WO2017194568A1

WO2017194568A1 - Treatment regimen using anti-muc1 maytansinoid immunoconjugate antibody for the treatment of tumors

Info

Publication number: WO2017194568A1
Application number: PCT/EP2017/061103
Authority: WO
Inventors: Sylvie ASSADOURIAN; Patrick Cohen; Nathalie FAGNIEZ; Alexia TELLIER
Original assignee: Sanofi
Priority date: 2016-05-11
Filing date: 2017-05-09
Publication date: 2017-11-16
Also published as: TW201808336A

Abstract

The present invention concerns a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer, wherein the method comprises at least two cycles, wherein, for each cycle, the conjugate is administered at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle, and wherein the conjugate is administered at least two times during each cycle.

Description

Treatment regimen using anti-Mud maytansinoid immunoconjugate antibody for the treatment of tumors

Field of the invention

The present invention concerns a treatment regimen using anti-Mud maytansinoid immunoconjugate for treating tumors while inducing low toxicity.

Background of the invention

There have been numerous attempts to develop anti-cancer therapeutic agents that specifically destroy target cancer cells without harming surrounding, non-cancerous cells and tissues. Such therapeutic agents have the potential to vastly improve the treatment of cancer in human patients.

One promising approach has been to link cell binding agents, such as monoclonal antibodies, with cytotoxic drugs. Depending on the selection of the cell binding agent, these cytotoxic conjugates can be designed to recognize and bind only specific types of cancerous cells, based on the expression profile of molecules expressed on the surface of such cells.

The international patent application WO 02/16401 described a murine monoclonal antibody DS6 which reacts with an antigen, CA6 that is expressed by human serous ovarian carcinomas. This murine monoclonal antibody DS6 can therefore target cancerous cells.

The CA6 antigen was more specificaffy characterized in U.S. Patent No. 7,834, 155, as a sialoglycotope on the MUC1 mucin receptor expressed by cancerous cells. This patent also provided antibodies, in particular humanized antibodies such as the humanized hDS6 antibody, capable of recognizing this CA6 sialoglycotope of the MUC1 mucin receptor.

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been used in cytotoxic conjugates, linked to a variety of murine monoclonal antibodies. In some cases, the drug molecules were linked to the antibody molecules through an intermediary carrier molecule such as serum albumin.

Such cytotoxic conjugates include conjugates comprising antibodies, preferably humanized antibodies, that recognize the CA6 sialoglycotope of the Mud mucin receptor expressed by cancerous cells and that may be used to inhibit the growth of a cell expressing the CA6 glycotope in the context of a cytotoxic agent.

One of these conjugates is huDS6-DM4, an immunoconjugate consisting of a humanized monoclonal antibody against the tumor-associated sialoglycotope CA6 (huDS6) conjugated to the cytotoxic maytansinoid DM4.

However, it is known that ocular toxicities such as keratopathies and blurred vision are observed with the use of antibody drug conjugates. Unfortunately, the occurrence of such effects which are not serious and reversible, limits the exposure of some of the patients to effective optimal doses, since it necessitates, in some cases, a dose decrease and/or a dose delay at the next administration of the conjugate.

For example, after a phase 1 escalation phase concerning the huDS6-DM4 immunoconjugate, it was first considered that the recommended dose of huDS6-DM4 should be 190 mg/m² administered every three weeks (q3w) (WO2015/014879). However during the expansion phase of this study (data not published) a late occurrence of ocular events leading to dose modification or drug discontinuation was observed. Indeed, while the efficacy of the huDS6-DM4 at that dose was considered promising (13.0% of ORR), the percentage of ocular toxicity leading to either a dose reduction of the conjugate or a dose delay was considered high (60.9%). A dose of 150 g/m² every three weeks was then used. Unfortunately, while the safety was improved (the percentage of ocular event leading to dose delay and/or dose reduction was 24.2%), the efficacy was reduced (3.1 % ORR).

There is thus an important need of new treatment regimens/schedules of cytotoxic conjugates enabling limiting toxicities, in particular ocular toxicities, while maintaining a good activity on tumors.

Summary of the invention

The present invention arises from the unexpected finding from the inventors that, by administrating the cytotoxic conjugate over at least two cycles of administration, at a dose corresponding to an administration schedule of 60 mg/m² every week of the cycle, it was possible to maintain a good efficacy on tumors, while limiting toxicity, in particular limiting ocular toxicity necessitating a reduction of the dose of administered conjugate or a delay in the beginning of the following cycle of administration. Indeed, the inventors showed that, when the huDS6-DM4 immunoconjugate was administered at a dose of 120 mg/m² repeated as a new cycle every two weeks (q2w) (the dose administered every cycle of such administration schedule corresponds to that of an administration schedule of 60 mg/m² every week of the cycle), the ocular toxicity leading dose modification or drug discontinuation was of only 6.3% while maintaining an acceptable probability of non-progression after 12 weeks of treatment (36.1 %). Most surprisingly, the inventors even showed that, when the huDS6-DM4 immunoconjugate was administered according to a scheme that comprises a first administration at a dose of 90 mg/m² at day 1 of a cycle and a second administration at a dose of 90 mg/m² at day 8 of the cycle, the scheme being repeated as a new cycle every three weeks (D1 D8q3w), the efficacy of the treatment was at /east similar compared to an administration at 190 mg/m² every three weeks (comparable ORR) and the ocular toxicity was simultaneously reduced (29.4% vs. 60.9%). Furthermore, observing that an ocular prophylaxis treatment may help reducing the ocular toxicity that might be associated with some elevated dose of the conjugate, the inventors designed a protocol wherein the conjugate is administered at a dose of 120 mg/m² at day 1 and at day 8 repeated as a new cycle every 3 weeks (q3w) (the dose administered every cycle of such administration schedule corresponds to that of an administration schedule of 80 mg/m² every week of the cycle) and wherein an ocular prophylaxis treatment is administered.

In certain aspects, the present invention thus concerns a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the method comprises at least two cycles,

wherein, for each cycle, the conjugate is administered at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle, and

wherein the conjugate is administered at least two times during each cycle.

In certain embodiments, in each cycle, the conjugate is administered at a dose corresponding to that of an administration schedule of from 60 to 80 mg/m² every week of the cycle. In certain embodiments, the cycle is a period of two or three weeks.

In certain embodiments, the cycle is a period of three weeks, and, for each cycle, the conjugate is administered at a dose of 90 mg/m² at day 1 of the cycle and at a dose of 90 mg/m² at day 8 of the cycle. In certain embodiments, the cycle is a period of three weeks, and, for each cycle, the conjugate is administered at a dose of 120 mg/m² at day 1 of the cycle and at a dose of 120 mg/m² at day 8 of the cycle.

In certain embodiments, the number of cycles is 2.

In certain embodiments, prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered. In certain embodiments, the prophylactic treatment for preventing eye disorders comprises administration of a vasoconstrictor and/or a corticosteroid, and/or use of a cold eye mask. In certain embodiments, the corticosteroid is administered three times a day starting the day of the conjugate administration and for 2 days. In certain embodiments, a cold eye mask is used during each administration of the conjugate.

In certain embodiments, said cell binding agent binds the extracellular domain of the UC1 glycoprotein. In certain embodiments, said cell binding agent recognizes and binds the CA6 glycotope on the MUC1 glycoprotein. In certain embodiments, said cell binding agent is an antibody or an epitope-binding fragment thereof.

In certain embodiments, said antibody or epitope-binding fragment thereof comprises one or more complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. In certain embodiments, said antibody or epitope-binding fragment thereof comprises a CDR1 -heavy chain (CDR1-H) sequence of SEQ ID NO: 1 , a CDR2-heavy chain (CDR2-H) sequence of SEQ ID NO: 2, a CDR3-heavy chain (CDR3-H) sequence of SEQ ID NO: 3, a CDR1-!ight chain (CDR1-L) sequence of SEQ ID NO: 4, a CDR2-light chain (CDR2-L) sequence of SEQ ID NO: 5 and a CDR3-light chain (CDR3-L) sequence of SEQ ID NO: 6.

In certain embodiments, said antibody or epitope-binding fragment thereof comprises a heavy chain variable region of sequence SEQ ID NO: 7 or a sequence at least 85% identical thereto. In certain embodiments, said antibody or epitope-binding fragment thereof comprises a light chain variable region of sequence SEQ ID NO: 8 or a sequence at least 85% identical thereto.

In certain embodiments, the epitope-binding fragment is selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabody and VHH. In certain embodiments, said cell binding agent is a monoclonal antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10 or a sequence at least 85% identical thereto.

In certain embodiments, said at least one cytotoxic agent is selected from the group consisting of a maytansinoid, a small drug, a tomaymycin derivative, a leptomycin derivative, a prodrug, a taxoid, CC-1065 and a CC-1065 analog.

In certain embodiments, said at least one cytotoxic agent is the maytansine DM1 of formula (I)

In certain embodiments, said at least one cytotoxic agent is the maytansine DM4 of formula (II)

In certain embodiments, the cell binding agent is covalently linked via a cleavable or non- cleavable linker to the at least one cytotoxic agent. In certain embodiments, said linker is selected from the group consisting of N-succinimidyl pyridyldithiobutyrate (SPDB), 4- (Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl (N~ maleimidomethyl) cyclohexane-1-carboxylate (SMCC). In certain embodiments, said linker is N-succinimidyl pyridyldithiobutyrate (SPDB) and said cytotoxic agent is DM4. In certain embodiments, said linker is 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB) and said cytotoxic agent is DM4.

In certain embodiments, said conjugate is characterized by a drug-to-antibody ratio (DAR) ranging from 3 to A, the DAR being calculated from the ratio of the cytotoxic agent concentration (c_D) to that of the cell binding agent (c_A);

wherein

^CD ~ ¾2Ββ ^X ^ 252) ~~ (¾252 ^X ^ 2 )]/ [(¾252 ^X ¾2 ) ~ (¾252. ^X ¾28o)l

C_A = [^y¾2B0 ~~ (¾ X ¾28o)l/ ¾280

and

and s_D28o are respectively the molar extinction coefficients of the cytotoxic agent at 252 nm and 280 nm,

ε_Α252 and ε_Α28ο are respectively the molar extinction coefficients of the cell binding agent at 252 nm and 280 nm, and

A252 and A₂8o are respectively the absorbances for the conjugate at 252 nm (A₂₅₂) and at 280 nm (A₂₈o), measured using a classic spectrophotometer apparatus.

In certain embodiments, said conjugate is administered intravenously. In certain embodiments, said conjugate is administered at a rate of 1 mL/min for 30 min and then increased to a maximal rate of 2 mL/min in the absence of hypersensitivity reactions.

In certain embodiments, the cancer is a solid tumor.

In certain embodiments, the cancer is a CA6-positive tumor. In certain embodiments, the tumor is qualified as CA 6-positive when at least 30% of the cells of the sample exhibit a level of intensity of 2+/3+ as determined using an immunohistochemistry (IHC) assay. In certain embodiments, the IHC assay utilizes the murine monoclonal antibody produced by the hybridoma cell line DS6 deposited at the American Type Culture Collection (ATCC) with the deposit number PTA-4449.

In certain aspects, the cancer is selected from the group consisting of breast cancer, lung cancer and bladder cancer. In certain aspects, the cancer is breast cancer. In certain aspects, the breast cancer is a triple negative breast cancer, not positive to receptors for estrogen, progesterone or HER2. An aspect of the present invention also concerns a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the method comprises at least two cycles, a cycle being a period of three weeks, and, for each cycle :

- the conjugate is administered at a dose of 120 mg/m² at day 1 of the cycle and at a dose of 120 mg/m² at day 8 of the cycle, and

- prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered.

An aspect of the invention also concerns a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the method comprises at least two cycles,

wherein the cycle is a period of two weeks, and

wherein the conjugate is administered at a dose of 120 mg/m² at day 1 of the cycle.

In some embodiments of the invention, the cell binding agent is a humanized anti- CA6 antibody and the cytotoxic agent is a maytansinoid.

In further embodiments, the cell binding agent is the humanized anti-CA6 antibody huDS6 comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10 and the cytotoxic agent is a maytansine compound such as DM1 or DM4.

In a particular embodiment, the conjugate used in the context of the invention is the compound huDS6-DM4 of the following formula (XXI)

I). An aspect of the present invention also concerns a conjugate comprising (i) a cell binding agent which specifically binds to tumor cells, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the cytotoxic agent is a tubulin binder; and

wherein, the method comprises the administration of the conjugate in a patient in need thereof, and at each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered, the prophylactic treatment consisting in two or more of:

- administration of an ocular vasoconstrictor,

- administration of an ocular corticosteroid, and

- use of a cold eye mask.

In certain embodiments, the prophylactic treatment consisting in two or more of:

- administration in each eye of three drops of 2.5% neosynephrine,

- administration of an ocular gel of dexamethasone (0.16%) three times a day for two days starting the day of the administration of the conjugate, and

- use of a cold eye mask during the administration of the conjugate.

An aspect of the present invention also concerns an article of manufacture comprising:

a) a packaging material;

b) a conjugate comprising (i) a cell binding agent which binds to the human mucin-

1 (MUC1) glycoprotein, linked to (ii) at least one cytotoxic agent; and

c) a label or package insert contained within said packaging material indicating that, for at least two cycles, said conjugate is administered, for each cycle, at a dose corresponding to that of an administration schedule of 60 mg/m² every week of the cycle.

In certain embodiments, the cycle is a period of three weeks, and, for each cycle, the conjugate is administered:

(i) at a dose of 90 mg/m² at day 1 of the cycle and at a dose of 90 mg/m² at day 8 of the cycle; or

(ii) at a dose of 120 mg/m² at day 1 of the cycJe and at a dose of 20 mg/m² at day 8 of the cycle.

In certain embodiments, the label or package insert further indicates that prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered. Detailed description of the invention

Definitions

In the context of the invention, the term "mucin-1 glycoprotein" or "MUC1 glycoprotein" refers to a mucin encoded by the MUC1 gene in humans. MUC1 is a glycoprotein with extensive O-linked glycosylation of its extracellular domain. MUC1 has a core protein mass of 120-225 kDa which increases to 250-500 kDa with glycosylation. It extends 200-500 nm beyond the surface of the cell. The protein is anchored to the apical surface of many epithelia by a transmembrane domain. Beyond the transmembrane domain is a SEA domain that contains a cleavage site for release of the large extracellular domain. The extracellular domain includes a 20 amino acid variable number tandem repeat (VNTR) domain, with the number of repeats varying from 20 to 120 in different individuals. These repeats are rich in serine, threonine and proline residues which permits heavy O-glycosylation.

In the context of the invention, the term "CA6 glvcotope" or "CA6 sialoqlycotope" refers to a tumor-associated antigen present on the extracellular domain of the MUC1 glycoprotein, which was identified by Kearse et al. (2000) Int. J. Cancer 88:866-872, as bearing a carbohydrate epitope that is sialic acid-dependent, and more particularly characterized in US2007/0041980.

As used herein, a sequence "at least 85% identical to a reference sequence" is a sequence having, on its entire length, 85% or more, in particular 90%, 91 %; 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the entire length of the reference sequence.

A percentage of "sequence identity" may be determined by comparing the two sequences, optimally aligned over a comparison window, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison is conducted by global pairwise alignment, e.g. using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443. The percentage of sequence identity can be readily determined for instance using the program Needle, with the BLOSU 62 matrix, and the following parameters gap-open=10, gap-extend=0.5.

In the context of the invention, a "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoieuci^'ne; 2) aliphati^'c-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. In particular a subject according to the invention is a human.

