied Biochem., 56-63 (1984); and Liu et al., Biochem., 18: 690-697 (1979)). The modified antibody is then reacted with a thiol-containing maytansinoid derivative to produce a conjugate. The conjugate can be purified by gel filtration on a Sephadex G25 column or by dialysis or tangential flow filtration. The antibody modified is processed with the maytansinol (1 to 2 molar equivalents / maleimide) containing thiol, and the antibody-maytansinol conjugate is purified by Sephadex G-25 column gel filtration, ceramic hydroxyapatite column chromatography, dialysis or tangential flow filtration or the combination of these methods. Usually, each antibody is connected to 1-10 maytansinol on average. One method is to modify the antibody with 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) to introduce maleimide, and then the antibody modified is reacted with the maytansinol containing thiol to obtain the conjugate connected by thioether. Similarly, each antibody molecule is obtained containing 1 to 10 drug molecules. The maytansinol conjugates of antibodies, antibody fragments and other proteins are prepared in the same manner.

[0128] In another aspect of the invention, the FOLR1 antibody is linked to the drug via a non-cleavable bond through an intervening PEG spacer. Suitable cross-linking reagents comprising a hydrophilic PEG chain that forms a linker between the drug and the anti-FOLR1 antibody or fragment are also well known in the art, or are commercially available (e.g., from Quanta Biodesign, Powell, Ohio). Suitable PEG-containing cross-linking agents can also be synthesized from commercially available PEG itself using standard chemical synthesis techniques known to those skilled in the art. The drug can be reacted with a bifunctional PEG-containing cross-linking agent by the methods detailed in U.S. Patent Publication Nos. 20090274713 and WO2009 / 0134976 to obtain a compound of the formula: Z-X l –(–CH2–CH2–O–)n –Y p -D, which can then be reacted with a cell-binding agent to provide a conjugate. Alternatively, the cell-binding agent can be modified with a bifunctional PEG cross-linker to introduce a thiol-reactive group (such as a maleimide or a haloacetamide), which can then be treated with a thiol-containing maytansinoid to provide a conjugate. In another approach, the cell-binding agent can be modified with a bifunctional PEG cross-linker to introduce a thiol moiety, which can then be treated with a thiol-reactive maytansinoid (such as a maytansinoid with a maleimide or a haloacetamide) to provide a conjugate.

[0129] Examples of suitable PEG-containing linkers include linkers having N-succinimidyl ester or N-sulfosuccinimidyl ester moieties that react with anti-FOLR1 antibodies or fragments thereof, and maleimido or haloacetyl-based moieties that react with compounds. The PEG spacer can be incorporated into any cross-linking agent known in the art by the methods described herein.

[0130] In some embodiments, the linker is a linker comprising at least one charged group, such as described in U.S. Patent Publication No. 2012 / 0282282, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the charged or pre-charged cross-linking agent is one containing a sulfonate substituent, a phosphate substituent, a carboxyl substituent, or a quaternary ammonium substituent, which significantly improves the solubility of the modified cell-binding agent and the cell-binding agent-drug conjugate, particularly for monoclonal antibody-drug conjugates with 2 to 20 drugs attached to each antibody. The conjugate prepared from the linker containing the pre-charged part will produce one or more charged parts after the conjugate is metabolized in the cell. In some embodiments, the linker is selected from: 4-(2-pyridyldithio)-2-sulfopentanoic acid N-succinimidyl ester (sulfo-SPP) and 4-(2-pyridyldithio)-2-sulfobutyric acid N-succinimidyl ester (sulfo-SPDB).

[0131] Many of the linkers disclosed herein are described in detail in US Patent Publication Nos. 2005 / 0169933, 2009 / 0274713, and 2012 / 0282282, and in WO 2009 / 0134976; the contents of which are incorporated herein by reference in their entireties.

[0132] The present invention includes aspects in which about 2 to about 8 drug molecules ("drug load"), e.g., maytansine, are attached to an anti-FOLR1 antibody or fragment thereof. As used herein, "drug load" refers to the number of drug molecules (e.g., maytansine) that can be attached to a cell-binding agent (e.g., an anti-FOLR1 antibody or fragment thereof). In one aspect, the number of drug molecules that can be attached to a cell-binding agent can be in the range of about 2 to about 8 on average (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7. .2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). N2'-deacetyl-N2'-(3-mercapto-1-oxypropyl)-maytansine (DM1) and N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl) maytansine (DM4) can be used.

