TW201808336A - Treatment regimen using anti-MUC1 maytansinoid immunoconjugate antibody for the treatment of tumors - Google Patents

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 of tumors with anti-Muc1 class maytansin immunoconjugate antibodies   

The present invention relates to the use of anti-Muc1 maytansinoid immunoconjugates for the treatment of tumors while inducing low toxicity.

There have been many attempts to develop anti-cancer therapeutics that specifically destroy target cancer cells without damaging surrounding, non-cancerous cells and tissues. This therapeutic agent has the potential to greatly improve cancer treatment in human patients.

One promising way is to link cell binding agents such as monoclonal antibodies to cytotoxic drugs. Depending on the choice of cell binding agent, these cytotoxic conjugates can be designed to recognize and bind only specific cancer cell types based on the molecular expression profile expressed on the surface of these cells.

International patent application WO 02/16401 describes the murine monoclonal antibody DS6, which reacts with the antigen CA6 expressed by human serous ovarian cancer. The murine monoclonal antibody DS6 can therefore target cancer cells.

The CA6 antigen is more specifically characterized as the sialic acid sugar position on the MUC1 mucin receptor expressed by cancer cells in U.S. Patent No. 7,834,155. The patent also provides antibodies, especially humanized antibodies such as humanized hDS6 antibodies, which are capable of recognizing this CA6 sialic acid sugar position of the MUC1 mucin receptor.

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C and chlorambucil have been linked to various murine monoclonal antibodies for cytotoxicity Conjugate. In some cases, the drug molecule and antibody molecule are linked by a vehicle carrier molecule such as hemoglobin.

Such cytotoxic conjugates include antibody-containing conjugates, preferably humanized antibodies, which recognize the Muc1 mucin receptor CA6 sialic acid sugar position expressed by cancer cells and can be used in the context of cytotoxic agents Inhibits the growth of cells expressing the CA6 sugar site.

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

However, it is known that ocular toxicity such as corneal disease and blurred vision are observed with the use of antibody drug conjugates. Unfortunately, the occurrence of this less severe and reversible effect limits the exposure of some patients to the effective optimal dose, because in some cases a dose reduction and / or dose delay is required at the next administration of the conjugate .

For example, after the phase 1 escalation phase for the huDS6-DM4 immunoconjugate, first consider that the recommended dose of huDS6-DM4 should be 190 mg / m ² administered every three weeks (q3w) (WO2015 / 014879). However, during the expansion phase of the study (data not published), late occurrences of ocular events that caused dose modification or drug discontinuation were observed. In fact, although the efficacy of huDS6-DM4 at this dose is considered promising (13.0% ORR), it is believed that the percentage of ocular toxicity caused a lower dose or a higher dose delay of the conjugate (60.9%). As a first way, the inventors reduced the recommended dose of huDS6-DM4. A dose of 150 mg / m ^{2 was} selected every three weeks. However, although safety was improved (the percentage of ocular events that caused dose delay and / or dose reduction was 24.2%), the efficacy was reduced (ORR of 3.1%).

Therefore, there is an important need for new treatments / schedules for cytotoxic conjugates that can maintain good activity on tumors and limit toxicity, especially ocular toxicity.

The present invention comes from the unexpected discovery of the present inventors that by administering the cytotoxic conjugate at a dose corresponding to the weekly dose of 60 mg / m ² during the cycle for at least two cycles of administration, It is possible to maintain a good therapeutic effect on tumors and limit toxicity, especially ocular toxicity, which forces the dose of conjugate administered to be reduced or delayed at the beginning of the next administration cycle.

In fact, the inventors have shown that when the huDS6-DM4 immunoconjugate is administered repeatedly at a dose of 120 mg / m ² every two weeks (q2w) as a new cycle (the cycle is scheduled to be administered every cycle Dose, corresponding to the planned weekly dose of 60 mg / m ² during the cycle), the dose change due to ocular toxicity or drug discontinuation was only 6.3%, and the acceptable Probability of non-progression (36.1%). Most surprisingly, the inventors even showed that when the huDS6-DM4 immunoconjugate is administered according to the following strategy, the therapeutic effect is at least similar to that administered at 190 mg / m ² every three weeks (comparable ORR) , And at the same time reduce ocular toxicity (29.4% vs. 60.9%), the regimen includes: first administration at a dose of 90 mg / m ² on the first day of the cycle, and 90 mg / m ² on the eighth day of the cycle The second dose is administered, and the regimen is repeated every three weeks as a new cycle (D1D8q3w). In addition, it was observed that preventive treatment of the eye can help reduce the ocular toxicity that may be associated with some elevated doses of the conjugate. The inventors devised a scheme in which the conjugate was taken on day 1 and day 8 120mg / m ² dose administration, repeated every 3 weeks as a new cycle (q3w) (the dose planned for each period of the administration plan corresponds to the weekly 80mg / m ² administration of the cycle Planned dose), and in which preventive treatment of the eye is administered.

The present invention relates to a conjugate for use in a method for treating cancer, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, which is (ii) at least one cytotoxic Dosing, wherein the method includes at least two cycles, wherein, in each cycle, the conjugate is administered at a dose corresponding to a weekly planned dose of at least 60 mg / m ² of the cycle, and wherein The conjugate is administered at least twice during each cycle.

In some embodiments, the cycle is a three-week period, and in each cycle the conjugate is administered as follows: at a dose of 90 mg / m ² on the first day of the cycle, at the At a dose of 90 mg / m ² on the 8th day of the cycle.

The present invention also relates to a conjugate for use in a method for treating cancer, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, and (ii) at least one cell Toxic agent connection, wherein the method includes at least two cycles, one cycle is a three-week period, and in each cycle:-the conjugate is administered at a dose of 120 mg / m ² on the first day of the cycle, It is also administered at a dose of 120 mg / m ² on the 8th day of the cycle, and-before, at the same time, or immediately after each administration of the conjugate, prophylactic treatment to prevent ocular disorders.

The present invention also relates to a conjugate for use in a method for treating cancer, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, and (ii) at least one cell Toxic agent connection, wherein the method includes at least two cycles, wherein the cycle is a two-week period, and wherein the conjugate is administered at a dose of 120 mg / m ² on the first day 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 a further embodiment, the cell binding agent is a humanized anti-CA6 antibody huDS6 comprising the heavy chain of sequence SEQ ID NO: 9 and the light chain of sequence SEQ ID NO: 10, and the cytotoxic agent is US Dentin compounds such as DM1 or DM4.

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

The present invention also relates to a conjugate used in a method for treating cancer, comprising: (i) a cell binding agent that specifically binds to tumor cells, which is connected to (ii) at least one cytotoxic agent, wherein The cytotoxic agent is a tubulin-binding agent; and wherein the method includes administering the conjugate in a patient in need thereof, and each time the conjugate is administered, administering Prophylactic treatment for the prevention of ocular disorders, which includes two or more of the following:-administration of ocular vasoconstrictors,-administration of ocular corticosteroids, and-use of cold eye masks.