As used herein, "conjugate", "immunoconjuqate", "antibody-drug conjugate" or "ADC" have the same meaning and are interchangeable.

Throughout the instant application, the term "comprising" is to be interpreted as encompassing ail specifically mentioned features as well optional, additional, unspecified ones. As used herein, the use of the term "comprising" also discloses the embodiment wherein no features other than the specifically mentioned features are present (i.e. "consisting of).

Cell binding agent

As used herein, the term "cell binding agent" refers to an agent that specifically recognizes and binds the human mucin-1 (MUC1) glycoprotein on the cell surface. In a particular embodiment, the ceil binding agent binds, more particularly specifically binds, the extracellular domain of the MUC1 glycoprotein as defined in the section "Definition" supra. In another embodiment, the cell binding agent recognizes and binds the CA6 glycotope on the MUC1 glycoprotein as defined in the section "Definition" supra.

In one embodiment, the cell binding agent specifically recognizes the human MUC1 glycoprotein, in particular the extracellular domain of the MUC1 glycoprotein, more particularly the CA6 glycotope on the MUC1 glycoprotein, such that it allows the conjugates to act in a targeted fashion with little side-effects resulting from non-specific binding.

In another embodiment, the cell binding agent of the present invention also specifically recognizes the human MUC1 glycoprotein, in particular the extracellular domain of the MUC1 glycoprotein, more particularly the CA6 glycotope on the MUC1 glycoprotein, so t at the conjugate wil) be in contact with the target eel! for a sufficient period of time to allow the cytotoxic agent portion of the conjugate to act on the cell, and/or to allow the conjugates sufficient time in which to be internalized by the cell.

The effectiveness of the conjugates of the present invention as therapeutic agents depends on the careful selection of an appropriate cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, in particular to the extracellular domain of the MUC1 glycoprotein, more particularly to the CA6 glycotope on the MUC1 glycoprotein. Cell binding agents may be of any kind presently known, or that become known and includes peptides and non-peptides, as long as they bind to the human MUC1 glycoprotein, in particular to the extracellular domain of the MUC1 glycoprotein, more particularly to the CA6 glycotope on the MUC1 glycoprotein. Generally, these can be antibodies (especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient-transport molecules (such as transferrin), or any other cell binding molecule substance.

More specific examples of cell binding agents that can be used include:

- polyclonal antibodies;

- monoclonal antibodies;

- epitope-binding fragments of antibodies such as Fab, Fab', F(ab')₂ or Fv.

Selection of the appropriate cell binding agent is a matter of choice that depends upon the particular cell population that is to be targeted, but in general, antibodies or epitope-binding fragments thereof are preferred if an appropriate one is available or can be prepared, more preferably a monoclonal antibody. An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1 , CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placentai mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domain structure and hence the combining site.

"Complementarity Determining Regions" or "CDRs" refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. A conventional antibody antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.

"Framework Regions" (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.

As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody. In the context of the invention, CDR/FR definition in an immunoglobulin light or heavy chain is to be determined based on I GT definition (Lefranc et al. (2003) Dev Comp Immunol. 27(1):55-77; www.imgt.org).

As used herein, the term "antibody" denotes conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanised, bispecific or multi-specific antibodies.

As used herein, antibody or immunoglobulin also includes "single domain antibodies" which have been more recently described and which are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples of singie domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit and bovine. Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibody devoid of light chains. In particular, Camelidae species, for example camel, dromedary, llama, alpaca and guanaco, produce heavy chain antibodies naturally devoid of light chain. Camelid heavy chain antibodies also lack the CH1 domain.

The variable heavy chain of these single domain antibodies devoid of light chains are known in the art as "VHH" or "nanobody". Similar to conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are about ten times smaller than IgG molecules, and as a consequence properly folded functional nanobodies can be produced by in vitro expression while achieving high yield. Furthermore, nanobodies are very stable, and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard (Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77:13-22).

The term "monoclonal antibody" or "mAb" as used herein refers to an antibody molecule of a single amino acid composition that is directed against a specific antigen, and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or hybridoma, but may also be recombinant, i.e. produced by protein engineering. The term "chimeric antibody" refers to an engineered antibody which in its broadest sense contains one or more region(s) from one antibody and one or more regions from one or more other antibody(ies). In particular a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, in association with a CH domain and a CL domain of another antibody, in particular a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit or the like can be used. A chimeric antibody may also denote a multi-specific antibody having specificity for at least two different antigens. In an embodiment, a chimeric antibody has variable domains of mouse origin and constant domains of human origin.

The term "humanized antibody" refers to an antibody which is initially wholly or partially of non-human origin and which has been modified to replace certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most of the time human CH and CL domains. In an embodiment, a humanized antibody has constant domains of human origin,

"Fragments" of (conventional) antibodies comprise a portion of an intact antibody, in particular the antigen binding region or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')₂, Fab', dsFv, (dsFv)₂, scFv, sc(Fv)₂, diabodies, bispecific and multi-specific antibodies formed from antibody fragments. A fragment of a conventional antibody may also be a single domain antibody, such as a heavy chain antibody or VHH.

The term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 Da and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papain, are bound together through a disulfide bond.

The term "F(ab')_?" refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.

The term "Fab"' refers to an antibody fragment having a molecular weight of about

50,000 Da and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')₂ fragment. A single chain Fv ("scF ") polypeptide is a covalently linked VH::VI_ heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker. The human scFv fragment of the invention includes CDRs that are held in appropriate conformation, in particular by using gene recombination techniques. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)₂.

"dsFv" is a VH::VL heterodimer stabilised by a disulphide bond.

"(dsFv)?" denotes two dsFv coupled by a peptide linker.

The term "bispecific antibody" or "BsAb" denotes an antibody which combines the antigen-binding sites of two antibodies within a single mo/ecule. Thus, BsAbs are able to bind two different antigens simultaneously. Genetic engineering has been used with increasing frequency to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions as described for instance in EP 2 050 764 A .

The term "multi-specific antibody" denotes an antibody which combines the antigen-binding sites of two or more antibodies within a single molecule.

The term "diabodv" refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen- binding sites.

In a particular embodiment, the epitope-binding fragment is selected from the group consisting of Fv, Fab, F(ab')₂, Fab', dsFv, (dsFv)₂, scFv, sc(Fv)₂, diabody and VHH.

In a particular embodiment, the conjugate of the invention comprises an antibody or epitope-binding fragment thereof which comprises one or more CDR(s), in particular one, two, three, four, five or six CDR(s), having an amino acid sequence selected from the group consisting of SYN H (SEQ ID NO: 1 ), YIYPGNGATNYNQKFQG (SEQ ID NO: 2), GDSVPFAY (SEQ ID NO: 3), SAHSSVSFMH (SEQ ID NO: 4), STSSLAS (SEQ ID NO: 5) and QQRSSFPLT (SEQ ID NO: 6). In a further embodiment, the conjugate of the invention may comprise an antibody or epitope-binding fragment thereof which comprises a CDR1 -H of sequence SEQ ID NO: 1 , a CDR2-H of sequence SEQ ID NO: 2 and a CDR3-H of sequence SEQ ID NO: 3.

In a further embodiment, the conjugate of the invention may comprise an antibody or epitope-binding fragment thereof which comprises a CDR1-L of sequence SEQ ID NO: 4, a CDR2-L of sequence SEQ ID NO: 5 and a CDR3-L of sequence SEQ ID NO: 6.

In a further embodiment, the conjugate of the invention may comprise an antibody or epitope-binding fragment thereof which comprises a CDR1-H of sequence SEQ ID NO: 1 , a CDR2-H of sequence SEQ ID NO: 2, a CDR3-H of sequence SEQ ID NO: 3, a CDR1- L of sequence SEQ ID NO: 4, a CDR2-L of sequence SEQ ID NO: 5 and a CDR3-L of sequence SEQ ID NO: 6.

Also provided is a conjugate which comprises an antibody or epitope-binding fragment which comprises a heavy chain variable region of sequence

QAQLVQSGAEWKPGASVK SCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYP GNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYW

GQGTLVTVSA (SEQ ID NO: 7) or a sequence at least 85%, more particularly at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, preferably provided that said sequence contains the sequences SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3.

Still provided is a conjugate which comprises an antibody or epitope-binding fragment which comprises a light chain variable region of sequence

EIVLTQSPATMSASPGERVTfTCSAHSSVSFMHWFQQKPGTSPKLWfYSTSSLAS

GVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAGTKLELKR (SEQ ID NO: 8)

or a sequence at least 85%, more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, preferably provided that said sequence contains the sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

Still provided is a conjugate which comprises an antibody or epitope-binding fragment which comprises a heavy chain of sequence

QAQLVQSGAEWKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGA TNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDSVPFAYWGQGTLVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKP KDTLM I S RTP E VTCWVD VS H E D P EVKFN WYVDG VEVH N AKTKP RE E QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9),

or a sequence at least 85%, more particularly at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, preferably provided that said sequence contains the sequences SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3.

Still provided is a conjugate which comprises an antibody or epitope-binding fragment which comprises a light chain of sequence

EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPAR FGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAGT LELKRTVAAPSVFIFPPS DEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10)

In another embodiment, humanized anti-MUC1 antibodies and epitope-binding fragments thereof are provided having a humanized or resurfaced heavy chain variable region having an amino acid sequence corresponding to SEQ ID NO: 7.

Similarly, humanized anti-MUC1 antibodies and epitope-binding fragments thereof are provided having a humanized or resurfaced light chain variable region having an amino acid sequence corresponding to SEQ (D NO: 8.

As used herein, the term "humanized antibody" refers to a chimeric antibody which contain minimal sequence derived from non-human immunoglobulin.

A "chimeric antibody", as used herein, is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged, so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass. "Chimeric antibody" also refers to an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged, so that the constant region is linked to a variable region of a different species, or belonging to another antibody class or subclass.

The goal of humanization is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted for non-rejection by other mammals, may be produced using several technologies such as resurfacing and CDR grafting. As used herein, the resurfacing technology uses a combination of molecular modeling, statistical analysis and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host.

Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed in U.S. Patent No. 5,639,641. Briefly, in a particular method, (1 ) position alignments of a pool of antibody heavy and light chain variable regions is generated to give a set of heavy and light chain variable region framework surface exposed positions wherein the alignment positions for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) a set of heavy and light chain variable region framework surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified; (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5 A of any atom of any residue of the complementarity-determining regions of the rodent antibody; and (5) the humanized rodent antibody having binding specificity is produced.

Antibodies can be humanized using a variety of other techniques including CDR- grafting (EP0239400; WO91/09967; U.S. Patent Nos. 5,530, 101 and 5,585,089), veneering or resurfacing (EP0592106; EP0519596; Padlan (1991) Molecular immunology

28(4/5):489-498; Studnicka et al. (1994) Protein Engineering 7 (6): 805-814; Roguska ef a/.

(1994) Proc. Natl. Acad. Sci U.S.A. 91 :969-973), and chain shuffling (U.S. Patent No.

5,565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods. See also U.S. Patent Nos. 4,444,887, 4,716, 111 , 5,545,806, and 5,814,318; and International patent application W098/46645,

WO98/50433, W098/24893, W098/16654, WO96/34096, W096/33735, and

WO91/10741. An embodiment of such a humanized antibody is a humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, or an epitope-binding fragment thereof, or a sequence at least 85%, more particularly at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto, preferably provided that said sequence contains the sequences SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

Cytotoxic agent

The term "cytotoxic agent" as used herein refers to a substance that reduces or blocks the function or growth, of cells andlor causes destruction of cells. Accordingly, the cytotoxic agent used in the conjugate of the present invention may be any compound that results on the death of a cell, or induces cell death, or in some manner decreases cell viability. Examples of cytotoxic agents include maytansinoids and maytansinoids analogs, a prodrug, tomamycin derivatives, toxoids, a leptomycin derivative, CC-1065 and CC- 1065 analogs, as defined below.

Among the cytotoxic agents that may be used in the present invention to form a conjugate, are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include maytansinol and maytansinol analogs. Maytansinoids are drugs that inhibit microtubule formation and that are highly toxic to mammalian cells.

Examples of suitable maytansinol analogues include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patents 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; 6,333,4 0; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogues of maytansinol having a modified aromatic ring include:

(1 ) C~19-dechloro (U.S. Patent No 4,256,746), prepared by LAH reduction of ansamytocin P2;

(2) C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro (U.S. Patent Nos.

4,361 ,650 and 4,307,016), prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH; and (3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Patent No.

4,294,757), prepared by acylation using acyl chlorides.

Specific examples of suitable analogues of maytansinol having modifications of other positions include:

(1 ) C-9-SH (U.S. Patent No. 4,424,219), prepared by the reaction of maytansinol with H₂S or P₂S₅;

(2) C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Patent No. 4,331 ,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Patent No.

4,450,254), prepared from Nocardia;

(4) C-15-hydroxy/acyloxy (U.S. Patent No. 4,364,866), prepared by the conversion of maytansinol by Streptomyces;

(5) C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929), isolated from Trewia nudiflora;

(6) C- 8-A/-demethyl (U.S. Patent Nos. 4,362,663 and 4,322,348), prepared by the demethylation of maytansinol by Streptomyces; and

(7) 4,5-deoxy (U.S. Patent No. 4,371 ,533), prepared by the titanium trichloride/LAH reduction of maytansinol.

In a particular embodiment, the conjugates of the present invention utilize the thiol- containing maytansinoid DM1 , formally termed A/²-deacetyl-A/²-(3-mercapto-1-oxopropyl)- maytansine, as the cytotoxic agent. DM1 is represented by the following structural formula

In another embodiment, the conjugates of the present invention utilize the thiol- containing maytansinoid DM4, formally termed A/^2'-deacetyl-/V^2'-(4-methyl-4-mercapto-1 - oxopentyl)-maytansine, as the cytotoxic agent. DM4 is represented by the following structural formula (I

In further embodiments of the invention, other maytansines, including thiol and disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the carbon atom bearing the sulfur atom, may be used. These include a maytansinoid having, at C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl, at C-3 an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl group, wherein the carbon atom of the acyl group bearing the thiol functionality has one or two substituents, said substituents being CH₃, C₂H₅, linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and further wherein one of the substituents can be H, and wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl functionality and the sulphur atom.

Such additional maytansines include compounds represented by formula (III):

wherein:

Y' represents (CR₇R₈)_l(CR₉=C ₁₀)p(C_≡C)_qA_r(CR₅R₆)_mD_ll(CR₁₁=CR₁₂)_r(C_≡C)_sB_t(CR₃R4)nCR₁R2SZ, wherein

R-, and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂ can be H;

A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical;

3, ₄, R₅, e, R7, Re, Rg, R10, R11 and Ri₂ are each independently H, CH₃,

C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic aikyl or aikenyi having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

I, m, n, o, p, q, r, s and t are each independently 0 or an integer of from 1 to

5, provided that at least two of l, m, n, o, p, q, r, s and t are not zero at any one time; and

Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (III) include compounds of formula (III) wherein:

Ri is methyl, R₂ is H and Z is H;R-, and R₂ are methyl and Z is H;

Ri is methyl, R₂ is H and Z is -SCH₃;

Ri and R₂ are methyl and Z is -SCH₃.