[0133] Thus, in one aspect, the immunoconjugate comprises 1 maytansine per antibody. In another aspect, the immunoconjugate comprises 2 maytansines per antibody. In another aspect, the immunoconjugate comprises 3 maytansines per antibody. In another aspect, the immunoconjugate comprises 4 maytansines per antibody. In another aspect, the immunoconjugate comprises 5 maytansines per antibody. In another aspect, the immunoconjugate comprises 6 maytansines per antibody. In another aspect, the immunoconjugate comprises 7 maytansines per antibody. In another aspect, the immunoconjugate comprises 8 maytansines per antibody.

[0134] In one aspect, the immunoconjugate comprises about 1 to about 8 maytansines per antibody. In another aspect, the immunoconjugate comprises about 2 to about 7 maytansines per antibody. In another aspect, the immunoconjugate comprises about 2 to about 6 maytansines per antibody. In another aspect, the immunoconjugate comprises about 2 to about 5 maytansines per antibody. In another aspect, the immunoconjugate comprises about 3 to about 5 maytansines per antibody. In another aspect, the immunoconjugate comprises about 3 to about 4 maytansines per antibody.

[0135] In one aspect, a composition comprising an immunoconjugate has an average of about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.4, 7.5 In one aspect, the composition comprising the immunoconjugate has an average of about 1 to about 8 drug molecules (e.g., maytansine) per antibody. In one aspect, the composition comprising the immunoconjugate has an average of about 2 to about 7 drug molecules (e.g., maytansine) per antibody. In one aspect, the composition comprising the immunoconjugate has an average of about 2 to about 6 drug molecules (e.g., maytansine) per antibody. In one aspect, the compositions comprising the immunoconjugates have an average of about 2 to about 5 drug molecules (e.g., maytansine) per antibody. In one aspect, the compositions comprising the immunoconjugates have an average of about 3 to about 5 drug molecules (e.g., maytansine) per antibody. In one aspect, the compositions comprising the immunoconjugates have an average of about 3 to about 4 drug molecules (e.g., maytansine) per antibody.

[0136] In one aspect, the composition comprising the immunoconjugate has an average of about 2±0.5, about 3±0.5, about 4±0.5, about 5±0.5, about 6±0.5, about 7±0.5, or about 8±0.5 attached drug molecules (e.g., maytansine) per antibody. In one aspect, the composition comprising the immunoconjugate has an average of about 3.5±0.5 drug molecules (e.g., maytansine) per antibody.

[0137] Anti-FOLR1 antibodies or fragments thereof can be modified by reacting a bifunctional cross-linking agent with an anti-FOLR1 antibody or fragment thereof, thereby causing a linker molecule to be covalently attached to the anti-FOLR1 antibody or fragment thereof. As used herein, a "bifunctional cross-linking agent" is any chemical moiety that covalently attaches a cell binding agent to a drug (such as a drug described herein). In another method, a portion of a connecting moiety is provided by a drug. In this aspect, the drug comprises a connecting moiety that is a portion of a larger linker molecule for attaching a cell binding agent to a drug. For example, in order to form maytansine DM1, the side chain at the C-3 hydroxyl of maytansine is modified to have a free sulfhydryl group (SH). The thiolated form of maytansine can react with a modified cell binding agent to form a conjugate. Therefore, the final linker is assembled by two components, one of which is provided by a cross-linking agent and the other is provided by the side chain of DM1.

[0138] Drug molecules can also be linked to antibody molecules via intervening carrier molecules such as serum albumin.

[0139] As used herein, the expression "linked to a cell binding agent" or "linked to an anti-FOLR1 antibody or fragment" refers to a conjugate molecule comprising at least one drug derivative linked to a cell binding agent anti-FOLR1 antibody or fragment via a suitable linker or a precursor thereof. An exemplary linker is SPDB or Sulfo-SPDB.