The invention also relates to an article of manufacture comprising: a) packaging material; b) a conjugate comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, which is at least equal to (ii) A cytotoxic agent linked; and c) a label or package insert contained in the packaging material indicating that in at least two cycles, in each cycle, the conjugate corresponds to each week of the cycle The planned dose of 60 mg / m ² was administered.

Another object of the present invention relates to an article of manufacture comprising: a) packaging materials; b) a conjugate comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, which is ( ii) at least one cytotoxic agent is attached; and c) the label or package insert contained in the packaging material indicates that at least two three-week cycles, for each cycle, the conjugate is at the end of the cycle 1 day at a dose of 120 mg / m ² and on the 8th day of the cycle at a dose of 120 mg / m ² , before, at the same time or immediately after each administration of the conjugate, to prevent ocular disorders Preventive treatment.

definition

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

In the context of the present invention, the term " CA6 glycosite " or " CA6 sialic acid glycosite " refers to a tumor-associated antigen present on the extracellular domain of the MUC1 glycoprotein, which is carried by Kearse et al. ) Int. J. Cancer. 88: 866-872 identification, and more specifically characterized in US2007 / 0041980.

As used herein, a sequence that is “ at least 85% identical to a reference sequence ” has 85% or more of the full length of the reference sequence over its full length, specifically 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

The percentage of " sequence identity " can be determined by comparing the two sequences of the agent in an optimal manner in the comparison window, where the portion of the polypeptide sequence in the comparison window can contain, compared to the reference sequence (which does not include additions or deletions) Added or deleted (ie, gaps) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same amino acid residues appear in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain sequence identity percentage. The optimal alignment of the sequences used for comparison is performed by global pairwise alignment using an algorithm such as Needleman and Wunsch J. Mol. Biol. 48: 443 (1970). The percent sequence identity can be easily determined, for example, using the Needle program with the BLOSUM62 matrix and the following parameters: gap opening = 10, gap extension = 0.5.

In the context of the present invention, " conservative amino acid substitution " is a substitution in which an amino acid residue is replaced by another amino acid residue having side chain groups that are similar in chemical properties (eg, charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of proteins. Examples of the group of amino acids having chemically similar side chains include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine And threonine; 3) side chains containing amides: 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 acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamine Acid-aspartic acid, and asparagine-glutamine.

As used herein, the term " subject " refers to mammals, such as rodents, cats, dogs, and primates. In particular, the subject according to the invention is a human.

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

Throughout this application, the term " comprising " is understood to cover all specifically mentioned features as well as optional, other, unspecified features. As used herein, the use of the term " comprising " also discloses embodiments in which there are no features other than those specifically mentioned (ie " consisting of ").

Cell binding agent

The term " cell binding agent " as used herein refers to an agent that specifically recognizes and binds to human mucin-1 (MUC1) glycoprotein on the cell surface. In specific embodiments, the cell binding agent binds, and more specifically specifically binds to the extracellular domain of the MUCl glycoprotein as defined in the " Definitions " section above. In another embodiment, the cell binding agent recognizes and binds to the CA6 sugar position on the MUC1 glycoprotein as defined in the " Definitions " section above.

In one embodiment, the cell binding agent specifically recognizes the human MUC1 glycoprotein, in particular the extracellular domain of the MUC1 glycoprotein, more specifically the CA6 sugar position on the MUC1 glycoprotein, thereby it allows the conjugate to be targeted Effect, accompanied by few side effects caused by 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 specifically the CA6 sugar position on the MUC1 glycoprotein, whereby the conjugate will interact with The target cells are contacted for sufficient time to allow the cytotoxic agent portion of the conjugate to act on the cell and / or to allow sufficient time for the conjugate to be internalized by the cell.

The effectiveness of the conjugate of the present invention as a therapeutic agent depends on appropriate cell binding to human mucin-1 (MUC1) glycoprotein, in particular the extracellular domain of MUC1 glycoprotein, more specifically the CA6 sugar site on the MUC1 glycoprotein Careful selection of agents. The cell binding agent may be of any currently known species, or become known, and include peptides and non-peptides, as long as it binds to the human MUC1 glycoprotein, especially the extracellular domain of MUC1 glycoprotein, more specifically CA6 on the MUC1 glycoprotein Glucose binding. In general, these can be antibodies (particularly monoclonal antibodies), lymphokines, hormones, growth factors, vitamins, nutrient transport molecules (eg transferrin), or any other cell-binding molecular substance.

Examples of more specific 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.

The selection of a suitable cell-binding agent is a matter of selection that depends on the specific cell population targeted, but in general, if suitable or available for preparation, antibodies or epitope-binding fragments are preferred, more preferably monoclonal antibody.

An " antibody " may be a natural or conventional antibody in which two heavy chains are connected to each other through a disulfide bond and each heavy chain is connected to a light chain through a disulfide bond. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain types (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for the antigen. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental activity, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the immunoglobulin Fab fragment, and is composed of a light chain and a heavy chain variable part. The specificity of antibodies lies in the structural complementarity between antibody binding sites and antigenic determinants. Antibody binding sites are composed of residues mainly derived from hypervariable or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable or framework regions (FR) affect the overall domain structure, and therefore the binding site.

" Complementarity determining region " or " CDR " refers to amino acid sequences that collectively define the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, called CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, the conventional antibody antigen-binding site contains six CDRs, including a collection of CDRs from each V region of the heavy and light chains.

" Framework region " (FR) refers to the amino acid sequence inserted between the CDRs, that is, those parts of the immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of immunoglobulins each have four FRs, called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H.

As used herein, a " human framework region " is substantially the same framework as a naturally occurring human antibody framework region (about 85%, or more, especially 90%, 95%, 97%, 99%, or 100%) Area.

In the context of the present invention, the definition of CDR / FR in the immunoglobulin light or heavy chain is based on the definition of IMGT (Lefranc et al. (2003) Dev Comp Immunol. 27 (1): 55-77; www.imgt.org) .

The term " antibody " as used herein refers to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, especially the variable heavy chains of single domain antibodies, and chimeric, humanized, bispecific or multispecific Sexual antibody.

As used herein, antibodies or immunoglobulins also include " single domain antibodies " which have recently been described and which are antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples of single-domain antibodies include heavy-chain antibodies, antibodies that do not naturally contain light chains, single-domain antibodies derived from conventional four-chain antibodies, and engineered single-domain antibodies. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. The single domain antibody may be a naturally occurring single domain antibody, which is considered to be a heavy chain antibody that does not contain a light chain. Specifically, camelids (Camelidae) species, for example, camel, dromedary (Dromedary), llama (Llama), alpaca (Alpaca) and guanaco (Guanaco) produce heavy chain antibodies naturally devoid of light chains. The Camelidae heavy chain antibody still lacks the CH1 domain.