Such additional maytansines also include compounds represented by formula (IV- , (IV-D) or (IV-D, L):

(IV-L) (IV-D)

wherein:

Y represents (CR₇R₈)i(CR₅R6)_m(CR₃R4)nCR₁R₂SZ,

wherein: Ri and R₂ are each independently CH₃, C₂H₅, linear alkyl or akenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂ can be H;

R3, R₄, R5, e, R7 and R₈ are each independently H, CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical;

I, m and n are each independently an integer of from 1 to 5, and in addition n can be 0;

Z is H, SR, -COR wherein R is linear or branched alkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocyclic radical; and

May represents a maytansinoid which bears the side chain at C-3, C-14 hydroxymethyJ, C-15 hydroxy or C-20 desmethyl. Particular embodiments of formulae (IV-L), (IV-D) and (IV-D,L) include compoundsulae (IV-L), (IV-D) and (IV-D.L) wherein:

Ri is methyl, R₂ is H, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , n is 0, and Z is H;

Ri and R₂ are methyl, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , n is 0, and Z is H;

Ri is methyl, R₂ is H, R₅, Re, R7 and R₈ are each H, I and m are each 1 , n is

0, and Z is -SCH₃;

Ri and R₂ are methyl, R₅, R₈, R₇ and R₈ are each H, I and m are each 1 , n is 0, and Z is -SCH₃.

In one embodiment, the cytotoxic agent is represented by formula (IV-L).

Such additional maytansines also include compounds represented by formula (V): wherein:

Y repr

wherein:

and R₂ are each independently CH₃, C₂H₅, linear alky! or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂ can be H;

R₃, RJ, 5, e, R7 and R₈ are each independently H, CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radicaf;

I, m and n are each independently an integer of from 1 to 5, and in addition n can be 0; and

Particular embodiments of formula (V) incJude compounds of formuJa (V) wherein:

Ri is methyl, R₂ is H, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , n is

0 and Z is H;

R and R₂ are methyl, R₅, R₆, R₇ and R₈ are each H, I and m are 1 , n is 0 and Z is H; is methyl, R₂ is H, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , n is 0 and Z is -SCH₃;

Ri and R₂ are methyl, R₅, R₆, R₇ and R₈ are each H, I and m are 1 , n is 0 and Z is -SCH₃.

Such additional maytansines further include compounds represented by formula (Vl-L), (Vl-D) or (VI-D.L):

(Vl-L) (Vl-D) (VI-D.L) wherein:

Y₂ represents (CR₇R₈)i(CR₅R₆)_m(CR₃R4)_nCRi ₂SZ2,

wherein:

R, and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂ can be H;

R3, R4, R5, Re, R7 and R₈ are each independently H, CH₃, C₂H₅, linear cyclic alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

Z₂ is SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from

3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical; and

May is a maytansinoid.

Such additional maytansines also include compounds represented by formula (VII):

wherein:

Υ₂' represents

(CR₇R₈)_l(CR₉=CR₁o)p(C_≡C)_qA_r(CR₅R₆)_mD_u(CR₁₁=CR_†2)_r(C_≡C)_sB_f(CR_{3 4})nCR₁R₂SZ₂, wherein:

Ri and R₂ are each independently CH₃, C₂H₅, linear branched or alkyi or alkenyl having from 1 to 10 carbon atoms, cyclic alkyi or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂ can be H;

A, B and D are each independently cycloalkyi or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical;

R₃, R₄, R₅, R₆, R₇, R₈, Rg, Rio, Rn and R₁₂ are each independently

H, CH₃₎ C₂H₅, linear alkyi or aikenyl having from 1 to 10 carbon atoms, branched or cyclic alkyi or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

I, m, n, o, p, q, r, s and t are each independently 0 or an integer of from 1 to 5, provided that at least two of I, m, n, o, p, q, r, s and t are not zero at any one time; and

2₂ is SR or -COR, wherein R is linear alkyi or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyi or alkenyl having from 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical. Particular embodiments of formula (VII) include compounds of formula (VII) wherein Ri is methyl and R₂ is H.

The above-mentioned maytansinoids can be conjugated to the cell binding agent defined in the section "Cell binding agent' above, in particular to the humanized antibody huDS6 comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, wherein the cell binding agent, in particular the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, is linked to the maytansinoid using the thiol or disulfide functionality that is present on the acyl group of an acylated amino acid chain found at C- 3, C-14 hydroxymathyl, C-15 hydroxy or C-20 desmethyl of the maytansinoid, and wherein the acyi group of the acyiated amino acid side chain has its thiol or disulfide functionality located at a carbon atom that has one or two substituents, said substituents being CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and in addition one of the substituents can be H, and wherein the acyl group has a linear chain length of at least three carbon atoms between the carbonyl functionality and the sulfur atom.

In one embodiment of the present invention, the conjugate is the one that comprises a cell binding agent as defined in the section "Cell binding agent" above, in particular the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, conjugated to a maytansinoid of formula (VIII):

wherein:

Yi' represents

(CR₇R₈),(CR₉=CR_i0)_p(C_≡C)_qA_r(CR₅R₆)_mD_u(CR„=CR₁₂)_r(C_≡C)_sB_f(CR₃R₄)_nCR₁R₂S-, wherein

A, B and D are each independently cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical;

R3, R4, R5, Re, R7, Re, Rg, R10, R11 and R12 are each independently

H, CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical; and

I, m, n, o, p, q, r, q and t are each independently 0 or an integer of from 1 to 5, provided that at least two of I, m, n, o, p, q, r, s and t are not zero at any one time.

In particular, R is methyl, R₂ is H, or and R₂ are methyl.

In a further embodiment of the present invention, the conjugate is the one that comprises a cell binding agent as defined in the section "Cell binding agent" above, in particular the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, conjugated to a maytansinoid of

(IX-L) (IX-D) (IX-D,L) wherein:

Yi represents (CR₇R8)i(CR₅R₆)_m(CR₃R₄)nCR₁ R₂S-,

wherein

Ri and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, heterocyclic aromatic or heterocycloakenyl radical, and in addition R₂ can be H;

R₃, 4, R5, e, 7 and R₈ are each independently H, CH₃, C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

I, m and n are each independently an integer from 1 to 5, and in addition n can be 0; and

May represents a maytansinol which bears the side chain at C-3, C- 14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Particular embodiments of formulae (IX-L), (IX-D) and (IX-D.L) include compounds of formulae (IX-L), (IX-D) and (IX-D.L) wherein:

Ri is methyl and R₂ is H, or and R₂ are methyl,

R! is methyl, R₂ is H, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , and n is 0,

Ri and R₂ are methy), R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , and n is 0.

More particularly, the cytotoxic agent is represented by formula (IX-L).

In a further embodiment of the present invention, the conjugate is the one that comprises a cell binding agent as defined in the section "Cell binding agent" above, in particular the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, conjugated to a maytansinoid of formula (X):

wherein the substituents are as defined for formula (IX) above.

In a further embodiment, in the above-described compounds, is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , and n is 0.

In further embodiments, in the above-described compounds, Ri and R₂ are methyl, R₅, R₆, R₇ and R₈ are each H, I and m are each 1 , and n is 0.

Further, the L-aminoacyl stereoisomer is preferred.

Each of the maytansinoids taught in U.S. Patent application No. 2004/0235840, may also be used as cytotoxic agent in the conjugate of the invention. Conjugates of cell binding agents as defined in the section "Cell binding agent' above, in particular of antibodies, with maytansinoid drugs can be evaluated for their ability to suppress proliferation of various unwanted cell lines in vitro. For example, cell lines such as the human epidermoid carcinoma line A-431 , the human small cell lung cancer cell line SW2, the human breast tumor line SKBR3 and the Burkitt's lymphoma cell line Namalwa can easily be used for the assessment of cytotoxicity of these compounds. Cells to be evaluated can be exposed to the compounds for 24 h and the surviving fractions of cells measured in direct assays by known methods. IC₅o values can then be calculated from the results of the assays.

The cytotoxic agent used in the conjugates according to the present Invention may also be a taxane or derivative thereof.

Taxanes are a family of compounds that includes paclitaxel (taxol), a cytotoxic natural product, and docetaxel (Taxotere), a semi-synthetic derivative, two compounds that are widely used in the treatment of cancer. Taxanes are mitotic-spindle poisons that inhibit the depolymerization of tubulin, resulting in cell death. While docetaxel and paclitaxel are useful agents in the treatment of cancer, their antitumor activity is limited because of their non-specific toxicity towards normal cells.

A particular taxane for use in the preparation of conjugates is the taxane of formula

(XI):

Methods for synthesizing taxanes that may be used in the cytotoxic conjugates of the present invention, along with methods for conjugating the taxanes to a cell binding agent as defined in the section "Cell binding agent above, such as the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, are described in detail in U.S. Patent Nos. 5,416,064, 5,475,092, 6,340,701 , 6,372,738, 6,436,931 and 6,596,757, and in U.S. Application Nos. 2004/0001838, 2003/0004210, 2004/0024049 and No 10/210,112.

The cytotoxic agent according to the present invention may also be a tomaymycin derivative. Tomaymycin derivatives are pyrrolo[1 ^benzodiazepines (PBDs), a known class of compounds exerting their biological properties by covalently binding to the N2 of guanine in the minor groove of DNA. PBDs include a number of minor groove binders such as anthramycin, neothramycin and DC-81.

Novel tomaymycin derivatives that retain high cytotoxicity and that can be effectively linked to cell binding agents as defined in the section "Cell binding agent above are described in the International Application No. WO2007/085930. The cell binding agent-tomaymycin derivative complexes permit the full measure of the cytotoxic action of the tomaymycin derivatives to be applied in a targeted fashion against unwanted cells only, therefore avoiding side effects due to damage to non-targeted healthy cells.

The cytotoxic agent according to the present invention may comprise one or more tomaymycin derivatives, linked to a cell binding agent as defined in the section "Cell binding agent" above, such as the humanized huDS6 antibody comprising a heavy chain of sequence SEQ JD NO: 9 and a light chain of sequence SEQ ID NO: 10, via a linking group. The linking group is part of a chemical moiety that is covalently bound to a tomaymycin derivative through conventional methods. In a particular embodiment, the chemical moiety can be covalently bound to the tomaymycin derivative via a disulfide bond.

The tomaymycin derivatives useful in the present invention have the formula (XII) shown belo

wherein

represents an optional single bond;

represents either a single bond or a double bond; provided that when represents a single bond, U and U', the same or different, independently represent H, and W and W, the same or different, are independently selected from the group consisting of OH, an ether such as -OR, an ester (e.g. an acetate), such as -OCOR, a carbonate such as -OCOOR, a carbamate such as -OCONRR', a cyclic carbamate, such that N10 and C11 are a part of the cycle, a urea such as -NRCONRR', a thiocarbamate such as - OCSNHR, a cyclic thiocarbamate such that N10 and C1 1 are a part of the cycle, - SH, a sulfide such as -SR, a sulphoxide such as -SOR, a sulfone such as -SOOR, a sulphonate such as -S03-, a sulfonamide such as -NRSOOR, an amine such as -NRR', optionally cyclic amine such that N10 and C1 1 are a part of the cycle, a hydroxylamine derivative such as -NROR', an amide such as -NRCOR', an azi^'do such as -N3, a cyano, a halo, a trialkyl or triarylphosphonium, an aminoacid- derived group.

Preferably W and W are the same or different and are OH, Ome, Oet, NHCONH₂, SMe;

and when represents a double bond, U and U' are absent and W and W represent H;

^■ R1 , R2, R1 '_r R2' are the same or different and independently chosen from Halide or Alkyl optionally substituted by one or more Halide, CN, NRR', CF₃, OR, Aryl, Het, S(0)_qR, or R1 and R2 and R1 ' and R2' form together a double bond containing group =B and =B' respectively.

In one embodiment, R1 and R2 and R1' and R2' form together a double bond containing group =B and =B' respectively.

^■ B and B' are the same or different and independently chosen from Alkenyl being optionally substituted by one or more Halide, CN, NRR', CF₃, OR, Aryl, Het, S(0)_qR or B and B' represent an oxygen atom.

In one embodiment, B=B'.

In a further embodiment, B=B'= =CH₂ or =CH-CH₃,

^■ X and X' are the same or different and independently chosen from one or more -O- , -NR-, -(C=0)-, -S(0)_q-.

In one embodiment, X=X'.

In a further embodiment, X=X'=0. ^■ A and A' are the same or different and independently chosen from AlkyI or Alkenyl optionally containing an oxygen, a nitrogen or a sulfur atom, each being optionally substituted by one or more Halide, CN, NRR', CF₃, OR, S(0)„R, Aryl, Het, AlkyI, Alkenyl.

In one embodiment, A=A'.

In a further embodiment, A=A'=linear unsubstituted alkyl.

^■ Y and Y' are the same or different and independently chosen from H, OR;

In one embodiment, Y=Y'.

In a further embodiment, Y=Y -OAIkyl, more preferably OMethyl.

^■ T is -NR-, -0-, -S(0)_q-, or a 4 to 10-membered aryl, cycloalkyl, heterocyclic or heteroaryi, each being optionally substituted by one or more Halide, CN, NRR', CF₃, R, OR, S(0)_qR, and/or linker(s), or a branched Alkyl, optionally substituted by one or more Hal, CN, NRR', CF₃, OR, S(0)_qR and/or linker(s), or a linear Alkyl substituted by one or more Hal, CN, NRR', CF₃, OR, S(0)_qR and/or linker(s).

In one embodiment, T is a 4 to 10-membered aryl or heteroaryi, more preferably phenyl or pyridyl, optionally substituted by one or more linker(s).

Said linker comprises a linking group. Suitable linking groups are well known in the art and include thiol, sulfide, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Preferred are disulfide groups and thioether groups.

When the linking group is a thiol-, sulfide (or so-called thioether -S-) or disulfide (- S-S-) -containing group, the side chain carrying the thiol, the sulfide or disulfide group can be linear or branched, aromatic or heterocyclic. One of ordinary skill in the art can readily identify suitable side chains.

In one embodiment, said linker is of formula -G-D-(Z)_P-S-Z'

wherein

G is a single or double bond, -0-, -S- or -NR-;

D is a single bond or -E-, -E-NR-, -E-NR-F-, -E-0-, -E-0-F-, -E-NR-CO-, -E- NR-CO-F-, -E-CO-, -CO-E-, -E-CO-F, -E-S-, -E-S-F-, -E-NR-C-S-, -E-NR- CS-F- ;

where E and F are the same or different and are independently chosen from linear or branched -(OCH₂CH2)iAlkyl(OCH₂CH2)j-, - Alkyl(OCH₂CH₂)rAlkyl-, -(OCH₂CH₂)_r, (OCH₂CH₂)iCycloalkyl(OCH₂CH₂)_r,

(OCH₂CH₂)iHeterocyclic(OCH₂CH₂)_r, -(OCH₂CH₂)Aryl(OCH₂CH₂)_fl -(OCH₂CH₂)iHeteroaryl(OCH₂CH₂)_r, -Alkyl- (OCH₂CH₂)iAlkyl(OCH₂CH₂)_r, -Alkyl-(OCH₂CH₂)_r, -Alkyl- (OCH₂CH₂)iCycloalkyl(OCH₂CH₂)_r,

Alkyl(OCH₂CH₂)iHeterocyclic(OCH₂CH₂)_r, -Alkyl- (OCH₂CH₂)Aryl(OCH₂CH₂)_r,

Alkyl(OCH₂CH₂)iHeteroaryl(OCH₂CH₂)_r, -Cycloalkyl-Alkyi-, -Alkyl- Cycloalkyl-, -Heterocyclic-Alkyl-, -Alkyl-Heterocyclic-, -Alkyl-Aryl-, - Aryl-Alkyl-, -Aikyl-Heteroaryl-, -Heteroaryl-Alkyl-;

where i and j, identical or different, are integers and independently chosen from 0, 1 to 2000;

Z is linear or branched -Alkyl-;

p is 0 or 1 ;

71 represents H, a thiol protecting group such as COR, R₂₀ or SR₂₀, wherein R₂₀ represents H, methyl, Alkyl, optionally substituted Cycloalkyl, aryl, heteroaryl or heterocyclic, provided that when Z' is H, said compound is in equilibrium with the corresponding compound formed by intramolecular cyclisation resulting from addition of the thiol group -SH on the imine bond - NH= of one of the PBD moieties.