[0140] In certain embodiments, the cytotoxic agents useful in the present invention are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Included are any drugs that inhibit microtubule formation like maytansinol and maytansinol analogs and are highly toxic to mammals.

[0141] Examples of suitable maytansinol esters include those having modified aromatic rings and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533;

[0142] In one embodiment, the immunoconjugates of the invention utilize the formal name N 2’-Deacetyl-N 2’ -(3-mercapto-1-oxypropyl)-maytansine containing thiol (DM1) as a cytotoxic agent. DM1 is represented by the following structural formula (I):

[0143]

[0144] In another embodiment, the conjugates of the invention utilize thiol-containing maytansinoid N 2’ -Deacetyl-N 2’ (4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) is used as a cytotoxic agent. DM4 is represented by the following structural formula (II):

[0145]

[0146] Another maytansinoid containing a side chain containing a sterically hindered thiol bond is N 2’ -Deacetyl-N- 2’ (4-Mercapto-1-oxopentyl)-maytansine (referred to as DM3), which is represented by the following structural formula (III):

[0147]

[0148] Each of the maytansinoids taught in U.S. Pat. Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the present invention. In this regard, the entire disclosures of 5,208,020 and 7,276,697 are incorporated herein by reference.

[0149] Many positions on maytansinol can be used as positions for chemically connecting the linking moiety. For example, the C-3 position with a hydroxyl group, the C-14 position modified with a hydroxyl group, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group are all expected to be used. In some embodiments, the C-3 position is used as the position for chemically connecting the linking moiety, and in some specific embodiments, the C-3 position of maytansinol is used as the position for chemically connecting the linking moiety.

[0150] Schematic diagrams of the structures of some conjugates are shown below:

[0151]

[0152]

[0153]

[0154] Also included within the present invention are any stereoisomers of any compound or conjugate depicted by any of the structures above, and mixtures thereof.

[0155] Various descriptions for the production of such antibody-maytansinoid conjugates are provided in US Pat. Nos. 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein in its entirety.

[0156] Generally speaking, the antibody solution in aqueous buffer can be incubated with a molar excess of maytansinoids with disulfide moieties with reactive groups. The reaction mixture can be quenched by adding excess amines (such as ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration.

[0157] The number of maytansinoid molecules bound by each antibody molecule can be determined by spectrophotometrically measuring the absorbance ratio at 252nm and 280nm. The average number of maytansinoid molecules / antibody can be, for example, 1-10 or 2-5. The average number of maytansinoid molecules / antibody can be, for example, about 3 to about 4. The average number of maytansinoid molecules / antibody can be about 3.5.

[0158] The ability of conjugates of antibodies and maytansinoids or other drugs to inhibit a variety of unwanted cell lines can be evaluated in vitro. For example, cell lines such as the human lymphoma cell line Daudi and the human lymphoma cell line Ramos can be easily used to assess the cytotoxicity of these compounds. The cells to be evaluated can be exposed to the compound for 4 to 5 days, and the survival fraction of the cells can be measured in a direct assay by known methods. The IC can then be calculated from the results of the assay. 50 value.

[0159] The immunoconjugates can be internalized into cells according to some embodiments described herein. Thus, the immunoconjugates can exert a therapeutic effect when taken up or internalized by FOLR1 expressing cells. In some specific embodiments, the immunoconjugates comprise an antibody, antibody fragment or polypeptide linked to a cytotoxic agent via a cleavable linker, and the cytotoxic agent is cleaved from the antibody, antibody fragment or polypeptide, wherein it is internalized by FOLR1 expressing cells.

[0160] In some embodiments, the immunoconjugate can reduce tumor volume. For example, in some embodiments, treatment with the immunoconjugate results in a %T / C value of less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some specific embodiments, the immunoconjugate can reduce the tumor size in KB, OVCAR-3, IGROV-1, and / or OV-90 xenograft models. In some embodiments, the immunoconjugate can inhibit metastasis.

[0161] III. Methods of Administering FOLR1 Binding Agents

[0162] The FOLR1 binding agents of the present invention (including antibodies, immunoconjugates and polypeptides) can be used for a variety of applications, including but not limited to therapeutic treatment methods, such as treating cancer. In certain embodiments, the agents can be used to inhibit tumor growth, induce differentiation, inhibit metastasis, reduce tumor volume and / or reduce tumorigenicity. The method of use can be an in vivo method.