These variable heavy chains that do not contain light chain single domain antibodies have been referred to in the art as " VHH " or " Nanobodies ." Similar to the conventional VH domain, VHH contains four FRs and three CDRs. Nanobodies have the advantage over conventional antibodies: they are about 10 times smaller than IgG molecules, and thus properly folded functional Nanobodies can be produced by in vitro expression while obtaining high yields. In addition, Nanobodies are very stable and resistant to proteases. The nature and production of Nanobodies have been reviewed by Harmsen and De Haard (Harmsen and De Haard (2007) Appl. Microbiol. Biotechnol. 77 (1): 13-22).

The term " monoclonal antibody " or " mAb " as used herein refers to an antibody molecule composed of a single amino acid, which is directed to a specific antigen and should not be understood that the antibody needs to be produced by any specific method. Monoclonal antibodies can be produced by monoclonal B cells or hybridomas, but can also be recombinant, that is, produced by protein engineering.

The term " chimeric antibody " refers to an engineered antibody, which in its broadest sense includes one or more regions from one antibody and one or more regions from one or more other antibodies. Specifically, the chimeric antibody comprises the VH domain and the VL domain of an antibody derived from a non-human animal, which is combined with the CH domain and the CL domain of another antibody, especially a human antibody. As the non-human animal, any animal such as mouse, rat, hamster, rabbit, etc. can be used. Chimeric antibodies can also refer to multispecific antibodies that are specific for at least two different antigens. In an embodiment, the chimeric antibody has a mouse-derived variable domain and a human-derived constant domain.

The term " humanized antibody " means an antibody that was originally completely or partially derived from a non-human source, which was modified to replace certain amino acids, especially in the framework regions of the heavy and light chains, to avoid or minimize human immunity reaction. The constant domains of humanized antibodies are mostly human CH and CL domains. The constant domains of humanized antibodies are mostly human CH and CL domains. In an embodiment, the humanized antibody has a constant domain of human origin.

(Conventional) " Fragments " of antibodies comprise a part of an intact antibody, especially the antigen-binding or variable regions 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 multispecific antibodies formed from antibody fragments . Fragments of conventional antibodies can also be single domain antibodies, such as heavy chain antibodies or VHH.

The term " Fab " refers to an antibody fragment having a molecular weight of about 50,000 Da and antigen-binding activity, wherein in the fragment obtained by treating IgG with protease papain, about half of the N-terminal side of the heavy chain and the entire light chain are bonded by disulfide bonds Together.

The term " F (ab ') ₂ " refers to an antibody fragment having a molecular weight of about 100,000 Da and antigen-binding activity, which is a fragment obtained by treating IgG with protease pepsin, which is higher than the Fab bonded via a disulfide bond in the hinge region Slightly larger.

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 cleaving the disulfide bond of the hinge region of the F (ab') ₂ fragment.

Single-chain Fv ("scFv") polypeptides are covalently linked VH :: VL heterodimers, which are usually expressed by a gene fusion comprising VH and VL encoding genes linked by a peptide encoding linker. The human scFv fragments of the present invention contain CDRs kept in proper conformation, especially by using genetic recombination techniques. Bivalent and multivalent antibody fragments can be formed spontaneously by the combination of monovalent scFvs or can be generated by peptide linkers by coupling monovalent scFvs, such as bivalent sc (Fv) ₂ .

" DsFv " is a VH :: VL heterodimer stabilized by disulfide bonds.

" (DsFv) ₂ " refers to two dsFvs coupled via a peptide linker.

The term " bispecific antibody " or "BsAb" refers to an antibody that combines the antigen binding sites of two antibodies in a single molecule. Therefore, BsAb can simultaneously bind two different antigens. As described in, for example, EP 2 050 764 A1, genetic engineering has been used more and more frequently to design, modify, and produce antibodies or antibody derivatives with an ideal combination of binding properties and effector functions.

The term " multispecific antibody " refers to an antibody that combines the antigen binding sites of two or more antibodies in a single molecule.

The term " diabodies " refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) ). By using a linker between two domains of the same chain that is short enough to not allow pairing, the domain is forced to pair with the complementary domain of the other chain and two antigen binding sites are created.

In a specific 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 specific embodiment, the conjugate of the invention comprises an antibody or epitope binding fragment thereof, which comprises one or more specifically one, two, three, four, five or six CDR of amino acid sequence: SYNMH (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 another embodiment, the conjugate of the invention may comprise an antibody or an epitope binding fragment thereof, which comprises the sequence CDR1-H of SEQ ID NO: 1, the sequence CDR2-H of SEQ ID NO: 2 and the sequence SEQ ID NO : CDR3-H of 3.

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

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

Also provided is a conjugate comprising an antibody or epitope binding fragment that contains or is at least 85% of the heavy chain variable region of the following sequence, more specifically at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences: (SEQ ID NO: 7), the preferred condition is that the sequence comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

Also provided are conjugates comprising an antibody or epitope binding fragment that contains or is at least 85% of the light chain variable region of the following sequence, more specifically at least 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences: EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSEPLTFGAGTKLELKR (SEQ ID NO: 8) sequence preferably includes the sequence: SEQ ID NO: 8 , SEQ ID NO: 5 and SEQ ID NO: 6.

Also provided is a conjugate comprising an antibody or epitope binding fragment that contains or is at least 85% of the heavy chain of the following sequence, more specifically at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences: (SEQ ID NO: 9), the preferred condition is that the sequence comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

Also provided is a conjugate comprising an antibody or epitope binding fragment that contains or is at least 85% of the light chain of the following sequence, more specifically at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences: (SEQ ID NO: 10), the preferred condition is that the sequence comprises the sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

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

Similarly, humanized anti-MUC1 antibodies and their epitope binding fragments possessing a humanized or resurfaced light chain variable region with an amino acid sequence corresponding to SEQ ID NO: 8 are provided.

The term " humanized antibody " as used herein refers to a chimeric antibody that contains minimal sequence derived from non-human immunoglobulin.

As used herein, a " chimeric antibody " is an antibody in which the constant region or part thereof is changed, replaced, or exchanged, and thus the variable region is linked to a different species or a constant region of another antibody species or subclass. "Chimeric antibody" also refers to an antibody in which the variable region or part thereof is changed, replaced, or exchanged so that the constant region is connected to a variable region of a different species or belongs to another antibody class or subclass.

The goal of humanization is to reduce the immunogenicity of heterologous antibodies such as murine antibodies for introduction into the human body, while maintaining the full antigen-binding affinity and specificity of the antibodies. Humanized antibodies or antibodies adapted to not be rejected by other mammals can be produced using several techniques, such as resurfacing and CDR grafting. As used herein, surface reforming techniques use a combination of molecular modeling, statistical analysis, and mutagenesis to change the non-CDR surface of the antibody variable region to mimic the surface of a known antibody of the target host.