^■ n, n', equal or different are 0 or 1.

^■ q is 0, 1 or 2.

■ R and R' are equal or different and independently chosen from H, Alkyl, Aryl, each being optionally substituted by Halide, CN, NRR', CF₃, R, OR, S(0)_qR, Aryl, Het; or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers.

The compounds of the general formula (XII) having geometrical and stereoisomers are also a part of the invention.

The N-10, C-1 1 double bond of tomaymycin derivatives of formula (XII) is known to be readily convertible in a reversible manner to corresponding imine adducts in the presence of water, an alcohol, a thiol, a primary or secondary amine, urea and other nucleophiles. This process is reversible and can easily regenerate the corresponding tomaymycin derivatives in the presence of a dehydrating agent, in a non-protic organic solvant, in vacuum or at high temperatures (Tozuka (1983) J. Antibiotics 36:276).

Thus, reversible derivatives of tomaymycin derivatives of general formula (XIII) can also be used in the present invention:

where A, X, Y, n, T, A\ X', Y', n\ R1 , R2, R1\ R2' are defined as in formula (XII) and W and W are the same or different and are selected from the group consisting of OH, an ether such as -OR, an ester (e.g. an acetate), such as -OCOR, -COOR, a carbonate such as -OCOOR, a carbamate such as -OCONRR', a cyclic carbamate, such that N10 and C11 are a part of the cycle, a urea such as -NRCONRR', a thiocarbamate such as - OCSNHR, a cyclic thiocarbamate such that N10 and C11 are a part of the cycle, -SH, a sulfide such as -SR, a sulphoxide such as -SOR, a sulfone such as -SOOR, a sulphonate such as -SO₃-, a sulfonamide such as -NRSOOR, an amine such as -NRR', optionally cyclic amine such that N10 and C1 1 are a part of the cycle, a hydroxylamine derivative such as -NROR', an amide such as -NRCOR, -NRCONRR', an azido such as -N₃, a cyano, a halo, a trialkyi or triarylphosphonium, an aminoacid-derived group. Preferably, W and W are the same or different and are OH, Ome, Oet, NHCONH₂, SMe.

Compounds of formula (XIII) may thus be considered as solvates, including water when the solvent is water; these solvates can be particularly useful.

In a further embodiment, the tomaymycin derivatives of the invention are selected from the group consisting in:

• 8,8'-[1 ,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy- 1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepin-5-one]

« 8,8'-[5-methoxy-1 ,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7- methoxy- ,2,3, 1 1 a-tetrahydro-5H-pyrrolo[2, 1 -cj[1 ,4Jbenzodiazepin-5-oneJ

» 8,8'-[1 ,5-pentanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1 ,2,3,11a- tetrahydro-5H-pyrrolo[2, 1 -c][ ,4]benzodiazepin-5-one]

• 8,8'-[ ,4-butanediylbis(oxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1 ,2,3, 1 1 a- tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one] 8, 8'-[3-methyl-1 , 5-penianediylbis(oxy)3-bis[(S)-2-eth-(E)-ylidene-7-methoxy- 1 ,2,3,11 a-tetrahydro-5H-pyrrolo[2,1-c][1 ,4]benzodiazepin-5-one3

8,8'-[2,6-pyridinediylbis(oxy)3-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1 ,2,3,11a- tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8 4-(3-tert-butoxycarbonylaminopropyloxy)-2,6-pyridinediylbis-(methyIeneoxy)] bis[(S)-2-eth-(E)-yIidene-7-methoxy-1 ,2,3,1 1 a-tetrahydro-5H-pyrrolo[2, 1 - c][1 ,4]benzodiazepin-5-one]

8,8'-[5-(3-aminopropyloxy)-1 ,3-benzenediylbis(methyleneoxy)]-bis[(S)-2-eth-(E)- ylidene-7-methoxy-1 ,2,3,1 1 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5- one]

8,8'-[5-(N-methyl-3-tert-butoxycarbonylaminopropyl)-1 ,3-benzenediylbis- (methyleneoxy)]-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1 ,2,3,1 1 a-tetrahydro-5H- pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-{5-[3-(4-methyl-4-methyldisulfanyl-pentanoylamino)propyloxy]-1 ,3- benzenediylbis(methyleneoxy)}-bis[(S)-2-eth-(E)-ylidene-7-methoxy-1 ,2,3,11 a- tetrahydro-5H-pyrrolo{2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[5-acetylthiomethyl-1 ,3-benzenediylbis(methyieneoxy)]-bis[(S)-2-methylene-7 met oxy- ,2,3, 11a-teirahydro-5H-pyrrolo[2, 1-c][1 ,4]benzodiazepin-5-one] bis-{2-[(S)-2-methylene-7-methoxy-5-oxo-1 ,3,1 1a-tetrahydro-5H-pyrrolo[2,1- c][1 ,4]benzodiazepin-8-yloxy]-ethyl}-carbamic acid tert-butyl ester

8,8'-[3-(2-acetylthioethyl)-1 ,5-pentanediylbis(oxy)]-bis[(S)-2-methylene-7-methoxy 1 ,2,3, 1 1 a-tetrahydro-5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[5-(N-4-mercapto-4,4-dimethylbutanoyl)amino-1 ,3- benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1 ,2,3, 1 1a-tetrahydro- 5H-pyrrolo[2, 1 -c][ ,4]benzodiazepin-5-one]

8,8'-[5-(N-4-methyldithio-4,4-dimethylbutanoyl)-amino-1 ,3- benzenediylbis(methyleneoxy)]-bis[7-methoxy-2-methylene-1 ,2,3, 1 1a-tetrahydro- 5H-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[5-(N-methyl-N-(2-mercapto-2,2-dimethylethyl)amino-1 ,3- benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1 ,2,3, 1 1 a-tetrahydro-5H- pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one] 8,8 5-(N-methyl-N-(2-methyldithio-2,2-dimethylethyl)amino-1 ,3- benzenediyl(methyleneoxy)]-bis[7-methoxy-2-methylene-1 ,2,3,1 1 a-tetrahydro-5H- pyrro!o[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[(4-(2-(4-mercapto-4-methyl)-pentanamido-ethoxy)-pyridin-2,6-dimethyl)- dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3, 1 1 a-tetrahydro-pyrrolo[2, 1 - c][1 ,4]benzodiazepin-5-one]

8,8'-[(1 -(2-(4-methyl-4-methyldisulfanyl)-pentanamido-ethoxy)-benzene-3,5- dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3, 1 a-tetrahydro- pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[(4-(3-(4-methyl-4-methyldisulfanyl)-pentanamido-propoxy)-pyridin-2,6- dimethyl)-dioxy]-bisf(S)-2-ei -(E)-y(idene-7-dirnet oxy-1 ,2,3, 1a-teirahydro- pyrrolo[2, 1-c][1 ,4] benzodiazepin-5-one]

8,8'-[(4-(4-(4-methyl-4-methyldisulfanyl)-pentanamido-butoxy)-pyridin-2,6- dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3, 1 a-tetrahydro- pyrrolo[2, -c][ ,4]benzodiazepin-5-one]

8,8'-[(4-(3-[4-(4-methyl-4-methyldisulfanyl-pentanoyl)-piperazin-1-yl]-propyl)- pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3, 1a- tetra ydro-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[(1-(3-[4-(4-methyl-4-methyidisulfanyl-pentanoyi)-piperazin-1-yl]-propyl)- benzene-3,5-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3,1 1 a- tetrahydro-pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

8,8'-[(4-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}- ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-

1 ,2,3,1 1 a-tetrahydro-pyrrolo[2,1-c][1 ,4]benzodiazepin-5-one]

8,8'-f(1-(2-{2-f2-(2-{2-[2-(4-methyI-4-methyldisulfanyl-pentanoylamino)-ethoxyj- ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzene-3,5-dimethyl)-dioxy]-bis[(S)-2- eth-(E)-ylidene-7-dimethoxy-1 ,2,3, 1 1 a-tetrahydro-pyrrolo[2,1-c][1 ,4]benzodiazepin-

5-oneJ

8,8'-[(1 -(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylamino)-ethoxy]-ethoxy}- ethoxy)-benzene-3,5~dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimeihoxy- 1 ,2,3,1 a-tetrahydro-pyrroio[2,1-c][1 ,4]benzodiazepin-5-one]

8,8'-[(4-(2-{2-[2-(2-{2-[2-(4-methyl-4-methyldisulfanyl-pentanoylannino)-ethoxy]- ethoxy}-ethoxy)-ethoxy3-ethoxy}-ethoxy)-pyridin-2,6-dimethyl)-dioxy]-bis[(S)-2-eth- (E)-ylidene-7-dimethoxy-1 ,2,3,11 a-tetrahydro-pyrrolo[2,1-c][1 ,4]ben2odiazepin-5- one]

• 8,8'-[(1-(2-[methyl-(2-methyl-2-m

3.5- dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3,1 1 a-tetrahydro- pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

• 8,8'-[(4-(3-[methyl-(4-methyl-4-methyldisulfanyl-pentanoyl)-amino]-propyl^

2.6- dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3,1 1 a-tetrahydro- pyrrolo[2, 1 -c][1 ,4]benzodiazepin-5-one]

• 8,8'-[(4-(3-[methyl-(2-methyi-2-methyldisulfanyl-propyl)-amino]-propy|)^yridm

dimethyl)-dioxy]-bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3,11a-tetrahydro- pyrrolo[2,1-c][1 ,4]benzodiazepin-5-one]

• 8,8'-[(1-(4-methyl-4-methyldisulfanyl)-pentanamido)-benzene-3,5-dimethyl)-dioxy^ bis[(S)-2-eth-(E)-ylidene-7-dimethoxy-1 ,2,3,1 1 a-tetrahydro-pyrrolo[2, 1 - c][1 ,4]benzodiazepin-5-one]

as well as the corresponding mercapto derivatives, or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers.

Particular compounds are those of formula (X)V) or (XV):

wherein X, X', A, A', Y, Y', T, n, n' are defined as in formula (XII).

The compounds of formula (XII) may be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by application or adaptation of the methods described below, or variations thereon as appreciated by the skilled artisan. The appropriate modifications and substitutions will be readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. In particular, such methods can be found in R.C. Larock, Comprehensive Organic Transformations, Wiley-VCH Publishers, 1999.

Methods for synthesizing the tomaymycin derivatives which may be used in the invention are described in the International Application No. WO2007/085930. Compounds of the present invention may be prepared by a variety of synthetic routes. The reagents and starting materials are commercially available, or readily synthesized by well-known techniques by one of ordinary skill in the arts (see, for example, WO00/12508, WO00/12507, WO2005/040170, WO2005/085260, FR1516743, Mori et al. (1986) Tetrahedron 42:3793-3806).

The cytotoxic agent according to the present invention may also be a \eptomycin derivative.

According to the present invention, "leptomycin derivatives" refer to members of the leptomycin family as defined in Kalesse ef al. (2002) Synthesis 8:981-1003, and includes: leptomycins, such as leptomycin A and leptomycin B, callystatins, ratjadones such as ratjadone A and ratjadone B, anguinomycins such as anguinomycin A, B, C, D, kasusamycins, leptolstatin, leptofuranins, such as leptofuranin A, B, C, D. Derivatives of leptomycin A and B are preferred.

More specifically, the leptomycin derivatives may be of formula (XVI):

wherein

Ra and Ra' are H or -Alk; preferably Ra is -Alk, preferably methyl and Ra' is H ; R 7 is alkyl optionaffy substituted by OR, CN, NRR', perfluoroalkyf; preferably, R 7 is alkyl, more preferably methyl or ethyl;

R9 is alkyl optionally substituted by OR, CN, NRR', perfluoroalkyl; preferably, R9 is alkyl, more preferably methyl;

X is -O- or -NR-; preferably, X is -NR-;

Y is -U-, -NR-U-, -0-U-, -NR-CO-U-, -U-NR-CO-, -U-CO-, -CO-U- ;

preferably, when X is -0-, Y is -U-, -NR-U-, -U-NR-CO-; where U is chosen from linear or branched -Alk-, -Alk(OCH₂CH₂)m-, - (OCH₂CH₂)_m-Alk-, -Alk(OCH₂CH₂)_m-Alk-, -(OCH₂CH₂)_m-, -CycloalkyI-, - Heterocyclic-, -Cycloalkyl-Alk-, -Alk-Cycloalkyl-, -Heterocyclic-Alk-, -Alk- Heterocyclic-;

where m is an integer chosen from 1 to 2000;

preferably, U is linear or branched -Alk-,

Z is -Alk-;

n is 0 or 1 ; preferably n is 0;

T represents H, a thiol protecting group such as Ac, Ri or SR^ wherein R-i represents H, methyl, Alk, CycloalkyI, optionally substituted aryl or heterocyclic, or

T represents

(XVII)

wherein:

Ra, Ra\ R17, R9, X, Y, Z, n are defined as above;

preferably, T is H or SR_{1 (} wherein R^ represents Alk, more preferably methyl;

R and R' identical or different are H or alkyl;

Alk represents a linear or branched alkyl; preferably Alk represents (-(CH_2- _q(CH₃)_q)_p-where p represents an integer from 1 to 10 and q represents an integer from 0 to 2; preferably, Alk represents -(CH₂)- or -C(CH₃)₂-.

or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the polymorphic crystalline structures of these compounds or their optical isomers, racemates, diastereomers or enantiomers.

Particular compounds may be chosen from:

• (2-Methylsulfanyl-ethyl)-amid of (2E,10E, 2E,16Z,18E)-(R)-6-Hydroxy- 3,5,7,9,11 ,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2- yl)-8-oxo-nonadeca-2, 10, 12, 16, 18-pentaenolc acid

• Bis-[(2-mercaptoethy[)-amid of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy- 3,5,7,9, 1 1 ,15,17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-clihyclro-2H-pyran- 2-yl)-8-oxo-nonadeca-2, 10, 2,16,18-pentaenoic acid] • (2-Mercapto-ethyl)-amid of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-3,5,7,9,11 ,15,17- heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-8-oxo- nonadeca-2, 10,12,16,18-pentaenoic acid

• (2-Methyldisulfanyl-ethyl)-amid of (2E, 10E,12E, 16Z, 18E)-(R)-6-hydroxy- 3,5,7,9, 1 1 , 15, 17-heptamethyl-19-((2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2- yl)-8-oxo-nonadeca-2, 0,12, 16, 18-pentaenoic acid

• (2-Methyl-2-methyldisulfanyl-propyl)-amid of (2E, 10E, 12E, 16Z, 18E)-(R)-6-hydroxy- 3,5,7,9, 1 1 , 15, 17-heptamethyl-19-((2S,3S)-3-nnethyl-6-oxo-3,6-dihydro-2H-pyran- 2-yl)-8-oxo-nonadeca-2, 10,12, 16, 18-pentaenoic acid

· (2-Mercapto-2-methyl-propy!)-amid of (2E,10E,12E,16Z,18E)-(R)-6-hydroxy-

3,5,7,9,11, 15, 17-heptamethyl-19-((2S,3S)-3-methyf-6-oxo-3,6-dihydro-2H-pyran-2- yl)-8-oxo-nonadeca-2, 10, 12, 16, 18-pentaenoic acid

in order to link the derivative to a cell-binding agent as defined in the section "Cell binding agent' above, the derivative must include a moiety (linking group) that allows the derivatives to be linked to a cell binding agent via a linkage such as a disulfide bond, a sulfide (or called herein thioether) bond, an acid-labile group, a photo-labile group, a peptidase-iabile group, or an esterase-labile group. The derivatives are prepared so that they contain a moiety necessary to link the leptomycin derivative to a cell binding agent via, for example, a disulfide bond, a thioether bond, an acid-labile group, a photo-labile group, a peptidase-iabile group, or an esterase-labile group. In order to further enhance solubility in aqueous solutions, the linking group can contain a polyethylene glycol spacer. In an embodiment, a sulfide or disulfide linkage is used because the reducing environment of the targeted cell results in cleavage of the sulfide or disulfide and release of the derivatives with an associated increase in cytotoxicity.