[0163] According to the methods described herein, the FOLR1 binding agent can be administered at a specific dose. For example, the FOLR1 binding agent (e.g., IMGN853) can be administered at a dose of about 0.15 mg / kg to about 7 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at a dose of about 3.0 mg / kg to about 6.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at a dose of about 3.3 mg / kg to about 6.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 0.15 mg / kg. Therefore, in some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 0.5 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 1.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 2.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 3.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 3.3 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 5.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 5.5 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 6.0 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 6.5 mg / kg. In some embodiments, the FOLR1 binding agent (e.g., IMGN853) is administered at about 7.0 mg / kg.

[0164] In addition, the FOLR1 binding agent can be administered at a specific dosing interval. For example, the FOLR1 binding agent can be administered from about four times per week to about once every four weeks. Thus, in some embodiments, the FOLR1 binding agent is administered about once every three weeks. In some embodiments, the FOLR1 binding agent is administered about once every two and a half weeks. In some embodiments, the FOLR1 binding agent is administered about once every two weeks. In some embodiments, the FOLR1 binding agent is administered about once every ten days. In some embodiments, the FOLR1 binding agent is administered about once per week. In some embodiments, the FOLR1 binding agent is administered about once every five days. In some embodiments, the FOLR1 binding agent is administered about once every four days. In some embodiments, the FOLR1 binding agent is administered about once every three days. In some embodiments, the FOLR1 binding agent is administered about once every two days. In some embodiments, the FOLR1 binding agent is administered about twice per week. In some embodiments, the FOLR1 binding agent is administered about three times per week.

[0165] FOLR1 binding agents can also be administered in cycles of about 3 weeks (i.e., about 21 days). For example, a FOLR1 binding agent can be administered twice in about 3 weeks. Thus, in some embodiments, a FOLR1 binding agent can be administered on about day 1 and day 8 of a 21 day cycle. In other embodiments, a FOLR1 binding agent can be administered three times in about 3 weeks. Thus, in some embodiments, a FOLR1 binding agent can be administered on about day 1, 8, and 15 of a 21 day cycle.

[0166] FOLR1 binding agents can also be administered in cycles of about 4 weeks (ie, about 28 days). For example, FOLR1 binding agents can be administered three times in about 4 weeks. Thus, in some embodiments, FOLR1 binding agents can be administered on about days 1, 8, and 15 of a 28-day cycle.

[0167] In some embodiments, the FOLR1 binding agent can be administered at a dose that results in a specific Cmax. For example, the FOLR1 binding agent can be administered at a dose that results in a Cmax of about 0.5 to about 250 μg / mL. Thus, in some embodiments, the FOLR1 binding agent is administered at a dose that results in a Cmax of about 50 to about 250 μg / mL. In some embodiments, the FOLR1 binding agent is administered at a dose that results in a Cmax of about 50 to about 200 μg / mL. Thus, in some embodiments, the FOLR1 binding agent is administered at a dose that results in a Cmax of about 50 to about 175 μg / mL. In some embodiments, the FOLR1 binding agent is administered at a dose that results in a Cmax of about 50 to about 150 μg / mL. Thus, in some embodiments, the FOLR1 binding agent is administered at a dose that results in a Cmax of about 100 to about 175 μg / mL. In some embodiments, the FOLR1 binding agent is administered at a dose that results in a Cmax of about 100 to about 150 μg / mL.

[0168] In certain embodiments, the FOLR1 binding agent may be administered at a dose that results in a particular AUC. For example, the FOLR1 binding agent may be administered at a dose that results in an AUC of about 50 h·μg / mL to about 18,000 h·μg / mL. In some embodiments, the FOLR1 binding agent may be administered at a dose that results in an AUC of about 10,000 h·μg / mL to about 18,000 h·μg / mL. In some embodiments, the FOLR1 binding agent may be administered at a dose that results in an AUC of about 10,000 h·μg / mL to about 17,500 h·μg / mL. In some embodiments, the FOLR1 binding agent may be administered at a dose that results in an AUC of about 10,000 h·μg / mL to about 17,000 h·μg / mL. In some embodiments, the FOLR1 binding agent may be administered at a dose that results in an AUC of about 10,000 h·μg / mL to about 16,000 h·μg / mL. In some embodiments, a FOLR1 binding agent can be administered at a dose that results in an AUC of about 10,000 h·μg / mL to about 15,000 h·μg / mL.