Strategies and methods of antibody surface reforming, and other methods of reducing antibody immunogenicity in different hosts, are disclosed in US Patent 5,639,641. In short, in the specific method, (1) generate the antibody heavy chain and light chain variable region position alignment, to obtain a set of heavy and light chain variable region framework surface exposed positions, of which all variable regions The alignment position of at least about 98% is the same; (2) define a set of amino acid residues exposed on the surface of the heavy and light chain variable region framework for rodent antibodies (or fragments thereof); (3) identify rodents with this group A set of heavy chain and light chain variable region framework surface exposed amino acid residues with the most similar surface exposed amino acid residues; (4) The set of heavy chain and light chain variable region framework surface exposed amino acids defined in step (2) Residues are substituted with exposed amino acid residues on the surface of the set of heavy and light chain variable region frameworks identified in step (3), except those within 5Å of any atoms of any residues located in the complementarity determining region of the rodent antibody Amino acid residues; and (5) to produce humanized rodent antibodies with binding specificity.

Various other techniques can be used to humanize antibodies, including CDR grafting (EP0239 400; W091 / 09967; US Patent Nos. 5,530,101 and 5,585,089), veneering or surface reforming (EP0592106; EP0519596; Padlan (1991) Molecular Immunology 28 (4/5): 489-498; Studnicka et al. (1994) Protein Engineering 7 (6): 805-814; Roguska et al. (1994) Proc. Natl. Acad. Sci USA 91 : 969-973), And chain shuffling (US Patent 5,565,332). Human antibodies can be prepared by a variety of methods known in the art, including phage display methods. See also US Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and International Patent Applications WO98 / 46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO91 / 10741.

An embodiment of such a humanized antibody is a humanized huDS6 antibody comprising the heavy chain of sequence SEQ ID NO: 9 and the light chain of sequence SEQ ID NO: 10, or an epitope binding fragment thereof, or at least 85 %, More specifically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical sequences, preferably if the sequence contains 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 cell function or growth and / or causes cell damage. Accordingly, the cytotoxic agent used in the conjugate of the present invention may be any compound that causes or induces cell death, or reduces cell viability in some way. Examples of cytotoxic agents include maytansinoid and maytansinoid analogs, prodrugs, tomycin derivatives, toxoids, micromycin derivatives, CC-1065 and CC-1065 analogs as defined below.

Cytotoxic agents that can be used in the present invention to form conjugates include maytansinoids and maytansinoids-like analogs . Examples of suitable maytansinoids include maytansinol and maytansinol analogues. Maytansinoids are drugs that inhibit microtubule formation and are highly toxic to mammalian cells.

Examples of suitable maytansinol analogs include those with modified aromatic rings and those with modifications at other positions. These appropriate maytansinoids are disclosed in U.S. Patent 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; 5,475,499; 5,585,5,5,5,333,410;

Specific examples of suitable analogs of maytansinol with a modified aromatic ring include: (1) C-19-dechlorination (US Patent No. 4,256,746), prepared by LAH reduction of ansamytocin P2; (2 ) C-20-hydroxyl (or C-20-demethyl) +/- C-19-dechlorination (US Patent Nos. 4,361,650 and 4,307,016), by using Streptomyces or Actinomyces Prepared by methylation or dechlorination using LAH; and (3) C-20-demethoxy, C-20-acetyl (-OCOR), +/- dechlorination (US Patent No. 4,294,757), by Preparation using acetyl chloride.

Specific examples of suitable analogs of modified maytansinol with other positions include: (1) C-9-SH (US Patent No. 4,424,219), prepared by the reaction of maytansinol with H ₂ S or P ₂ S ₅ ) ; (2) C-14-alkoxymethyl (demethoxy / CH ₂ OR) (US Patent No. 4,331,598); (3) C-14-hydroxymethyl or oxymethyl (CH ₂ OH or CH ₂ OAc) (U.S. Pat. No. 4,450,254), was prepared from Nocardia (Nocardia); (4) C -15- hydroxy / alkoxy acyl (U.S. Pat. No. 4,364,866), by transforming Streptomyces (of Streptomyces) It may be prepared from maytansinol; (5) C-15-methoxy (US Patent Nos. 4,313,946 and 4,315,929), isolated from Peach Tree ( Trewia nudiflora ); (6) C-18- N -desmethyl (US patent Nos. 4,362,663 and 4,322,348), prepared by the demethylation of maytansinol by Streptomyces ; and (7) 4,5-deoxy (US Patent No. 4,371,533), the reduction of beauty by titanium trichloride / LAH Prepared by alcohol.

In a specific embodiment, the conjugate of the present invention utilizes a thiol-containing maytansinoid (DM1), which is officially named N ^{2 '} -desacetyl- N ^2' -(3-mercapto-1-oxy Propyl) -Maytansine as a cytotoxic agent. DM1 is shown by the following structural formula (I):

(I) In another embodiment, the conjugate of the present invention utilizes a thiol-containing maytansinoid DM4, which is officially named N ^{2 '} -desacetyl- N ^2' -(4-methyl-4 -Mercapto-1-oxopentyl) -maytansine as a cytotoxic agent. DM4 is shown by the following structural formula (II):

In a further embodiment of the present invention, other maytansinoids may be used, including thiol and disulfide-containing maytansinoids that carry mono- or di-alkyl substituted carbon atoms carrying sulfur atoms. These include maytansinoids having hydroxymethyl groups, C-15 hydroxy groups or C-20 demethylation groups at C-3, C-14, and acetylated amino acid side chains with acetyl groups carrying hindered thiol groups, which carry thiols The carbon atom of the acetyl group of the functional group has one or two substituents, and the substituents are CH ₃ , C ₂ H ₅ , a linear or branched alkyl or alkenyl group having 1 to 10 carbon atoms, and a 3- Cyclic alkyl or alkenyl group of 10 carbon atoms, phenyl group, substituted phenyl group or heterocyclic aryl group or heterocycloalkyl group, and further, wherein one of the substituents may be H, and wherein the acyl group is The carbonyl functional group has a linear length of at least 3 carbon atoms between the sulfur atom.

Such other maytansine includes compounds represented by formula (III):

Where: Y 'stands for (CR ₇ R ₈ ) _l (CR ₉ = CR ₁₀ ) _p (C≡C) _q A _r (CR ₅ R ₆ ) _m D _u (CR ₁₁ = CR ₁₂ ) _r (C≡C) _s B _t (CR ₃ R ₄ ) _n CR ₁ R ₂ SZ, where R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl group with 1-10 carbon atoms, A branched or cyclic alkyl or alkenyl group having 3 to 10 carbon atoms, a phenyl group, a substituted phenyl group or a heterocyclic aryl or heterocycloalkyl group, and in addition R ₂ may be H; A, B, D is a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, unsubstituted or substituted aryl or heterocyclic aryl or heterocycloalkyl; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , a linear alkyl or alkenyl group having 1-10 carbon atoms, having 3-10 Branched or cyclic alkyl or alkenyl group with one carbon atom, phenyl group, substituted phenyl group or heterocyclic aryl group or heterocyclic alkyl group; l, m, n, o, p, q, r, s and t is independently an integer of 0 or 1-5, provided that at least two of l, m, n, o, p, q, r, s, and t are not simultaneously zero; and Z is H, SR, or- COR, where R is 1-10 carbon atoms Alkyl or alkenyl chain, branched or cyclic alkyl group having 3-10 carbon atoms or an alkenyl group, or an unsubstituted or substituted aryl or heterocyclic aromatic or heterocycloalkyl.