Compounds of the present invention may be prepared by a variety of synthetic routes. The reagents and starting materials are commerciaily available, or readily synthesized by well-known techniques by one of ordinary skill in the art. Methods for synthesizing leptomycin derivatives that may be used in the cytotoxic conjugates of the present invention, along with methods for conjugating said leptomycin derivatives to cell binding agents such as antibodies, are described in detail in European Patent Application No. EP 1864682.

The cytotoxic agent used in the cytotoxic conjugates according to the present invention may also be CC-1065 or a derivative thereof.

CC-1065 is a potent anti-tumor antibiotic isolated from the culture broth of Streptomyces zelensis. CC-1065 is about 1000-fold more potent in vitro than are commonly used anti-cancer drugs, such as doxorubicin, methotrexate and vincristine (Bhuyan er a/. (1982) Cancer Res. 42:3532-3537). CC-1065 and its analogs are disclosed in U.S. Patent Nos. 6,372,738, 6,340,701 , 5,846,545 and 5,585,499.

The cytotoxic potency of CC-1065 has been correlated with its alkylating activity and its DNA-binding or DNA-intercalating activity. These two activities reside in separate parts of the molecule. Thus, the alkylating activity is contained in the cyclopropapyrroloindole (CPI) subunit and the DNA-binding activity resides in the two pyrroloindoie subunits.

Although CC-1065 has certain attractive features as a cytotoxic agent, it has limitations in therapeutic use. Administration of CC-1065 to mice caused a delayed hepaiofoxicity leading to mortality on day 50 after a single intravenous dose of 12.5 g/kg (Reynolds et al. (1986) J. Antibiotics XXIX: 319-334). This has spurred efforts to develop analogs that do not cause delayed toxicity, and the synthesis of simpler analogs modeled on CC-1065 has been described (Warpehoski et al. (1988) J. Med. Chem. 31 : 590-603).

In another series of analogs, the CPi moiety was replaced by a cyclopropabenzindole (CBI) moiety (Boger et al. (1990) J. Org. Chem. 55:5823-5833; Boger et al. (1991 ) BioOrg. Med. Chem. Lett. 1 :115-120). These compounds maintain the high in vitro potency of the parental drug, without causing delayed toxicity in mice. Like CC-1065, these compounds are alkylating agents that bind to the minor groove of DNA in a covalent manner to cause cell death. However, clinical evaluation of the most promising analogs, Adozelesin and Carzelesin, has led to disappointing results (Foster ef al. (1996) Investigational New Drugs 13:321-326; Wolff et al. (1996) Clin. Cancer Res. 2:1717- 1723). These drugs display poor therapeutic effects because of their high systemic toxicity.

The therapeutic efficacy of CC-1065 analogs can be greatly improved by changing the in vivo distribution through targeted delivery to the tumor site, resulting in lower toxicity to non-targeted tissues, and thus, lower systemic toxicity. In order to achieve this goal, conjugates of analogs and derivatives of CC-1065 with cell-binding agents that specifically target tumor cells have been described (US Patents; 5,475,092; 5,585,499; 5,846,545). These conjugates typically display high target-specific cytotoxicity in vitro, and exceptional anti-tumor activity in human tumor xenograft models in mice (Chari et al. (1995) Cancer Res. 55:4079-4084).

Recently, prodrugs of CC-1065 analogs with enhanced solubility in aqueous medium have been described (European Patent Application EP1832577). In these prodrugs, the phenolic group of the alkylating portion of the molecule is protected with a functionality that renders the drug stable upon storage in acidic aqueous solution, and confers increased water solubility to the drug compared to an unprotected analog. The protecting group is readily cleaved in vivo at physiological pH to give the corresponding active drug. In the prodrugs described in EP1832577, the phenolic substituent is protected as a sulfonic acid containing phenyl carbamate which possesses a charge at physiological pH, and thus has enhanced water solubility. In order to further enhance water solubility, an optional polyethylene glycol spacer can be introduced into the linker between the indolyl subunit and the cleavable linkage such as a disulfide group. The introduction of this spacer does not alter the potency of the drug.

Methods for synthesizing CC-1065 analogs that may be used in the cytotoxic conjugates of the present invention, along with methods for conjugating the analogs to cell binding agents such as antibodies, are described in detail in EP 832577 and U.S. Patent Nos. 5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618 and in U.S. Application US2003/01995 9 and US2003/0195365. Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin, tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs, dolastatin and dolastatin analogs are also suitable for the preparation of conjugates of the present invention. The drug molecules can also be linked to the antibody moiecules through an intermediary carrier molecule such as serum albumin. Doxarubicin and Danorubicin compounds, as described, for example, in U.S. Patent No. 6,630,579, may also be useful cytotoxic agents. In a particular embodiment of the invention, the at least one cytotoxic agent is the maytansine DM1 of formula (I). In another particular embodiment of the invention, the at least one cytotoxic agent is the maytansine DM4 of formula (II). These cytotoxic agents are conjugated to the cell binding agents, antibodies, epitope-binding fragments of antibodies as disclosed herein.

Linker

"Linker", as used herein, means a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches a polypeptide to a drug moiety.

The conjugates may be prepared by in vitro methods. In order to link a drug or prodrug to the cell binding agent, in particular to the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.

Conjugation of a cell binding agent as defined in the section "Cell binding agent' above, in particular an antibody of the invention, with cytotoxic agents as defined in the section "Cytotoxic agent" above may be made using a variety of bifunctional protein coupling agents including but not limited to /V-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4- (Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), /V-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyi (/V-maieimidomefhyf) cyciohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)-hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethyienediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1 ,5- difluoro-2,4-dinitrobenzene).

In a particular embodiment, said linker is selected from the group consisting of N- succinimidyi pyridyldithiobutyrate (SPDB), 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyi (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). The cell binding agent of the conjugate of the invention may be covalently linked via a cleavable or non-cleavable linker to the at least one cytotoxic agent.

The linker may be a "cleavable linker" facilitating release of the cytotoxic agent in the cell. For example, an acid-labile linker, a peptidase-sensitive (inker, an esterase labile linker, a photolabile linker or a disulfide-containing linker (see e.g. U.S. Patent No. 5,208,020) may be used. The linker may be also a "non-cleavable linker" (for example SMCC linker) that might lead to better tolerance in some cases.

Alternatively, a fusion protein comprising the cell binding agent as defined in the section "Cell binding agent' above, in particular the antibody of the invention and a cytotoxic polypeptide may be made, by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

The cell binding agents, in particular the antibodies, of the present invention may also be used in Dependent Enzyme Mediated Prodrug Therapy by conjugating the polypeptide to a prodrug-activating enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01 145) to an active anti-cancer drug (see, for example, WO88/07378 and U.S. Patent No. 4,975,278). The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to convert it into its more active, cytotoxic form. Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic fluorocytosine into the anticancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D- amino acid substituents; carbohydrate-cleaving enzymes such as O-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; P-lactamase useful for converting drugs derivatized with P-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. The enzymes can be covafently bound to the polypeptides of the invention by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.

According to a particular embodiment, in the conjugate of the invention, the cytotoxic agent may be a maytansinoid, in particular DM1 or DM4.

In such a conjugate, the cell binding agent as defined in the section "Cell binding agent above, in particular the antibody, is conjugated to said at least one cytotoxic agent by a linking group. In particular said linking group may be a non-cleavable linker, such as SPDB, sulfo-SPDB, or SMCC.

In a particular embodiment, said linker is N-succinimidyl pyridyldithiobutyrate (SPDB) and said cytotoxic agent is DM4. In another particular embodiment, said linker is 4-(Pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB) and said cytotoxic agent is DM4.

More particularly, the conjugate may be selected from the group consisting of:

) an antibody-SPDB-DM4 conjugate of formula (XVIII);

(XVIII)

Ab-SPDB-DM4 ii) an antibody-sulfo-SPDB-DM4 conjugate of formula (XIX);

Ab-SulfoSPDB-DM4 antibody-S CC-DM1 conjugate of formula (XX)

Ab-SMCC-DM1

In general, the conjugate can be obtained by a process comprising the steps of:

(i) bringing into contact an optionally-buffered aqueous solution of a cell-binding agent (e.g. an antibody according to the invention) with solutions of a linker and a cytotoxic compound;

(ii) then optionally separating the conjugate which was formed in (i) from the unreacted cell-binding agent.

The aqueous solution of cell-binding agent can be buffered with buffers such as, e.g. potassium phosphate, acetate, citrate or N-2-Hydroxyethylpiperazine-N'-2- ethanesulfonic acid (Hepes buffer). The buffer depends upon the nature of the cell-binding agent. The cytotoxic compound is in solution in an organic polar solvent, e.g. dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

The reaction temperature is usually comprised between 20°C and 40°C. The reaction time can vary from 1 to 24 hours. The reaction between the cell-binding agent and the cytotoxic agent can be monitored by size exclusion chromatography (SEC) with a refractometric and/or UV detector. If the conjugate yield is too low, the reaction time can be extended.

A number of different chromatography methods can be used by the person skilled in the art in order to perform the separation of step (ii): the conjugate can be purified e.g. by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatograhy (HIC), affinity chromatography, mixed-support chromatography such as hydroxyapatite chromatography, or high performance liquid chromatography (HPLC). Purification by dialysis or diafiltration can also be used.

As used herein, the term "aggregates" means the associations which can be formed between two or more cell-binding agents, said agents being modified or not by conjugation. The aggregates can be formed under the influence of a great number of parameters, such as a high concentration of cell-binding agent in the solution, the pH of the solution, high shearing forces, the number of bonded dimers and their hydrophobic character, the temperature (see Wang and Gosh (2008) J. Membr Sci. 318: 31 1-316, and references cited therein); note that the relative influence of some of these parameters is not clearly established. In the case of proteins and antibodies, the person skilled in the art will refer to Cromwell et al. (2006) AAPS Jounal 8 :E572- E579. The content in aggregates can be determined with techniques well known to the skilled person, such as SEC (see Walter er a/. (1993) Anal. Biochem. 212:469-480.

After step (i) or (ii), the conjugate-containing solution can be submitted to an additional step (iii) of chromatography, ultrafiltration and/or diafiltration.

The conjugate is recovered at the end of these steps in an aqueous solution.

In the embodiments of the invention wherein the cytotoxic agent is a maytansinoid, in order to link the maytansinoid to the cell binding agent as defined in the section "Cell binding agent" above, such as the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, the maytansinoid may comprise a linking moiety. The linking moiety contains a chemical bond that allows for the release of fully active maytansinoids at a particular site. Suitable chemical bonds are well known in the art and include disulfide bonds, acid labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds. Preferred are disulfide bonds.

The linking moiety also comprises a reactive chemical group. In an embodiment, the reactive chemical group can be covalently bound to the maytansinoid via a disulfide bond linking moiety.

Particular reactive chemical groups are A/-succinimidyl esters and N- sulfosuccinimidyl esters.

Particular maytansinoids comprising a linking moiety that contains a reactive chemical group are C-3 esters of maytansinol and its analogs where the linking moiety contains a disulfide bond and the chemical reactive group comprises a W-succinimidyl or /\/-sulfosuccinimidyl ester.

Many positions on maytansinoids can serve as the position to chemically link the linking moiety. For example, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group are all expected to be useful. However the C-3 position is preferred and the C-3 position of maytansinol is especially preferred.

While the synthesis of esters of maytansinol having a linking moiety is described in terms of disulfide bond-containing linking moieties, one of skill in the art will understand that linking moieties with other chemical bonds (as described above) can also be used with the present invention, as can other maytansinoids. Specific examples of other chemical bonds include acid labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds. The disclosure of U.S. Patent No. 5,208,020 teaches the production of maytansinoids bearing such bonds.

The synthesis of maytansinoids and maytansinoid derivatives having a disulfide moiety that bears a reactive group is described in U.S. Patent Nos. 6,441 ,163 and 6,333,410, and U.S. Application No. 2003/0055226.

The reactive group-containing maytansinoids, such as DM1 , are reacted with a cell binding agent as defined in the section "Cell binding agent" above, in particular with an antibody, such as the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, to produce cytotoxic conjugates. These conjugates may be purified by HPLC or by gel-filtration. Several excellent schemes for producing such cell binding agent-maytansinoid, in particular antibody-maytansinoid conjugates are provided in U.S. Patents No. 6,333,410, and 6,441 ,163 and U.S. Application Nos. 2003/0055226 and 2004/0001838.

In general, a solution of an antibody in aqueous buffer may be incubated with a molar excess of maytansinoids having a disulfide moiety that bears a reactive group. The reaction mixture can be quenched by addition of excess amine (such as ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate may then be purified by gel-filtration.

The number of maytansinoid molecules bound per antibody molecule can be determined by measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm. An average of 1-10 maytansinoid molecules/antibody molecule is preferred.

Maytansinoids may also be linked to cell binding agents using PEG linking groups, as set forth in U.S. Application No. 2004/0001838. These PEG linking groups are soluble both in water and in non-aqueous solvents, and can be used to join one or more cytotoxic agents to a cell binding agent. Exemplary PEG linking groups include hetero-bifunctional PEG linkers that bind to cytotoxic agents and cell binding agents at opposite ends of the linkers through a functional sulfhydryl or disulfide group at one end, and an active ester at the other end.

As a general example of the synthesis of a cytotoxic conjugate using a PEG linking group, reference is again made to U.S. Application No. 2004/0001838 for specific details. Synthesis begins with the reaction of one or more cytotoxic agents bearing a reactive PEG moiety with a cell-binding agent, resulting in displacement of the terminal active ester of each reactive PEG moiety by an amino acid residue of the cell binding agent, such as the humanized huDS6 antibody comprising a heavy chain of sequence SEQ ID NO: 9 and a light chain of sequence SEQ ID NO: 10, to yield a cytotoxic conjugate comprising one or more cytotoxic agents covalently bonded to a cell binding agent through a PEG linking group.

The conjugate molecules of the Invention may be formed using any techniques. In particular, the tomaymycin derivatives of the invention may be linked to an antibody or other cell binding agent as defined in the section "Cell binding agent' above via an acid labile linker, or by a photolabile linker. The derivatives can be condensed with a peptide having a suitable sequence and subsequently linked to a cell binding agent to produce a peptidase labile linker. The conjugates can be prepared to contain a primary hydroxyl group, which can be succinylated and linked to a cell binding agent to produce a conjugate that can be cleaved by intracellular esterases to liberate free derivative. Preferably, the derivatives are synthesized to contain a free or protected thiol group, and then one or more disulfide or thiol-containing derivatives are each covalently linked to the cell binding agent via a disulfide bond or a thioether link.