[0169] In certain embodiments, the disease treated with a FOLR1 binding agent or antagonist (e.g., an anti-FOLR1 antibody) is cancer. In certain embodiments, the cancer is characterized by FOLR1 expressing cells to which the FOLR1 binding agent (e.g., an antibody) binds. In certain embodiments, the tumor overexpresses human FOLR1.

[0170] The present invention provides methods for treating cancer, comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of a FOLR1 binding agent. Cancers that can be treated by the methods encompassed by the present invention include, but are not limited to, neoplasms, tumors, metastases, or any disease or condition characterized by uncontrolled cell growth. The cancer can be a primary cancer or a metastatic cancer. Specific examples of cancers that can be treated by the methods encompassed by the present invention include, but are not limited to, ovarian cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, malignant glioma, and head and neck cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is lung cancer.

[0171] In some embodiments, the cancer is a cancer that expresses FOLR1 (polypeptide or nucleic acid). In some embodiments, a FOLR1 binding agent is administered to a patient with elevated FOLR1 expression levels, e.g., as described in U.S. Published Application No. 2012 / 0282175 or International Published Application No. WO 2012 / 135675, both of which are incorporated herein by reference in their entirety. Thus, in some embodiments, FOLR1 expression is measured by immunohistochemistry (IHC) and a staining intensity score and / or a staining uniformity score is given by comparison with a control (e.g., a calibrated control) that exhibits a defined score (e.g., an intensity score of 3 is given to the test sample if the intensity is comparable to a calibrated control of level 3, or an intensity of 2 is given to the test sample if the intensity is comparable to a calibrated control of level 2). Staining uniformity that is heterogeneous or homogeneous also indicates increased FOLR1 expression. Staining intensity and staining uniformity scores can be used alone or in combination (e.g., 2 homogeneous, 2 heterogeneous, 3 homogeneous, 3 heterogeneous, etc.). In another example, an increase in FOLR1 expression can be determined by detecting an increase of at least 2-fold, at least 3-fold, or at least 5-fold relative to a control value (e.g., expression levels in tissue or cells obtained from a subject without cancer or with cancer that does not have elevated FOLR1 values).

[0172] In some embodiments, the cancer is a cancer that expresses FOLR1 at a level of 2 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is a cancer that expresses FOLR1 at a level of 3 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is a lung cancer that expresses FOLR1 at a level of 2 heterogeneous or higher (determined by IHC). In some embodiments, the cancer is a lung cancer that expresses FOLR1 at a level of 3 heterogeneous or higher (determined by IHC).

[0173] In certain embodiments, the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a FOLR1 binding agent. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.

[0174] In addition, the present invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a FOLR1 binding agent. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administering the agent.

[0175] The present invention also provides pharmaceutical compositions comprising one or more FOLR1 binding agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical combinations can be used to inhibit tumor growth and treat cancer in human patients.

[0176] In certain embodiments, the preparation is prepared by combining the purified antibody or agent of the present invention with a pharmaceutically acceptable vehicle (e.g., carrier, excipient) for storage and use (Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to: non-toxic buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues) ; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates, such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants, such as TWEEN or polyethylene glycol (PEG).

[0177] The pharmaceutical compositions described herein can be administered in any number of ways for local or systemic treatment. Administration can be local (such as administration to mucous membranes, including vaginal and rectal delivery), such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral, including intravenous, arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In some specific embodiments, administration is intravenous administration.

[0178] The antibody or immunoconjugate can be combined with a second compound in a pharmaceutical co-formulation or dosing regimen as a combination therapy. In some embodiments, the second compound is a steroid. In some embodiments, the method encompasses the administration of a steroid and an immunoconjugate that results in a reduction in headache compared to the administration of the immunoconjugate alone.