Preferred embodiments of formula (III) include compounds of formula (III), wherein: R ₁ is methyl, R ₂ is H and Z is H; R ₁ and R ₂ are methyl, and Z is H; R ₁ is Methyl, R ₂ is H, and Z is -SCH ₃ ; R ₁ and R ₂ are methyl, and Z is -SCH ₃ .

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

Where: Y represents (CR ₇ R ₈ ) _l (CR ₅ R ₆ ) _m (CR ₃ R ₄ ) _n CR ₁ R ₂ SZ, where: R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ has Straight chain alkyl or alkenyl group of 1-10 carbon atoms, branched or cyclic alkyl or alkenyl group having 3-10 carbon atoms, phenyl group, substituted phenyl group or heterocyclic aryl group or heterocyclic ring Alkyl, and in addition R ₂ may be H; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C2H ₅ , linear with 1-10 carbon atoms Alkyl or alkenyl, branched or cyclic alkyl or alkenyl with 3-10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl; l, m and n each Independently an integer of 1-5, and in addition n may be 0; Z is H, SR, -COR, where R is a linear or branched alkyl or alkenyl group having 1-10 carbon atoms, having 3- 10 carbon atoms cycloalkyl or alkenyl, or unsubstituted or substituted aryl or heterocyclic aryl or heterocycloalkyl; and May represents maytansinoids, which carry side chains C-3, C-14 Hydroxymethyl, C-15 hydroxyl or C-20 demethylation.

Specific examples of formulae (IV-L), (IV-D) and (IV-D, L) include compounds of formulae (IV-L), (IV-D) and (IV-D, L), where: R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, n is 0, and Z is H.

R ₁ and R ₂ are methyl groups, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, n is 0, and Z is H.

R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, n is 0, and Z is -SCH ₃ .

R ₁ and R ₂ are methyl groups, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are respectively 1, n is 0, and Z is -SCH ₃ .

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

These other maytansine also include compounds represented by formula (V):

Where: Y represents (CR ₇ R ₈ ) _l (CR ₅ R ₆ ) _m (CR ₃ R ₄ ) _n CR ₁ R ₂ SZ, where: R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , Straight chain alkyl or alkenyl having 1-10 carbon atoms, branched or cyclic alkyl or alkenyl having 3-10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocyclic Alkyl, and additionally R ₂ may be H; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , having 1 to 10 carbon atoms Linear alkyl or alkenyl, branched or cyclic alkyl or alkenyl with 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl; l, m And n are each independently an integer of 1-5, and in addition, n may be 0; and Z is H, SR, or -COR, where R is a linear alkyl or alkenyl group having 1-10 carbon atoms, having 3-10 carbon atom branched or cyclic alkyl or alkenyl, or unsubstituted or substituted aryl or heterocyclic aryl or heterocycloalkyl.

Specific examples of formula (V) include compounds of formula (V), wherein: R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R _{8 are} each H, and l and m are each 1, n is 0 and Z is H.

R ₁ and R ₂ are methyl groups, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, n is 0, and Z is H.

R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, n is 0, and Z is -SCH ₃ .

R ₁ and R ₂ are methyl groups, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are 1, n is 0, and Z is -SCH ₃ .

These additional maytansine also include compounds represented by formula (VI-L), (VI-D) or (VI-D, L):

Where: Y ₂ represents (CR ₇ R ₈ ) _l (CR ₅ R ₆ ) _m (CR ₃ R ₄ ) _n CR ₁ R ₂ SZ ₂ , where: R ₁ and R ₂ are independently CH ₃ and C ₂ H ₅ , linear alkyl or alkenyl group having 1-10 carbon atoms, branched or cyclic alkyl or alkenyl group having 3-10 carbon atoms, phenyl group, substituted phenyl group or heterocyclic aryl group or Heterocycloalkyl, and in addition R ₂ may be H; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , having 1-10 Linear alkyl or alkenyl groups with carbon atoms, branched or cyclic alkyl or alkenyl groups with 3 to 10 carbon atoms, phenyl groups, substituted phenyl groups or heterocyclic aryl groups or heterocyclic alkyl groups; 1. m and n are each independently an integer of 1-5, and in addition n may be 0; Z ₂ is SR or COR, where R is a linear alkyl or alkenyl group having 1-10 carbon atoms, having 3-10 A branched or cyclic alkyl or alkenyl group of one carbon atom, or an unsubstituted or substituted aryl or heterocyclic aryl or heterocycloalkyl group; and May is a maytansinoid.

These additional maytansine also include compounds represented by formula (VII):

Where: Y ₂ 'represents (CR ₇ R ₈ ) _l (CR ₉ = CR ₁₀ ) _p (C≡C) _q A _r (CR ₅ R ₆ ) _m D _u (CR ₁₁ = CR ₁₂ ) _r (C≡C ) _s B _t (CR ₃ R ₄ ) _n CR ₁ R ₂ SZ ₂ , wherein: R ₁ and R ₂ are each independently CH ₃ , C ₂ H ₅ , straight or branched chain with 1-10 carbon atoms Alkyl or alkenyl, cyclic alkyl or alkenyl having 3-10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl, and additionally R ₂ may be H; A , B and D are each independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, an unsubstituted or substituted aryl or heterocyclic aryl or heterocycloalkyl group; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , a linear alkyl group or an alkene having 1-10 carbon atoms Radicals, branched or cyclic alkyl or alkenyl groups with 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl; l, m, n, o, p, q, r, s, and t are each independently an integer of 0 or 1-5, provided that at least two of l, m, n, o, p, q, r, s, and t are not simultaneously zero; and Z ₂ is SR or -COR, where R is Linear alkyl or alkenyl groups having 1-10 carbon atoms, branched or cyclic alkyl or alkenyl groups having 3-10 carbon atoms, or unsubstituted or substituted aryl or heterocyclic aryl groups or Heterocycloalkyl.

Particular embodiments of formula (VII) include compounds of formula (VII) wherein R ₁ is methyl and R ₂ is H.

The above maytansinoid may be conjugated with the cell binding agent defined in the section " Cell binding agent " above, especially a humanized antibody comprising the heavy chain of sequence SEQ ID NO: 9 and the light chain of sequence SEQ ID NO: 10 huDS6, wherein the cell-binding agent, in particular the humanized huDS6 antibody comprising the heavy chain of sequence SEQ ID NO: 9 and the light chain of sequence SEQ ID NO: 10, used with maytansinoid present in maytansinoid Thiol or disulfide functional group on the acetyl group of the acylated amino acid chain found in the C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 demethylation group, and wherein the acylated amino acid side The acyl group of the chain has its thiol or disulfide functional group at the carbon atom with one or two substituents, the substituents are CH ₃ , C ₂ H ₅ , straight chain with 1-10 carbon atoms Alkyl or alkenyl, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aryl or heterocycloalkyl, and additionally said substituents One may be H, and wherein the acetyl group has a linear length of at least 3 carbon atoms between the carbonyl functional group and the sulfur atom.