Numerous methods of conjugation are taught in U.S. Patent Nos. 5,416,064 and 5,475,092. The tomaymycin derivatives can be modified to yield a free amino group and then linked to an antibody or other cell binding agent via an acid labile linker or a photolabile linker. The tomaymycin derivatives with a free amino or carboxyl group can be condensed with a peptide and subsequently linked to a cell binding agent to produce a peptidase labile linker. The tomaymycin derivatives with a free hydroxyl group on the linker can be succinylated and linked to a cell binding agent to produce a conjugate that can be cleaved by intracellular esterases to liberate free drug. Most preferably, the tomaymycin derivatives are treated to create a free or protected thiol group, and then the disulfide- or thiol containing tomaymycin dimers are linked to the cell binding agent via disulfide bonds.

In one embodiment, monoclonal antibody- or cell binding agent-tomaymycin derivative conjugates are those that are joined via a disulfide bond, as discussed above, that are capable of delivering tomaymycin derivatives. Such cell binding conjugates are prepared by known methods such as by modifying monoclonal antibodies with succinimidyl pyridyl-dithiopropionate (SPDP) (Carlsson et af. (1978) Biochem. J. 173:723- 737). The resulting thiopyridyl group is then displaced by treatment with thiol-containing tomaymycin derivatives to produce disulfide linked conjugates. Alternatively, in the case of the aryldithio-tomaymycin derivatives, the formation of the cell binding conjugate is effected by direct displacement of the aryl-thiol of the tomaymycin derivative by sulfhydryl groups previously introduced into antibody molecules. Conjugates containing 1 to 10 tomaymycin derivative drugs linked via a disulfide bridge are readily prepared by either method.

More specifically, a solution of the dithio-nitropyridyl modified antibody at a concentration of 2.5 mg/ml in 0.05 M potassium phosphate buffer, at pH 7.5 containing 2 mM EDTA is treated with the thiol-containing tomaymycin derivative (1.3 molar eq./dithiopyridyl group). The release of thio-nitropyridine from the modified antibody is monitored spectrophotometrically at 325 nm and is complete in about 16 hours. The antibody-tomaymycin derivative conjugate is purified and freed of unreacted drug and other low molecular weight material by gel filtration through a column of Sephadex G-25 or Sephacryl S300. The number of tomaymycin derivative moieties bound per antibody molecule can be determined by measuring the ratio of the absorbance at 230 nm and 275 nm. An average of 1-10 tomaymycin derivative molecules/antibody molecule can be linked via disulfide bonds by this method.

The effect of conjugation on binding affinity towards the antigen-expressing cells can be determined using the methods previously described by Liu et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:8618-8623. Cytotoxicity of the tomaymycin derivatives and their antibody conjugates to cell lines can be measured by back-extrapolation of cell proliferation curves as described in Goldmacher et al. ( 985) J. Immunol. 135:3648-3651. Cytotoxicity of these compounds to adherent cell lines can be determined by clonogenic assays as described in Goldmacher ef al. (1986) J. Cell Biol. 102:1312-1319.

Drug-to-antibody ratio

According to an embodiment, the conjugate according to the invention is characterised by a "drug-to-antibody ratio" (or "DAR") as measured by DAR UV ranging from 1 to 10, for instance from 2 to 5, in particular from 3 to 4, more particularly of 3.5. This is generally the case of conjugates including maytansinoid molecules.

This DAR number can vary with the nature of the cell binding agent, in particular the antibody, and of the drug (i.e. the cytotoxic agent) used along with the experimental conditions used for the conjugation (like the ratio cytotoxic agent/cell binding agent, the reaction time, the nature of the solvent and of the co-solvent if any). Thus the contact between the cell binding agent and the cytotoxic agent leads to a mixture comprising several conjugates differing from one another by different drug-to-antibody ratios; optionally the naked cell binding agent; optionally aggregates. The DAR that is determined is thus a mean value.

A method which can be used to determine the DAR, herein called DAR UV, consists in measuring spectrophotometrically the ratio of the absorbance of a solution of substantially purified conjugate at λ₀ and 280 nm. 280 nm is a wavelength generally used for measuring protein concentration, such as antibody concentration. The wavelength A_D is selected so as to allow discriminating the drug from the antibody, i.e. as readily known to the skilled person, h_D is a wavelength at which the drug has a high absorbance and A_D is sufficiently remote from 280 nm to avoid substantial overlap in the absorbance peaks of the drug and antibody. A_D may be selected as being 252 nm in the case of maytansinoid molecules. A method of DAR calculation may be derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science:

The absorbances for the conjugate at λ₀ (A_AD) and at 280 nm (A₂₈o) are measured using a classic spectrophotometer apparatus (allowing to calculate the "DAR parameter"). The absorbances can be expressed as follows:

AID = (c_D x ε_ολΏ) + (¾ X ε^)

-^280 ⁼ (^CD ^x ¾2SQ) + (¾ ^x ¾28o)

wherein:

c_D and c_A are respectively the concentrations in the solution of the drug and of the antibody

8DAD and 8p28o are respectively the molar extinction coefficients of the drug at λ₀ and 280 nm

ε_Αλϋ and ε_Α28ο are respectively the molar extinction coefficients of the antibody at h_D and 280 nm.

Resolution of these two equations with two unknowns leads to the foliowing equations:

<¾ = [(¾280 X Am) - (¾LD X Λ₂8θ)]/ί(¾λΒ X ¾23θ3 ~

C_A = [4₂80 ^— (¾ ^x ¾28o)] ¾Z80

The average DAR is then calculated from the ratio of the drug concentration to that of the antibody: DAR = c_D / c_A.

In a particular embodiment, h_D is 252 nm.

Accordingly, in that particular embodiment, the conjugate is characterized by a drug-to-antibody ratio (DAR) ranging from 3 to 4, in particular of 3.5, the DAR being calculated from the ratio of the cytotoxic agent concentration (c_D) to that of the cell binding agent (c_A); wherein

^CD ~ [£¾2BQ ^X

^X ¾28o) ^— (¾252 ^X ¾2Βθ)1 and SD252 and SD28O are respectively the molar extinction coefficients of the cytotoxic agent at 252 nm and 280 nm,

SA252 and 8A28O are respectively the molar extinction coefficients of the cell binding agent at 252 nm and 280 nm, and

A₂₅₂ and A₂₈Q are respectively the absorbances for the conjugate at 252 nm (A252) and at 280 nm (A₂₈o), measured using a classic spectrophotometer apparatus.

Treatment

The inventors demonstrated that a patient suffering from cancer, in particular from breast cancer, lung cancer or bladder cancer, more particularly of breast cancer, showed at least non-progression of tumor or tumor regression but with low ocular toxicity, when he/she was administered with huDS6-DM4, over at least two cycles of administration, at a dose corresponding to an administration schedule of at least 60 mg/m² repeated as a new cycle every week.

The present invention thus concerns a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent" herein above, for use in a method for treating cancer, wherein the method comprises at least two cycles and wherein, for each cycle, the conjugate is administered at least two times during each cycle at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle.

The present invention also concerns the use of a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent" herein above, for the manufacture of a medicament intended to treat cancer, wherein said conjugate is administered over at least two cycles and wherein, for each cycle, said conjugate is administered at least two times during each cycle at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle.

The present invention also concerns a method for treating cancer in a patient in need thereof, said method comprising at least two cycles and wherein, for each cycle, a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' herein above, is administered to said patient in need thereof at least two times during each cycle at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

By the term "treating cancer" as used herein is meant the inhibition of the growth of malignant cells of a tumor and/or the progression of metastases from said tumor. Accordingly, such treatment can lead to the non-progression of the tumor, in particular over 12 weeks. Such treatment can also lead to the regression of tumor growth, i.e., the decrease in size of a measurable tumor. In a particular embodiment, such treatment leads to a partial regression of the tumor or metastasis. In another particular embodiment, such treatment leads to the complete regression of the tumor or metastasis.

According to the invention, the term "patient" or "patient in need thereof" is intended for a human or non-human mammal affected or likely to be affected with a malignant tumor.

In certain embodiments, Response Evaluation Criteria in Solid Tumors (RECIST) parameters can be used to assess the effectiveness of a cancer treatment. RECIST was jointly created by the National Cancer Institute of the United States, the National Cancer Institute of Canada Clinical Trials Group and the European Organisation for Research and Treatment of Cancer. It is designed to provide a clear set of criteria to evaluate the progression, stabilization or responsiveness of tumors.

A lesion is a localized change in a tissue or an organ. Tumors are types of lesions.

In the terminology of RECIST, "lesion" is generally used instead of "tumor." Target lesions are lesions that have been specifically measured. Non-target lesions are lesions whose presences have been noted, but whose measurements have not been taken.

Response assessment and evaluation criteria for target lesions are as follows: -Complete Response (CR) signifies that all target lesions have disappeared during the course of treatment;

-Partial Response (PR) signifies that decreases of at least 30% have been noted in the lesion that has the largest diameter (LD); -Stable Disease (SD) signifies that there has been no significant decrease or increase in the size of target lesions, based on the smallest sum LD; and

-Progressive Disease (PD) signifies that there has been an increase of at least 20% in the sum of the LD of targeted lesions.

Response assessment and evaluation criteria for non-target lesions are as follows:

-Complete Response (CR) signifies the disappearance of ail non-target lesions;

-Incomplete Response / Stable Disease (SD) signifies the continued presence of one or more non-target lesions and/or maintenance of tumor marker level above the normal limits; and

Progressive Disease (PD) signifies the appearance of at least one new lesion, or the increasing size of at least one existing non-target lesion.

CT and MRI are reproducible methods to measure target lesions selected for response assessment. Conventional CT and MRI is typically performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT is typically performed using a 5 mm contiguous reconstruction algorithm. This applies to tumors of the chest, abdomen and pelvis. Head and neck tumors and those of extremities usually require specific protocols.

Lesions on chest X-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, CT is preferable.

Ultrasound (US) is a possible alternative to clinical measurements of superficial palpable lymph nodes, subcutaneous lesions and thyroid nodules. US might also be useful to confirm the complete disappearance of superficial lesions usually assessed by clinical examination.

The utilization of endoscopy and laparoscopy for objective tumor evaluation can be useful in confirming complete pathological response when biopsies are obtained.

Cytology and histology can be used to differentiate between PR and CR in rare cases (e.g., after treatment to differentiate between residual benign lesions and residual malignant lesions in tumor types such as germ cell tumors).

In a particular embodiment, the patient to be treated may have been previously treated with other anti-cancer treatments. In particular, the patient to be treated may have been previously treated with an oxaliplatin-, cisplatin-, a carboplatin-, and/or a paclitaxel- docetaxel-based regimen.

The conjugate of the invention is administered at a therapeutically effective amount, i.e. a sufficient amount of the conjugate to treat said cancer disease, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the conjugate of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific conjugate employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific conjugate employed; the duration of the treatment; drugs used in combination or coincidental with the specific conjugate employed; and like factors well known in the medical arts.

In certain embodiments, a dose of a conjugate of the invention that is administered to a patient is about 10 mg/m², about 15 mg/m², about 20 mg/m², about 25 mg/m², about 30 mg/m², about 35 mg/m², about 40 mg/m², about 45 mg/m², about 50 mg/m², about 55 mg/m², about 60 mg/m², about 65 mg/m², about 70 mg/m², about 75 mg/m², about 80 mg/m², about 85 mg/m², about 90 mg/m², about 95 mg/m², about 100 mg/m², about 105 mg/m², about 1 10 mg/m², about 1 15 mg/m², about 120 mg/mm², about 125 mg/m², about 130 mg/m², about 135 mg/m², about 140 mg/m², or about 145 mg/m².

In certain embodiments, dosing occurs in two, three, four, five, six, seven, eight, nine or ten or more dosing cycles. In certain embodiments, a dose is administered to a patient in a dosing cycle of once weekly, once every two weeks (q2w), once every three weeks (q3w), or once every four weeks (q4w). In certain embodiments, a dose is administered to a patient two or more times during a q2w dosing cycle, a q3w dosing cycle or a q4w dosing cycle. In certain embodiments, a dose is administered once every week of a q2w dosing cycle, a q3w dosing cycle or a q4w dosing cycle. In certain embodiments, a dose is administered at day 1 (D1 ) and at day 8 (D8) of a q2w dosing cycle. In certain embodiments, a dose is administered at day 1 (D1 ) and at day 8 (D8) of a q3w dosing cycle. In a particular embodiment, said therapeutically effective amount of the conjugate administered to the patient is a dose corresponding to that of an administration schedule of dose ranging from 60 to 80 mg/m² every week of each cycle of the treatment method. In a particular embodiment, said therapeutically effective amount of the conjugate administered to the patient is a dose corresponding to that of an administration schedule of 60 mg/m² every week of each cycle of the treatment method. In an embodiment, the cycle is a period of three weeks and said therapeutically effective amount of the conjugate administered to the patient is a dose of 90 mg/m² at day 1 of each cycle of the treatment method and a further dose of 90 mg/m² at day 8 of each cycle of the treatment method.

It should further be noted that said therapeutically effective amounts are also safe doses regarding in particular eyes disorders.

In another particular embodiment, the cycle is a period of three weeks and said therapeutically effective amount of the conjugate administered to the patient is a dose of 120 mg/m² at day 1 of each cycle of the treatment method and a further dose of 120 mg/m² at day 8 of each cycle of the treatment method.

According to the invention, the conjugate is administered repeatedly during the treatment method according to a protocol including at least two cycles, the conjugate being administered as least two times during each cycle.

In a particular embodiment, the number of cycles is of 2. In a more particular embodiment, the number of cycles is 3, 4 or 6.

In a particular embodiment, the cycle is a period of two or three weeks.

In a more particular embodiment, the cycle is a period of two weeks, and, for each cycle, the conjugate is administered at a dose of 120 mg/m² at day 1 of the cycle.

The conjugate of the invention may be administered in the form of a pharmaceutical composition including pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions including the conjugate of the invention and the route of administration naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc.

The conjugates of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like. In a particular embodiment, the conjugate of the invention is administered intravenously.

In particular, the pharmaceutical compositions including the conjugate of the invention may contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

To prepare pharmaceutical compositions, an effective amount of the conjugate of the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, stabilizing agents, cryoprotectants or antioxidants. The prevention of the action of microorganisms can be brought about by antibacterial and antifungal agents. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media w ich can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCI solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In a particular embodiment, the conjugate of the invention is suitably administered intravenously at a rate of 1 mL/min for 30 min and then increased to a maximal rate of 2 mL/min in the absence of hypersensitivity reactions.

Cancers to be treated according to the invention include malignancy of any type, in particular solid tumors, for example breast cancer, lung cancer and more particularly non- small cells lung cancer (NSCLC), and bladder cancer.

In one embodiment, the cancer to be treated according to the invention is a CA6- positive tumor. In particular, the tumor is qualified by immunohistochemistry (IHC) using a scale of 0 to 3+. A score of 0 (i.e., no or little staining in < 10% of cells) or 1 + (i.e., faint, partial staining in > 10% of cells) is considered negative for CA-6. A score of 2+ (i.e., weak to moderate, complete staining in > 30% of cells) or 3+ (i.e., strong, complete membrane staining in > 30% of cells) is considered positive for CA-6. More particularly, the immunohistochemistry (IHC) assay utilizes the murine monoclonal antibody produced by the hybridoma cell line DS6 deposited at the American Type Culture Collection (ATCC) with the deposit number PTA-4449.

In a further embodiment, the cancer to be treated is a breast cancer, more particularly a triple negative breast cancer (TNBC), not positive to receptors for estrogen, progesterone or HER2.

The conjugate of the invention may be administered in combination with a medication to prevent or control keratitis, in particular with a keratitis prophylactic or curative ocular composition.

In a particular embodiment, a prophylactic treatment for preventing eye disorders, a prophylactic treatment for preventing keratitis, is administered prior to, simultaneously with, or immediateiy after each administration of the conjugate.