[0179] The administration of steroids can be carried out simultaneously with the immunoconjugate, before the administration of the immunoconjugate and / or after the administration of the immunoconjugate. In some embodiments, the steroid is administered within about one week, about five days, about three days, about two days, or about one day or 24 hours before the administration of the immunoconjugate. In some embodiments, the steroid is administered within the same day of the administration of the immunoconjugate. In some embodiments, the steroid is administered multiple times. In some embodiments, the steroid is administered about the day before the administration of the immunoconjugate and on the day of the administration of the immunoconjugate. Steroids can be administered via a variety of ways, including, for example, local, pulmonary, oral, parenteral or intracranial administration. In some embodiments, administration is oral administration. In some embodiments, administration is intravenous administration. In some embodiments, administration is oral and intravenous administration.

[0180] The antibody or immunoconjugate may also be combined with an analgesic or other drug for preventing or treating headaches in a pharmaceutical compound formulation or dosing regimen as a combination therapy. For example, in addition to administering the antibody or immunoconjugate, acetaminophen and / or diphenhydramine may also be administered. The analgesic may be administered before, simultaneously with, or after the administration of the immunoconjugate and may be administered via an appropriate route of administration. In some embodiments, the analgesic is administered orally.

[0181] In some embodiments, the method comprises administering a first compound that is an antibody or immunoconjugate, a second compound that is a steroid, and a third compound that is an analgesic. In some embodiments, the method comprises administering a first compound that is IMGN388, a second compound that is dexamethasone, and a third compound that is acetaminophen and / or diphenhydramine.

[0182] The antibody or immunoconjugate can be combined with a second compound having anticancer properties in a pharmaceutical co-formulation or dosing regimen as a combination therapy. The second compound of the pharmaceutical co-formulation or dosing regimen can have complementary activities to the ADC of the co-formulation so that they do not adversely affect each other. Also provided is a pharmaceutical composition comprising a FOLR1 binder and a second anticancer agent.

[0183] ***

[0184] Embodiments of the present disclosure are further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods of using the antibodies of the present disclosure. It will be apparent to those skilled in the art that many changes can be made to both materials and methods without departing from the scope of the present disclosure. Example

[0185] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art will appreciate various modifications or changes thereto, which will also be included within the spirit and scope of the present application.

[0186] Example 1

[0187] IMGN853 dosing trial in human cancer patients

[0188] IMGN853 is an antibody-drug conjugate (ADC) comprising a folate receptor 1 (FOLR1)-binding antibody and a potent maytansinoid DM4. IMGN853 has been previously described in International Published Application Nos. WO 2011 / 106528, WO 2012 / 135675, and WO 2012 / 138749, and U.S. Published Application Nos. 2012 / 0009181, 2012 / 0282175, and 2012 / 0282282, each of which is incorporated herein by reference in its entirety. IMGN853 is huMov19-sSPDB-DM4, and the huMov19 antibody contains a variable heavy chain having the amino acid sequence of SEQ ID NO: 3 and a variable light chain having the amino acid sequence of SEQ ID NO: 5. FOLR1 protein is expressed at elevated levels on many solid tumors, particularly epithelial ovarian cancer (EOC), endometrial cancer, non-small cell lung cancer (NSCLC), and clear cell renal cell carcinoma.

[0189] The study was conducted to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of IMGN853, as well as to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. The study consisted of two components: an accelerated dose titration component, in which IMGN853 immunoconjugate was administered to patients with any type of refractory solid tumor expressing FOLR1, including epithelial ovarian cancer (EOC) and other FOLR1-positive solid tumors; and a dose escalation component.

[0190] For the accelerated adjustment part of the study, IMGN853 was given intravenously (IV) on Day 1 of each 21-day (3-week) cycle. Eighteen patients were enrolled at seven dose levels ranging from 0.15 to 7.0 mg / kg IMGN853: 11 patients with EOC, 5 patients with endometrial cancer, and 2 patients with clear cell renal cell carcinoma (see Table 1). Of these 18 patients, 8 patients reported adverse events (AEs) that were considered to be related to the study drug. Most AEs were mild or moderate.