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

Where: Y ₁ 'represents (CR ₇ R ₈ ) _l (CR ₉ = CR ₁₀ ) _p (C≡C) _q A _r (CR ₅ R ₆ ) _m D _u (CR ₁₁ = CR ₁₂ ) _r (C≡C ) _s B _t (CR ₃ R ₄ ) _n CR ₁ R ₂ S-, wherein A, B and D are each independently a cycloalkyl or cycloalkenyl group having 3 to 10 carbon atoms, unsubstituted or substituted Aryl or heterocyclic aryl or heterocycloalkyl; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ and R ₁₂ are each independently H, CH ₃ , C ₂ H ₅ , linear alkyl or alkenyl group having 1-10 carbon atoms, branched or cyclic alkyl or alkenyl group having 3-10 carbon atoms, phenyl group, substituted phenyl group or hetero group Cycloaryl or heterocycloalkyl; and l, m, n, o, p, q, r, s, and t are each independently an integer of 0 or 1-5, provided that l, m, n, o, p At least two of, q, r, s and t are not zero at the same time.

Specifically, R ₁ is methyl, R ₂ is H, or R ₁ and R ₂ are methyl.

In another embodiment of the present invention, the conjugate is a conjugate comprising a cell binding agent as defined in the " Cell Binding Agent " section above, in particular a heavy chain comprising the sequence SEQ ID NO: 9 and the sequence SEQ ID NO: 10 light chain humanized huDS6 antibody, the cell binding agent is conjugated to a maytansinoid of formula (IX-L), (IX-D) or (IX-D, L):

Where: Y ₁ represents (CR ₇ R ₈ ) _l (CR ₅ R ₆ ) _m (CR ₃ R ₄ ) _n CR ₁ R ₂ S-, where R ₁ and R ₂ are independently CH ₃ , C ₂ H ₅ , A linear alkyl or alkenyl group having 1-10 carbon atoms, a branched or cyclic alkyl or alkenyl group having 3-10 carbon atoms, a phenyl group, a substituted phenyl group, a heterocyclic aryl group or Heterocyclenyl, and additionally R ₂ may be H; R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently H, CH ₃ , C ₂ H ₅ , having 1-10 Linear alkyl or alkenyl groups with carbon atoms, branched or cyclic alkyl or alkenyl groups with 3 to 10 carbon atoms, phenyl groups, substituted phenyl or heterocyclic aryl groups or heterocyclic alkyl groups; l , M and n are each independently an integer of 1-5, and in addition n may be 0; and May represents maytansinol, which carries a side chain C-3, C-14 hydroxymethyl, C-15 hydroxy or C -20 to methyl.

Specific embodiments of formulas (IX-L), (IX-D) and (IX-D, L) include compounds of formula (IX-L), (IX-D) and (IX-D, L), where: R ₁ is methyl and R ₂ is H, or R ₁ and R ₂ are methyl, R ₁ is methyl, R ₂ is H, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m Each is 1, n is 0, R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, n is 0.

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

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

The substituents are as defined in formula (IX) above.

In a further embodiment, in the above compound, R ₁ is H, R ₂ is methyl, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, and n is 0.

In a further embodiment, in the above compound R ₁ and R ₂ are methyl, R ₅ , R ₆ , R ₇ and R _{8 are} each H, l and m are each 1, and n is 0.

In addition, L -aminoacyl stereoisomers are preferred.

Each type of maytansin taught by US Patent Application No. 2004/0235840 can also be used as a cytotoxic agent in the conjugate of the present invention.

The conjugates of cell binding agents, especially antibodies and maytansinoid-like drugs, as defined in the section " Cell binding agents " above, can be evaluated for their ability to inhibit the proliferation of various undesirable cell lines in vitro. For example, cell lines such as human epidermal cancer cell line A-431, human small cell lung cancer cell line SW2, human breast tumor cell line SKBR3, and Burkitt's lymphoma cell line Namalwa can be easily used to evaluate these compounds Cytotoxicity. The cells to be evaluated can be exposed to the compound for 24 hours, and the viable cell fraction can be measured by a direct test by known methods. The IC ₅₀ value can then be calculated from the results of the test.

The cytotoxic agent used in the conjugate according to the invention may also be a taxane or a derivative thereof.

Taxanes are a family of compounds that include the cytotoxic natural product paclitaxel (Taxol) and the semi-synthetic derivative docetaxel (Taxotere), both compounds It is widely used in cancer treatment. Taxanes are mitotic spindle poisons that inhibit the depolymerization of tubulin and cause cell death. Although docetaxel and paclitaxel are both agents that can be used for cancer treatment, their anti-tumor activity is limited due to their non-specific toxicity to normal cells.

The specific taxane used in the preparation of the conjugate is the taxane of formula (XI):

A method for synthesizing a taxane compound that can be used in the cytotoxic conjugate of the present invention, together with a method for conjugating the taxane compound to a cell binding agent as defined in the " Cell Binding Agent" section above , including a sequence The methods of humanizing huDS6 antibodies of the heavy chain of SEQ ID NO: 9 and the light chain of sequence SEQ ID NO: 10 are described in detail in US Patent Nos. 5,416,064, 5,475,092, 6,340,701, 6,372,738, 6,436,931, and 6,596,757 and US Patent Application No. 0001838, 2003/0004210, 2004/0024049 and 10 / 210,112.

The cytotoxic agent according to the present invention may also be a tomycin derivative. Derivative of tomycin is pyrrole [1,4] benzodiazepine (PBD), a known type of compound that exhibits its biological properties by covalently binding N2 to guanine in the minor groove of DNA. PBD includes some minor groove binders such as anthramycin, neothramycin and DC-81.

A novel toyomycin derivative that maintains high cytotoxicity and can be effectively linked to a cell binding agent as defined in the " Cell Binding Agent " section above is described in International Application No. WO2007 / 085930. The cell-binding agent-domemycin derivative complex allows the full measure of the cytotoxic effect of the tomycin derivative applied in a targeted mode only for undesired cells, thus avoiding Side effects caused by damage to healthy cells.

The cytotoxic agent according to the present invention may comprise one or more toyamycin derivatives, which are connected via a linking group to a cell binding agent as defined in the " Cell Binding Agent " section above, such as a heavy chain comprising the sequence SEQ ID NO: 9 And the humanized huDS6 antibody of the light chain of sequence SEQ ID NO: 10. The linking group is part of the chemical moiety covalently bonded to the tomycin derivative by conventional methods. In a specific embodiment, the chemical moiety can be covalently bound to the tomycin derivative via a disulfide bond.