The invention further concerns a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent' herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent" herein above, for use in a method for treating cancer,

- prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders, more particularly for preventing keratitis, is administered.

The present invention also concerns the use of a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent herein above, for the manufacture of a medicament intended to treat cancer, wherein said conjugate is administered over at least two cycles, a cycle being a period of three weeks, and wherein, for each cycle:

- the conjugate is administered at a dose of 90 mg/m² at day 1 of the cycle and at a dose of 90 mg/m² at day 8 of the cycle, and

- prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders, more particularly for preventing keratitis, is administered. The present invention also concerns the use of a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 ( UC1 ) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' herein above, for the manufacture of a medicament intended to treat cancer, wherein said conjugate is administered over at least two cycles, a cycle being a period of three weeks, and wherein, for each cycle:

The present invention also concerns the use of a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent" herein above, for the manufacture of a medicament intended to treat cancer, wherein said conjugate is administered over at least two cycles, a cycle being a period of two weeks, and wherein, for each cycle:

- the conjugate is administered at a dose of 120 mg/m² at day 1 of the cycle, and

The present invention further concerns a method for treating cancer in a patient in need thereof, said comprising at least two cycles, a cycle being a period of three weeks, and, for each cycle:

- a conjugate comprising (i) a cell binding agent which binds to the human mucin-1

(MUC1) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' herein above, is administered to said patient in need thereof at a dose of 90 mg/m² at day 1 of the cycle and at a dose of 90 mg/m² at day 8 of the cycle, and

- prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders, more particularly for preventing occurrence of keratitis, is administered to said patient. The present invention also concerns a method for treating cancer in a patient in need thereof, said comprising at least two cycles, a cycle being a period of three weeks, and, for each cycle:

- a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent" herein above, linked to

(ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' herein above, is administered to said patient in need thereof at a dose of 120 mg/m² at day 1 of the cycle and at a dose of 120 mg/m² at day 8 of the cycle, and

- prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders, more particularly for preventing occurrence of keratitis, is administered to said patient.

The present invention further concerns a method for treating cancer in a patient in need thereof, said comprising at least two cycles, a cycle being a period of two weeks, and, for each cycle:

(MUC1 ) glycoprotein, as defined in the section "Cell binding agent' herein above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' herein above, is administered to said patient in need thereof at a dose of 120 mg/m² at day 1 of the cycle, and

By "prophylactic treatment for preventing eye disorders" is meant herein any therapy preventing or alleviating ocular adverse events due to an anti-tumoral treatment administered to a subject suffering from cancer. Ocular adverse events due to anti- tumoral treatments, due in particular to tubulin binders cytotoxic drugs, are well-known from the skilled person. Such ocular adverse events or eye disorders include keratitis, blurred vision, abnormal sensation in eye, dry eye, increased lacrimation, photophobia, corneal deposits, blepharitis, eye pain, astigmatism and conjunctival hyperae ia.

In a particular embodiment, said prophylactic treatment is for preventing keratitis.

Prophylactic treatments for preventing eye disorders, in particular for preventing keratitis, are known from the skilled person. In a particular embodiment, said prophylactic treatment for preventing eye disorders, in particular for preventing keratitis, comprises administration of a vasoconstrictor and/or a corticosteroid, and/or use of a cold eye mask. As used herein, a "cold eye mask" refers to a cold pack that is designed for use in the eye area. In certain embodiments of the invention, a cold compress can be used, e.g., a continuous cold therapy device, or a cold pack comprising a re-freezable substance (e.g., refrigerant or water) can be used. A variety of cold packs are commercially available and would be suitable for use.

By "vasoconstrictor" is meant herein any compound inducing vasoconstriction at the corneal level, i.e. inducing the narrowing of blood vessels resulting from contraction of the muscular wall of the vessels. Examples of vasoconstrictors are well-known from the skilled person and include antihistamines, caffeine, methylphenidate, oxymetazoline, phenylephrine (or neosynephrine), propylhexedrine, pseudoephedrine and tetrahydrozoline. In a particular embodiment, the vasoconstrictor is neosynephrine, more particularly neosynephrine in the form of eye drops.

The inventors demonstrated that a particularly effective prevention of ocular adverse events, such as keratitis, could be obtained by administering the vasoconstrictor, in particular neosynephrine, at the time of the conjugate administration.

Accordingly, in a particular embodiment, the vasoconstrictor, in particular neosynephrine, more particularly at less than 2.5%, still particularly in the form of drops, is administered at the time of the conjugate administration, in particular at a dose of 3 drops.

By "corticosteroid" is meant herein any compound belonging to the corticosteroid class of steroid hormones. Examples of corticosteroids are well-known from the skilled person and include corticosteroids of the hydrocortisone type, such as hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone and prednisolone; acetonides such as triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide and halcinonide; corticosteroids of the betamethasone type such as betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate and fluocortolone; halogenated esters of corticosteroids such as hydrocortisone-17-valerate, halometasone, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluorocortolone pivalate and fluprednidene acetate; and labile prodrug esters of corticosteroids such as hydrocortisone- 17-butyrate, hydrocortisone-17-aceponate, hydrocortisone-17-buteprate, ciclesonide and prednicarbate. In a particular embodiment, the corticosteroid is dexamethasone, more particularly dexamethasone in the form of ocular gel.

The inventors demonstrated that a particularly effective prevention of ocular adverse events, such as keratitis, could be obtained by administering the corticosteroid, in particular dexamethasone, more particularly in the form of ocular gel, three times a day starting the day of the conjugate administration and for two days.

Accordingly, in a particular embodiment, the corticosteroid, in particular dexamethasone, more particularly at 0.16%, still particularly in the form of ocular gel, is administered three times a day starting the day of the conjugate administration and for 2 days.

The inventors also showed that a particularly effective prevention of ocular adverse events, such as keratitis, could be obtained when a cold eye mask is used during the whole administration of the conjugate.

Accordingly, in a particular embodiment, a cold eye mask is used during the whole administration of the conjugate.

The inventors also showed that the most effective prevention of ocular adverse events, such as keratitis, could be obtained when the vasoconstrictor, the corticosteroid and the cold eye mask were used together.

Accordingly, in a particular embodiment, (i) the vasoconstrictor, in particular neosynephrine, more particularly at less than 2.5%, still particularly in the form of drops, is administered at the time of the conjugate administration, in particular at a dose of 3 drops, (ii) the corticosteroid, in particular dexamethasone, more particularly at 0.16%, still particularly in the form of ocular gel, is administered three times a day starting the day of the conjugate administration and for 2 days, and (iii) a cold eye mask is used during the whole administration of the conjugate.

The present invention thus also concerns a combination of a vasoconstrictor, as defined above, and a corticosteroid, as defined above, for simultaneous, separate or sequential use for preventing ocular adverse events, in particular keratitis, in a patient suffering from cancer and under anti-cancerous therapy, in particular with the conjugate of the invention, optionally in combination with a cold eye mask. The present invention also concerns a vasoconstrictor, as defined above, for use in combination with a corticosteroid, as defined above, and optionally a cold eye mask, in a method for preventing ocular adverse events, in particular keratitis, in a patient suffering from cancer and under anti-cancerous therapy, in particular with the conjugate of the invention.

The present invention also concerns a corticosteroid, as defined above, for use in combination with a vasoconstrictor, as defined above, and optionally a cold eye mask, in a method for preventing ocular adverse events, in particular keratitis, in a patient suffering from cancer and under anti-cancerous therapy, in particular with the conjugate of the invention.

The invention also concerns a method for preventing ocular adverse events, in particular keratitis, in a patient suffering from cancer and under anti-cancerous therapy, in particular with the conjugate of the invention, comprising simultaneously, separately or sequentially administering to said patient a vasoconstrictor, as defined above, and a corticosteroid, as defined above, the patient further preferably wearing a cold eye mask.

In a particular embodiment, the vasoconstrictor, in particular neosynephrine, more particularly at less than 2.5%, still particularly in the form of drops, in particular at a dose of 3 drops, is administered at the time of the administration of the anti-cancerous therapy, in particular of the conjugate of the invention.

In another particular embodiment, the corticosteroid, in particular dexamethasone, more particularly at 0.16%, still particularly in the form of ocular gel, is administered three times a day starting the day of the administration of the anti-cancerous therapy, in particular of the conjugate of the invention, and for 2 days.

In a particular embodiment, a cold eye mask is used during the whole administration of the anti-cancerous therapy, in particular of the conjugate of the invention.

In still a particular embodiment, (i) the vasoconstrictor, in particular neosynephrine, more particularly at less than 2.5%, still particularly in the form of drops, in particular at a dose of 3 drops, is administered at the time of the administration of the anti-cancerous therapy, in particular of the conjugate of the invention, (ii) the corticosteroid, in particular dexamethasone, more particularly at 0.16%, still particularly in the form of ocular gel, is administered three times a day starting the day of the administration of the anti-cancerous therapy, in particular of the conjugate of the invention, and for 2 days, and (iii) a cold eye mask is used during the whole administration of the anti-cancerous therapy, in particular during administration of the conjugate of the invention.

The invention also concerns a conjugate comprising (i) a cell binding agent which specifically binds to tumor cells, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the cytotoxic agent is a tubulin binder; and

- administration of an ocular vasoconstrictor,

- administration of an ocular corticosteroid, and

- the use of a cold eye mask.

In a particular embodiment said cell binding agent binds an antigen on the surface of the tumor cells that is also naturally present in the eye, for example in the corneal epithelium, the basal cells of the conjunctiva, and/or the cells of the lacrimal gland.

In a particular embodiment, said cell binding agent is a monoclonal antibody, and said tubulin binder is a maytansinoid such as DM1 or DM4.

In a particular embodiment, said prophylactic treatment consists in two or more of:

- administration in each eye of three drops of 2.5% neosynephrine,

- use of a cold eye mask during the administration of the conjugate.

More particularly, said prophylactic treatment consists in the combination of administration in each eye of three drops of 2.5% neosynephrine; administration of an ocular gel of dexamethasone (0.16%) three times a day for two days starting the day of the administration of the conjugate, and the use of cold eye mask during the administration of the conjugate.

The present invention further concerns an article of manufacture comprising:

a) a packaging material;

b) a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, as defined in the section "Cell binding agent' above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' above; and c) a label or package insert contained within said packaging material indicating that, for at least two cycles, said conjugate is administered at least two times during each cycle and, for each cycle, at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle.

In an embodiment the label or package insert indicates that for at least two cycles of three weeks, said conjugate is administered, for each cycle, at a dose of 90 mg/m² at day 1 (D1) of the cycle and at a dose of 90 mg/m² at day 8 (D8) of the cycle.

In an alternative embodiment the label or package insert indicates that for at least two cycles of three weeks, said conjugate is administered, for each cycle, at a dose of 120 mg/m² at day 1 of the cycle and at a dose of 120 mg/m² at day 8 of the cycle.

The present invention also concerns an article of manufacture comprising:

a) a packaging material;

b) a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent" above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent' above; and c) a label or package insert contained within said packaging material indicating that, for at least two cycles of three weeks, said conjugate is administered, for each cycle, at a dose of 120 mg/m² at day 1 of the cycJe and at a dose of 120 mg/m² at day 8 of the cycle, and prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered.

The present invention also concerns an article of manufacture comprising:

a) a packaging material;

b) a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, as defined in the section "Cell binding agent" above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent above; and c) a label or package insert contained within said packaging material indicating that, for at least two cycles of three weeks, said conjugate is administered, for each cycle, at a dose of 90 mg/m² at day 1 of the cycle and at a dose of 90 mg/m² at day 8 of the cycle, and prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered.

The present invention further concerns an article of manufacture comprising:

a) a packaging material; b) a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1) glycoprotein, as defined in the section "Cell binding agent" above, linked to (ii) at least one cytotoxic agent, as defined in the section "Cytotoxic agent" above; and c) a label or package insert contained within said packaging material indicating that, for at least two cycles of two weeks, said conjugate is administered, for each cycle, at a dose of

120 mg/m² at day 1 of the cycle and optionally prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered.

Brief description of the sequences

SEQ Sequence Description

ID

NO:

1 SYNMH CDR1-H of huDS6

2 YIYPGNGATNYNQKFQG CDR2-H of huDS6

3 GDSVPFAY CDR3-H of huDS6

4 SAHSSVSFMH CDR1-L of huDS6

5 STSSLAS CDR2-L of huDS6

6 QQRSSFPLT CDR3-L of huDS6

7 QAQLVQSGAEWKPGASVKMSCKASGYTFTSYN Heavy chain variable MHWVKQTPGQGLEWIGYIYP region of huDS6

GNGATNYNQKFQGKATLTADPSSSTAYMQISSLTS EDSAVYFCARGDSVPFAYW GQGTLVTVSA

8 EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWF Light chain variable region QQKPGTSPKLWIYSTSSLAS of huDS6

GVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQ RSSFPLTFGAGTKLELKR

9 QAQLVQSGAEWKPGASVK SCKASGYTFTSYN Heavy chain of huDS6 HWVKQTPGQGLEWIGYIYPGNGATNYNQKFQG

KATLTADPSSSTAYMQISSLTSEDSAVYFCARGDS

VPFAYWGQGTLVTVSAASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

FPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP

SVFLFPPKPKDTL ISRTPEVTCWVDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT

VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS

PGK

10 EIVLTQSPATMSASPGERVTITCSAHSSVSF HWF Light chain of huDS6

QQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTS

YSLTISS EAEDAATYYCQQRSSFPLTFGAGTKLE

LKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYP

REAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGEC

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following example, which is included for purposes of illustration only and is not intended to be limiting.

Example

A First-in-human (FIH) study was conducted to assess the safety, dose limiting toxicities (DLTs)/ recommended dose (RD) and pharmacokinetics (PK), following huDS6- DM4 intravenous administration given: every 3 weeks (10-240 mg/m²); every 2 weeks (120 mg/m²); and at D1 , D8 every 3 weeks (90 mg/m²).

Pre-screening on CA6 expression was performed on 1067 patients: 60% were CA6 positive (>1 %) and 24% were CA6 positive (>30% tumor cells with intensity 2+/3+). CA6 antigen expression was assessed by immunohistochemistry (IHC) on paraffin embedded tumor tissue. The IHC assay uses the murine monoclonal antibody DS6 produced by the hybridoma cell line deposited at the American Type Culture Collection (ATCC) with the deposit number PTA-4449.

1 14 patients with heavily pretreated solid tumors expressing mostly CA6 in≥30% tumor cells with intensity 2+/3+ by immunohistochemistry were enrolled.

In the dose escalation phase, 34 heavily pre-treated patients were enrolled, with CA6 positive solid tumors including pancreas, ovarian and breast tumors. Dose limiting toxicities (DLTs) were observed in 2 patients out of 8 treated at the highest dose of 240 mg/m² every three weeks (q3w) [grade 3 diarrhea (cycle 1 ) & grade 3 keratitis (cycle 2)]. Overall, huDS6-DM4 was well tolerated with late occurrence of reversible corneal adverse event (known with previous maytansin-ADCs), few hematological events and some peripheral neuropathies, observed from the dose of 150 mg/m² every 3 weeks.

The initial recommended dose (RD) of 190 mg/m² q3w was used in an expansion phase (in breast, ovarian and pancreatic cancer patients). However, a high incidence of keratopathy was observed mainly at cycle 2. A decision was made to decrease the dose to 150 mg/m² q3w, at which dosage the corneal toxicity decreased but, unfortunately, the activity was impacted.