[0191] Table 1: Recruitment by tumor type

[0192]

[0193]

[0194] At a dose of 7.0 mg / kg, 4 patients have experienced ocular toxicity. One patient reported grade 3 dose-limiting punctate keratitis and grade 2 blurred vision, which were considered to be definitely related to the study treatment. In addition, one patient each had grade 3, grade 2, and grade 1 blurred vision; all events were considered to be possibly or definitely related to IMGN853 treatment. Therefore, the maximum tolerated dose in this administration plan (i.e., once every three weeks) was considered to have exceeded the dose level of 7.0 mg / kg, and all patients who remained at the 7.0 mg / kg dose level had their doses reduced to the previous dose level (5.0 mg / kg).

[0195] Drug exposure was measured in 14 patients and was found to generally increase linearly. > The half-life is approximately 4 days at a dose of 3.3 mg / kg. Confirmed CA125 responses were reported in two patients: one patient with serous ovarian cancer and one patient with serous endometrial cancer. In addition, an unconfirmed partial response was achieved in a patient with endometrial cancer. Patients receiving IMGN853 at a dose greater than or equal to 5.0 mg / kg received dexamethasone 10 mg IV (or similar steroid equivalent) 30 to 60 minutes prior to administration of the anti-FOLR1 immunoconjugate (e.g., IMGN853).

[0196] Pharmacokinetic (PK) parameters from cycle 1 (first dosing cycle per patient only) of the Phase 1 trial of IMGN853 are reported. Figure 1 ) It was demonstrated that the clearance of IMGN853 was rapid at low doses (CL = 1.1 mL / h / kg) with a half-life of approximately 35.4 hours or 1.5 days. The clearance was reduced at higher doses (CL = 0.4 mL / h / kg) and the half-life increased to approximately 4 days at 7.0 mg / kg. It was demonstrated that exposure (AUC) and Cmax were also generally increased at higher doses.

[0197] The dose titration study demonstrated that IMGN853 was well tolerated at doses up to 5.0 mg / kg. Enrollment continued at the 5.0 mg / kg dose level. All patients previously treated at 7.0 mg / kg and continuing on study had their dose reduced to 5.0 mg / kg. Additional patients were also enrolled at 5.0 mg / kg to further confirm the safety profile seen in the 3 patients initially assigned to this dose.

[0198] Once the MTD is defined, the study will move to the dose escalation phase. Three escalation cohorts will evaluate patients with FOLR1 protein-positive (1) platinum-resistant epithelial ovarian cancer, (2) recurrent or refractory epithelial ovarian cancer, and (3) recurrent or refractory non-small cell lung cancer (NSCLC). Cohorts 2 and 3 will be evaluated for IMGN853 PD by tumor biopsy before and after administration and / or by FLT-PET imaging, respectively. IMGN853 will be administered at a dose of at least 3.3 mg / kg and may include a dose of 5.0 mg / kg or up to 6.0 mg / kg. Initially, IMGN853 should be administered at a rate of 1 mg / min; after 30 minutes, if well tolerated, the rate can be increased to 3 mg / min. If well tolerated after 30 minutes at 3 mg / min, the rate can be increased to 5 mg / min. Subsequent infusions can be delivered at a tolerated rate.

[0199] For all IMGN853 doses at 3.3 mg / kg or higher, prophylactic steroid therapy will be included using the regimen described in Example 2 (e.g., steroid therapy will be included with 10 mg dexamethasone IV (or similar steroid equivalent) 30 to 60 minutes before the required IMGN853 administration, and prophylactic diphenhydramine HCl and acetaminophen are recommended prior to IMGN853 administration). Cycles will be repeated until (i) the patient's disease worsens, (ii) the patient experiences unacceptable toxicity, (iii) the patient withdraws consent, (iv) the patient develops a concomitant condition that would prevent further study treatment, or (v) the patient discontinues for noncompliance or administration reasons.

[0200] Responses were assessed using RECIST and Gynecologic Cancer International Group (GCIG) criteria when appropriate.

[0201] Example 2

[0202] IMGN853 steroid-based prevention of infusion reactions

[0203] To reduce the likelihood of infusion reactions, any of the following steroid-based prophylaxis regimens may be used.