The tomycin derivatives that can be used in the present invention have the formula (XII) shown below:

Where --- represents an optional single bond; Represents a single or double bond; the condition is when When representing a single bond, U and U 'are the same or different, independently represent H, and W and W' are the same or different, independently selected from OH, ethers such as -OR, esters (such as acetate) such as -OCOR, Carbonates such as -OCOOR, carbamates such as -OCONRR ', cyclic carbamates such that N10 and C11 are part of the ring, ureas such as -NRCONRR', thiocarbamates such as -OCSNHR, cyclothioamino Formate so that N10 and C11 are part of the ring, -SH, sulfide such as -SR, sulfonate such as -SOR, sulfone such as -SOOR, sulfonate such as -SO _3- , sulfonamide such as -NRSOOR, amine Such as -NRR ', optionally cyclic amines such that N10 and C11 are part of the ring, hydroxylamine derivatives such as -NROR', amides such as -NRCOR ', azido groups such as -N ₃ , cyano, halogen, trioxane Radicals or triarylphosphonium, amino acid derived groups.

Preferably W and W 'are the same or different, and are OH, Ome, Oet, NHCONH ₂ , SMe; and when When representing a double bond, U and U 'do not exist, and W and W' represent H; ￭R1, R2, R1 ', R2' are the same or different and are independently selected from one or more of Hal, CN, NRR ', CF ₃ , OR, aryl, Het, S (O) _q R optionally substituted alkyl or halide, or R ₁ and R ₂ and R1 ′ and R2 ′ together form a group containing = B and = B '' S double bond.

In one embodiment, R ₁ and R ₂ and R _{1 ′} and R ₂ ′ together form a double bond containing the groups = B and = B ', respectively.

￭B and B 'are the same or different and are independently selected from alkenyl optionally substituted by one or more of Hal, CN, NRR', CF ₃ , OR, aryl, Het, S (O) _q R, or B And B 'represent oxygen atoms.

In one embodiment, B = B '.

In a further embodiment, B = B '== CH ₂ or = CH-CH ₃ , ￭X, X' are the same or different and are independently selected from one or more -O-, -NR-,-(C = O)-, -S (O) _q- .

In one embodiment, X = X '.

In another embodiment, X = X '= 0.

￭A and A 'are the same or different and are independently selected from alkyl or alkenyl groups optionally containing oxygen, nitrogen or sulfur atoms, the alkyl or alkenyl groups are respectively one or more of Hal, CN, NRR', CF _3. OR, S (O) _q R, aryl, Het, alkyl, alkenyl are optionally substituted.

In one embodiment, A = A '.

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

￭Y and Y 'are the same or different and are independently selected from H and OR; in one embodiment, Y = Y'.

In a further embodiment, Y = Y '= O alkyl, more preferably O methyl.

￭T is -NR-, -O-, -S (O) _q or 4-10 membered aryl, cycloalkyl, heterocyclic or heteroaryl, the group is respectively one or more Hal, CN, NRR ', CF ₃ , R, OR, S (O) _q R and / or linker are optionally substituted, or branched alkyl, the branched alkyl is substituted by one or more Hal, CN, NRR' , CF ₃ , OR, S (O) qR and / or a linker are optionally substituted, or a linear alkyl group, the linear alkyl group is one or more of Hal, CN, NRR ′, CF ₃ , OR, S ( O) _q R and / or joint substitution.

In one embodiment, T is a 4-10 membered aryl or heteroaryl group, more preferably phenyl or pyridine, which is optionally substituted with one or more linkers.

The linker contains a linking group. Suitable linking groups are well known in the art and include thiols, sulfides, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups And esterase instability groups. Preferred are disulfide groups and thioether groups.

When the linking group is a group containing a thiol-, sulfide (or so-called thioether-S-) or disulfide bond (-SS-)-, the thiol, sulfide or disulfide group is carried The side chain of can be linear or branched, aromatic or heterocyclic. Those of ordinary skill in the art can easily identify the appropriate side chain.

In one embodiment, the linker has the formula -GD- (Z) _p -SZ '

Where G is a single or double bond, -O-, -S- or -NR-; D is a single bond or -E-, -E-NR-, -E-NR-F-, -EO-, -EOF -, -E-NR-CO-, -E-NR-CO-F-, -E-CO-, -CO-E-, -E-CO-F, -ES-, -ESF-, -E- NR-CS-, -E-NR-CS-F-; wherein E and F are the same or different and are independently selected from linear or branched chain; branched- (OCH ₂ CH ₂ ) _ialkyl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) OCH ₂ CH ₂ ) _i -alkyl-,-(OCH ₂ CH ₂ ) _i -,-(OCH ₂ CH ₂ ) _i cycloalkyl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i heterocyclyl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i aryl (OCH ₂ CH ₂ ) _j -,-(OCH ₂ CH ₂ ) _i Heteroaryl (OCH ₂ CH ₂ ) _j- , -alkyl- (OCH ₂ CH ₂ ) _i alkyl (OCH ₂ CH ₂ ) _j- , -alkyl- (OCH ₂ CH ₂ ) _i- , -alkane Group-(OCH ₂ CH ₂ ) _i cycloalkyl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i heterocyclic group (OCH ₂ CH ₂ ) _j- , -alkyl- (OCH ₂ CH ₂ ) _i aryl (OCH ₂ CH ₂ ) _j- , -alkyl (OCH ₂ CH ₂ ) _i heteroaryl (OCH ₂ CH ₂ ) _j- , -cycloalkyl-alkyl-, -alkyl- Cycloalkyl-, -heterocyclic-alkyl-, -alkyl-heterocyclic-, -alkyl-aryl-, -aryl-alkyl-, -alkyl-heteroaryl-, -heteroaryl -Alkyl-; where i and j The same or different and integers independently selected from 0, 1-2000; Z is linear or branched-alkyl-; p is 0 or 1; Z 'represents H, a thiol protecting group such as COR, R ₂₀ Or SR ₂₀ , wherein R ₂₀ represents H, methyl, alkyl, optionally substituted cycloalkyl, aryl, heteroaryl or heterocyclic ring, provided that when Z ′ is H, the compound is passed through The corresponding compound formed by intramolecular cyclization by adding a thiol group -SH to the imine bond -NH = of one of the PBD parts.

￭n, n 'are the same or different, 0 or 1.

￭q is 0, 1, or 2.

￭R and R 'are the same or different, and are independently selected from H, alkyl, and aryl, and each group is selected from Hal, CN, NRR', CF ₃ , R, OR, S (O) _q R, aryl Group, Het optionally substituted; or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or the polymorphic crystal structure of these compounds or its optical isomers, racemates, diastereomers and Enantiomers.

Compounds of general formula (XII) with geometric and stereoisomers are also part of the present invention.

It is known that the N-10 and C-11 double bonds of tomycin derivatives of formula (XII) are easily reversible in the presence of water, alcohols, thiols, primary or secondary amines, urea and other nucleophiles Converted to the corresponding imine adduct. This process is reversible and can easily regenerate the corresponding tomycin derivative in an aprotic organic solvent, vacuum or high temperature in the presence of a dehydrating agent (Tozuka (1983) J. Antibiotics 36 : 276) .