Two new alternative schedules were tested in the ongoing phase I study: 90 mg/m² at day 1 and day 8 every 3 weeks (D1 D8 q3w) and 120 mg/m² every two weeks (q2w). These alternative schedules were selected to increase benefit/risk ratio by maintaining lower C_max but similar AUC over 12 weeks and higher C_min compared to the 190 mg/m² schedule.

Go no Go criteria based on efficacy and safety parameters were defined for the selection of the RD: ≤25% of ocular events leading to dose modification (delay and/or dose reduction) or drug discontinuation and ≥ 15% overall response rate (ORR) or probability of non-progression at 12 weeks in≥ 50%. A total of 33 patients were included in the two alternative schedules (17 and 16 patients, in 90 mg/m² D1 D8 q3w and 120 mg/m² q2w, respectively).

The characteristics of the enrolled patients are summarized in the Table 1 below.

Table 1

The safety results of this study are resumed in the above Tables 2 and 3.

All results presented below are reported on the patients treated at the recommended doses in the expansion part (150 mg/m² Q3w and 190 mg/m² Q3w and the two alternative schedules).

Laboratory tests

Hematological n(%) 90 mg/m² 120mg/m² 150 mg/m2 190 mg/m 2

D D8 q3w q2w q3w q3w N= N=16 N=33 N=23

Anemia

All grades 13 (76%) 11 (69%) 26 (79%) 9 (83%)

Grade 3 0/17 0/16 1/33 0/23

White Blood cell decreased

All grades 7 (41%) 3 (19%) 13 (39%) (48%)

Grade 3 0/17 0/16 0/33 0/23

Neutrophil count decreased

All grades 5 (29%) 1 (6%) 5 ( 5%) 4 ( %)

Grade 3 0/17 0/16 1/33 1/23

Platelet count decreased

All grades 3 (18%) 2 (13%) 0 (30%) 11 (48%)

Grade 3 0/17 0/16 0/33 0/23 Liver and renal n(%) 90 mg/m² 120mg/m² 150 mg/m 2 190 mg/m2

D1D8q3w q2w q3w q3w

N=17 N=16 N=33 N=23

ALT increased

Alt grades 10 (59%) 8 (50%) 16 (48%) 6 (70%)

Grade 3 1/17 1/16 1/33 1/23

AST increased

All grades 13 (76%) 11 (69%) 26 (79%) 21 (91 %)

Grade 3 1/17 2/16 4/33 1/23

Alkaline phosphatase increased

All grades 8 (47%) 9 (56%) 21 (64%) (48%)

Grade 3 1/17 2/16 5/33 3/23

Blood bilirubin increased

All grades 0/17 3 (19%) 7 (21%) 3 (13%)

Grade 3 0/17 1/16 3/33 0/23

Creatinine increased

All grades 4 (24%) 4 (25%) 7 (21%) 3 (13%)

Grade 3 0/17 0/16 0/33 0/23

No grade 4 laboratory tests abnormality was observed. Hematological toxicity was limited and no difference was observed between the q3w schedules except for platelets. No severe liver or renal function tests abnormalities related to treatment was observed.

Treatment-emergent adverse events

Table 3

Almost all patients presented at least one adverse event. 48% had grade 3 adverse events and 12% has a related grade 3 adverse event. 7% had an adverse event leading to permanent treatment discontinuation (including 2 patients with keratitis). Ocular corneal events were the main adverse event and were globally dose/schedule dependent. The main ocular corneal event was keratitis, with a higher incidence at 190 mg/m² q3w (65%), similar incidence (about 35%) at 150 mg/m² q3w and 90 mg/m² D1 D8 q3w and a lower incidence (13%) at 120 mg/m^z q2w.

As shown in Table 4 below, eye disorders leading to dose modification represented 68.2% (28/41) of all TEAEs leading to dose modification.

Table 4

Eye disorders leading to dose modification was observed in 31% of patients treated with high doses:

- Highest incidence (61%) at 190 mg/m² q3w;

- Intermediate (approximately 24-29%) for 150 mg/m² q3w and 90mg/m² D1D8 q3w; and

- Lowest incidence (6%) at 120 mg/m² q2w.

Keratitis represented 93% of the eye disorders leading to dose modification.

Ocuiar adverse events are summarized in Table 5 beiow. Table 5

Eye disorders were observed in 55% of treated patients. More particularly grade 3 eye disorders were observed in 11% (18% in 90 mg/m² D1 D8 q3w).

Keratitis was observed in 38% of patients, with a higher incidence (>60%) at 190 mg/m² q3w and a lower incidence at 20 mg/m² q2w.

Blurred vision was observed in 17% of patients.

A total of 10 patients (6 in the 90 mg/m² D1 D8 q3w schedule and 4 in the 120 mg/m² q2w schedule) received primary ocular prophylaxis and no eye disorders leading dose modification was observed. Only 1 patient presented a keratitis at 90 mg/m² D1 D8 q3w. In this latter alternative schedule, the incidence of ocular corneal events (mainly keratitis) being the main AE, was lower or similar to 150 mg/m² q3w and lower to 190 mg/m² q3w.

This prophylactic treatment was a preventive (eg, primary) prophylaxis for keratitis, around the time of each infusion:

- Vasoconstrictor (≤ 2.5%; 3 drops) (i.e. neosynephrine)

- Corticosteroid ocular gel (Sterdex/dexamethasone 0.16%) tid (morning, midday and night), starting the day of infusion, and for 2 days - Cold eye mask (unless patient does not tolerate it) from start of infusion till its end.

Other overall TEAEs were fatigue (32.6%), peripheral neuropathy (31.6%), Gl disorders [(nausea (29%), abdominal pain (26%), diarrhea (25%)] and neutropenia (2.6%). Low grade liver and renal abnormalities were noted.

109 patients on the 114 were evaluated for ORR. As shown in Table 6, similar ORR was obtained at 90 mg/m² D1 D8 q3w (2 partial responses / 15) and 190 mg/m² q3w (1 complete response and 2 partial responses / 23).

Table 6

Tumor regression was noted in about 60% patients at 190 mg/m² q3w and 90 mg/m² D D8 q3w and about 35% patients at 150 mg/m² q3w and 120 mg/m² q2w.

Considering all tested doses, one complete response (ovary), 8 partial responses (3 breast, 2 ovary, 1 NSCLC, 1 cervix and 1 bladder) and 39% stable disease were observed. Similar objective response rate (ORR) was observed in 13.3% (2/15) at 90 mg/m² D1 D8 q3w and 3% (3/23) at 190 mg/m² q3w.

Table 7 below summarizes the results obtained with the different schedules tested.

Table 7

Conclusion

huDS6-DM4 is an active drug with responses seen notably in breast, lung and bladder cancers. Both schedules 90 mg/m² D1 D8 q3w and 120 mg/m² q2w lead to an acceptable safety profile for eyes disorders, better than the schedule 190 mg/m² q3w.

Similar ORR at 90 mg/m² D1 D8 q3w compared to 190 mg/m² q3w was observed. Primary prophylaxis of eye disorders was determined to be beneficial and lead to further improve the ocular safety profile of huDS6-DM4.

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings failing within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the method comprises at least two cycles,

wherein said conjugate is administered at least two times during each cycle.

2. The conjugate for its use according to ciai^'m 1 , wherein in each cycle the conjugate is administered at a dose corresponding to that of an administration schedule of from 60 to 80 mg/m² every week of the cycle.

3. The conjugate for its use according to claim 1 or 2, wherein the cycle is a period of two or three weeks.

4. The conjugate for its use according to any one of claims 1 to 3, wherein the cycle is a period of three weeks, and, for each cycle, the conjugate is administered at a dose of 90 mg/m² at day 1 of the cycle and at a dose of 90 mg/m² at day 8 of the cycle.

5. The conjugate for its use according to any one of claims 1 to 4, wherein, prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered.

6. The conjugate for its use according to claim 5, wherein the cycle is a period of three weeks, and, for each cycle, the conjugate is administered at a dose of 120 mg/m² at day 1 of the cycle and at a dose of 120 mg/m² at day 8 of the cycle.

7. The conjugate for its use according to claim 5 or 6, wherein the prophylactic treatment for preventing eye disorders comprises administration of a vasoconstrictor and/or a corticosteroid, and/or use of a cold eye mask.

8. The conjugate for its use according to claim 7, wherein the corticosteroid is administered three times a day starting the day of the conjugate administration and for 2 days.

9. The conjugate for its use according to claim 7, wherein the cold eye mask is used during the whole administration of the conjugate.

10. The conjugate for its use according any one of claims 1 to 9, wherein said cell binding agent binds the extracellular domain of the UC1 glycoprotein.

11. The conjugate for its use according to any one of claims 1 to 10, wherein said cell binding agent recognizes and binds the CA6 glycotope on the MUC1 glycoprotein.

12. The conjugate for its use according to any one of claims 1 to 1 1 , wherein said cell binding agent is an antibody or an epitope-binding fragment thereof.

13. The conjugate for its use according to claim 12, wherein said antibody or epitope- binding fragment thereof comprises one or more complementarity-determining region (CDR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

14. The conjugate for its use according to claim 13, wherein said antibody or epitope- binding fragment thereof comprises a CDR1 -heavy chain (CDR1 -H) sequence of SEQ ID NO: 1 , a CDR2-heavy chain (CDR2-H) sequence of SEQ ID NO: 2, a CDR3-heavy chain (CDR3-H) sequence of SEQ ID NO: 3, a CDR1-light chain (CDR1-L) sequence of SEQ ID NO: 4, a CDR2-light chain (CDR2-L) sequence of SEQ ID NO: 5 and a CDR3-light chain (CDR3-L) sequence of SEQ ID NO: 6.

15. The conjugate for its use according to claim 13 or 14, wherein said antibody or epitope-binding fragment thereof comprises a heavy chain variable region of sequence

SEQ ID NO: 7 or a sequence at least 85% identical thereto.

16. The conjugate for its use according to any one of claims 13 to 15, wherein said antibody or epitope-binding fragment thereof comprises a light chain variable region of sequence SEQ ID NO: 8 or a sequence at least 85% identical thereto.

17. The conjugate for its use according to any one of claims 12 to 16, wherein the epitope- binding fragment is selected from the group consisting of Fv, Fab, F(ab')₂, Fab', dsFv, (dsFv)₂, scFv, sc(Fv)₂, diabody and VHH.

18. The conjugate for its use according to any one of claims 1 to 16, wherein said cell binding agent is a monoclonal antibody comprising a heavy chain of sequence SEQ ID

NO: 9 and a fight chain of sequence SEQ ID NO: 10 or a sequence at least 85% identical thereto.

19. The conjugate for its use according to any one of claims 1 to 18, wherein said at least one cytotoxic agent is selected from the group consisting of a maytansinoid, a small drug, a tomaymycin derivative, a leptomycin derivative, a prodrug, a taxoid, CC-1065 and a CC- 1065 analog.

20. The conjugate for its use according to claim 19, wherein said at least one cytotoxic agent is the maytansine DM1 of formula (I)

21. The conjugate for its use according to claim 19, wherein said at least one cytotoxic agent is the maytansine DM4 of formula (II)

22. The conjugate for its use according to any one of claims 1 to 21 , wherein the cell binding agent is covalently linked via a cleavable or non-cleavable linker to the at least one cytotoxic agent.

23. The conjugate for its use according to claim 22, wherein said linker is selected from the group consisting of N-succinimidyl pyridyldithiobutyrate (SPDB), 4-(Pyridin-2- yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), and succinimidyl (N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC).

24. The conjugate for its use according to claim 22, wherein said linker is N-succinimidyl pyridyldithiobutyrate (SPDB) and said cytotoxic agent is DM4.

25. The conjugate for its use according to claim 22, wherein said linker is 4-(Pyridin-2- yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB) and said cytotoxic agent is DM4.

26. The conjugate for its use according to any one of claims 1 to 25, wherein said conjugate is characterized by a drug-to-antibody ratio (DAR) ranging from 3 to 4, the DAR being calculated from the ratio of the cytotoxic agent concentration (c_D) to that of the cell binding agent (c_A);

wherein

C_D = [C¾2S0 X ^252) - (¾2S2 ^X ^ 28o)]/ ¾252 ^X ¾2So) ~ (¾252 X ¾2Bo)l

C_A = f t₂80 ^— (^CD ^X ¾2Bo)J/¾280 and

ε₀252 and s_D28o are respectively the molar extinction coefficients of the cytotoxic agent at 252 nm and 280 nm,

ε_Α252 and ε_Α28ο are respectively the molar extinction coefficients of the cefl binding agent at 252 nm and 280 nm, and

A₂₅₂ and A₂₈o are respectively the absorbances for the conjugate at 252 nm (A₂5₂) and at 280 nm (A₂₈₀), measured using a classic spectrophotometer apparatus.

27. The conjugate for its use according to any one of claims 1 to 26, wherein the number of cycles is 2.

28. The conjugate for its use according to any one of claims 1 to 27, wherein said conjugate is administered intravenously.

29. The conjugate for its use according to claim 28, wherein said conjugate is administered at a rate of 1 mL/min for 30 min and then increased to a maximal rate of 2 mL/min in the absence of hypersensitivity reactions.

30. The conjugate for its use according to any one of claims 1 to 29, wherein the cancer is a solid tumor.

31. The conjugate for its use according to any one of claims 1 to 30, wherein the cancer is a CA6-positive tumor.

32. The conjugate for its use according to claim 31 , wherein the tumor is qualified as CA6- positive when at least 30% of the cells of the sample exhibit a level of intensity of 2+/3+ as determined using an immunohistochemistry (IHC) assay.

33. The conjugate for its use according to claim 32, wherein the IHC assay comprises the murine monoclonal antibody produced by the hybridoma cell line DS6 deposited at the American Type Culture Collection (ATCC) with the deposit number PTA-4449.

34. The conjugate for its use according to any one of claims 30 to 33, wherein the cancer is selected from the group consisting of breast cancer, lung cancer and bladder cancer.

35. The conjugate for its use according to claim 34, wherein the cancer is breast cancer.

36. The conjugate for its use according to claim 35, wherein the breast cancer is a triple negative breast cancer, not positive to receptors for estrogen, progesterone or HER2.

37. A conjugate comprising (i) a cell binding agent which specifically binds to tumor cells, linked to (ii) at least one cytotoxic agent, for use in a method for treating cancer,

wherein the cytotoxic agent is a tubulin binder; and

- administration of an ocular vasoconstrictor,

- administration of an ocular corticosteroid, and

use of a cold eye mask.

38. The conjugate according to claim 37, wherein the prophylactic treatment consisting in two or more of:

- administration in each eye of three drops of 2.5% neosynephrine,

- use of a cold eye mask during the administration of the conjugate.

39. An article of manufacture comprising:

a) a packaging material;

b) a conjugate comprising (i) a cell binding agent which binds to the human mucin-1 (MUC1 ) glycoprotein, linked to (ii) at least one cytotoxic agent; and

c) a label or package insert contained within said packaging material indicating that, for at least two cycles, said conjugate is administered at least two times per cycle and is administered, for each cycle, at a dose corresponding to that of an administration schedule of at least 60 mg/m² every week of the cycle.

40. The article of manufacture according to claim 39, wherein the label or package insert further indicates that the cycle is a period of three weeks, and, for each cycle, the conjugate is administered:

(ii) at a dose of 120 mg/m² at day 1 of the cycle and at a dose of 120 mg/m² at day 8 of the cycle.

41. The article of manufacture according to claim 39 or 40, wherein the label or package insert further indicates that prior to, simultaneously with, or immediately after each administration of the conjugate, a prophylactic treatment for preventing eye disorders is administered.