[0204] (1) Patients receive dexamethasone 10 mg IV (or similar steroid equivalent) 30 to 60 minutes prior to administration of the anti-FOLR1 immunoconjugate (eg, IMGN853).

[0205] (2) Patients receive dexamethasone 10 mg IV (or similar steroid equivalent) and diphenhydramine hydrochloride (25-50 mg IV or PO) 30 to 60 minutes prior to administration of the anti-FOLR1 immunoconjugate (e.g., IMGN853), with or without acetaminophen (325-650 mg IV or PO). This prophylactic regimen is recommended and is at the discretion of each investigator.

[0206] (3) Patients received dexamethasone 8 mg (or similar steroid equivalent) orally BID on the day prior to administration of the anti-FOLR1 immunoconjugate (e.g., IMGN853). On the day of administration of the anti-FOLR1 immunoconjugate (e.g., IMGN853), patients received dexamethasone 10 mg IV (or similar steroid equivalent), diphenhydramine hydrochloride (25-50 mg IV or PO), with or without acetaminophen (325-650 mg IV or PO) 30-60 minutes prior to administration of the anti-FOLR1 immunoconjugate (e.g., IMGN853).

[0207] (4) Oral administration of steroids (e.g., dexamethasone) within 24 hours before infusion.

[0208] ****

[0209] It should be appreciated that the Detailed Description section, rather than the Summary and Abstract sections, is intended to be used to interpret the claims. As contemplated by the inventors, the Summary and Abstract sections set forth one or more but not all exemplary embodiments of the present invention, and therefore are not intended to limit the present invention and the appended claims in any way.

[0210] The present invention has been described above with the aid of functional building blocks that illustrate implementation of specified functions and relationships thereof. For ease of description, the boundaries of these functional building blocks are arbitrarily defined herein. Alternative boundaries may be defined as long as the specified functions and relationships thereof are appropriately performed.

[0211] The description of the foregoing specific embodiments will therefore fully reveal the general nature of the invention, and others can easily modify and / or adapt such specific embodiments to various applications without departing from the general concept of the invention by applying knowledge in the art without undue experimentation. Therefore, such adaptations and modifications are intended to fall within the meaning and scope of equivalent forms of the disclosed embodiments, in accordance with the teachings and guidance provided herein. It should be understood that the terms or terminology herein are used for illustrative purposes rather than restrictive purposes, so that the terms or terminology of this specification will be interpreted by the skilled person in accordance with the teachings and guidance.

[0212] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined in accordance with the following claims and their equivalents.

[0213]

[0214]

Claims

1. A method for treating a human patient suffering from cancer, comprising administering to said patient an effective dose of an immunoconjugate bound to a FOLR1 polypeptide, wherein said administration produces a Cmax of about 100-150 μg / mL.

2. The method of claim 1, wherein the immunoconjugate is administered at a dose of about 3 mg / kg to about 6 mg / kg.

3. The method of claim 2, wherein the immunoconjugate is administered at a dose of about 3.0 mg / kg.

4. The method of claim 2, wherein the immunoconjugate is administered at a dose of about 3.3 mg / kg.

5. The method of claim 2, wherein the immunoconjugate is administered at a dose of about 5.0 mg / kg.

6. The method of claim 2, wherein the immunoconjugate is administered at a dose of about 5.5 mg / kg.

7. The method of any one of claims 1-6, wherein the immunoconjugate comprises an antibody comprising a light chain CDR1 comprising SEQ ID NO: 6, a light chain CDR2 comprising SEQ ID NO: 7, a light chain CDR3 comprising SEQ ID NO: 8, a heavy chain CDR1 comprising SEQ ID NO: 9, a heavy chain CDR2 comprising SEQ ID NO: 10 or 11, and a heavy chain CDR3 comprising SEQ ID NO:

12.

8. The method of claim 7, wherein the immunoconjugate comprises an antibody comprising a variable light chain comprising SEQ ID NO: 5 and a variable heavy chain comprising SEQ ID NO:

3.

9. The method of claim 8, wherein the immunoconjugate is IMGN853.

10. The method of any one of claims 1-9, wherein the immunoconjugate is administered intravenously.

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