Therefore, the reversible derivatives of toyamycin 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 as defined in formula (VII) above, W and W 'are the same or different and are selected from The group consisting of: OH, ethers such as -OR, esters (such as acetates) such as -OCOR, -COOR, carbonates such as -OCOOR, carbamates such as -OCONRR ', cyclic carbamate to make N10 And C11 are part of the ring, urea such as -NRCONRR ', thiocarbamate such as -OCSNHR, cyclic thiocarbamate such that N10 and C11 are part of the ring, -SH, sulfide such as -SR, sub碸 如 -SOR, 碸 如 -SOOR, sulfonate such as -SO _3- , sulfonamide such as -NRSOOR, amine such as -NRR ', optionally cyclic amine so that N10 and C11 are part of the ring, hydroxylamine derivatives such as -NROR ', an amine such as -NRCOR, -NRCONRR', an azido such as -N _3, a cyano group, halo, a trialkyl or triarylphosphonium, an amino acid derived group. Preferably, W and W 'are the same or different and are OH, Ome, Oet, NHCONH ₂ , SMe.

The compound of formula (XIII) can therefore be considered as a solvate and contains water when the solvent is water; these solvates can be particularly useful.

In a further embodiment, the tomycin derivative of the present invention is selected from the group consisting of:

￭8,8 '-[1,3-phenylene bis (methyleneoxy)]-bis [(S) -2-ethylene- (E) -yl-7-methoxy-1,2 , 3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5-methoxy-1,3-phenylenebis (methyleneoxy)]-bis [(S) -2-ethylene- (E) -yl-7-methyl Oxy-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[1,5-pentylidenebis (oxy)]-bis [(S) -2-ethylene- (E) -yl-7-methoxy-1,2,3, 11a-Tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[1,4-Butylenebis (oxy)]-bis [(S) -2-ethylene- (E) -yl-7-methoxy-1,2,3,11a -Tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[3-Methyl-1,5-pentylidenebis (oxy)]-bis [(S) -2-ethylene- (E) -yl-7-methoxy-1 , 2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[2,6-pyridinylenebis (oxy)]-bis [(S) -2-ethylene- (E) -yl-7-methoxy-1,2,3, 11a-Tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[4- (3-tert-butoxycarbonylaminopropoxy) -2,6-pyridinylene bis- (methyleneoxy)]-bis [(S) -2-ethylene -(E) -yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5- (3-Aminopropoxy) -1,3-phenylene bis (methyleneoxy)]-bis [(S) -2-ethylene- (E)- Yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5- (N-methyl-3-tert-butoxycarbonylaminopropyl) -1,3-phenylenebis- (methyleneoxy)]-bis [(S)- 2-ethylene- (E) -yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-{5- [3- (4-methyl-4-methyldithio-pentylamino) propoxy] -1,3-phenylene bis (methyleneoxy )}-Bis [(S) -2-Ethylene- (E) -yl-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1 , 4] Benzodiazepine -5-ketone]

￭8,8 '-[5-Acetylthiomethyl-1,3-phenylene bis (methyleneoxy)]-bis [(S) -2-methylene-7-methoxy Yl-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭Bis- {2-[(S) -2-methylene-7-methoxy-5-oxo-1,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1 , 4] Benzodiazepine -8-yloxy] -ethyl} -carbamic acid tert-butyl ester

￭8,8 '-[3- (2-Ethylthioethyl) -1,5-pentylidenebis (oxy)]-bis [(S) -2-methylene-7-methyl Oxy-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5- (N-4-Mercapto-4,4-dimethylbutyryl) amino-1,3-phenylene bis (methyleneoxy)]-bis [7-methoxy Yl-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5- (N-4-methyldithio-4,4-dimethylbutyryl) -amino-1,3-phenylene bis (methyleneoxy)]-bis [7-Methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5- (N-methyl-N- (2-mercapto-2,2-dimethylethyl) amino-1,3-phenylene (methyleneoxy)]- Bis [7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[5- (N-methyl-N- (2-methyldithio-2,2-dimethylethyl) amino-1,3-phenylene (methyleneoxy Group)]-bis [7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[(4- (2- (4-Mercapto-4-methyl) -pentylamino-ethoxy) -pyridine-2,6-dimethyl) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1,4] benzene Diaza -5-ketone]

￭8,8 '-((1- (2- (4-methyl-4-methyldithio) -pentylamino-ethoxy) -benzene-3,5-dimethyl) -diox Radical] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1, 4] Benzodiazepine -5-ketone]

￭8,8 '-((4- (3- (4-methyl-4-methyldithio) -pentylamino-propoxy) -pyridine-2,6-dimethyl) -diox Radical] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1, 4] Benzodiazepine -5-ketone]

￭8,8 '-((4- (4- (4-methyl-4-methyldithio) -pentylamino-butoxy) -pyridine-2,6-dimethyl) -diox Radical] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1, 4] Benzodiazepine -5-ketone]

￭8,8 '-[(4- (3- [4- (4-methyl-4-methyldithio-pentyl) -pyrazin-1-yl] -propyl) -pyridine-2 , 6-dimethyl) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1,4] Benzodiazepine -5-ketone]

￭8,8 '-[(1- (3- [4- (4-Methyl-4-methyldithio-pentyl) -pentazin-1-yl] -propyl) -benzene-3 , 5-dimethyl) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1,4] Benzodiazepine -5-ketone]

￭8,8 '-[(4- (2- {2- [2- (4-methyl-4-methyldithio-pentylamino) -ethoxy] -ethoxy} -ethyl Oxy) -pyridine-2,6-dimethyl) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3, 11a-Tetrahydro-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[(1- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentylamino) -ethoxy Group] -ethoxy} -ethoxy) -ethoxy] -ethoxy} -ethoxy) -benzene-3,5-dimethyl) -dioxy] -bis [(S)- 2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1,4] benzodiazepines -5-ketone]

￭8,8 '-[(1- (2- {2- [2- (4-methyl-4-methyldithio-pentylamino) -ethoxy] -ethoxy} -ethyl Oxy) -benzene-3,5-dimethyl) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3, 11a-Tetrahydro-pyrrole [2,1-c] [1,4] benzodiazepine -5-ketone]

￭8,8 '-[(4- (2- {2- [2- (2- {2- [2- (4-methyl-4-methyldithio-pentylamino) -ethoxy Group] -ethoxy} -ethoxy) -ethoxy] -ethoxy} -ethoxy) -pyridine-2,6-dimethyl) -dioxy] -bis [(S)- 2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1,4] benzodiazepines -5-ketone]

￭8,8 '-[(1- (2- [methyl (2-methyl-2-methyldithio-propyl) -amino] -ethoxy) -benzene-3,5-dimethyl Group) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1- c] [1,4] Benzodiazepine -5-ketone]

￭8,8 '-[(4- (3- [Methyl (4-methyl-4-methyldithio-pentyl) -amino] -propyl) -pyridine-2,6-dimethyl Group) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1- c] [1,4] Benzodiazepine -5-ketone]

￭8,8 '-[(4- (3- [methyl- (2-methyl-2-methyldithio-propyl) -amino] -propyl) -pyridine-2,6-dimethyl Group) -dioxy] -bis [(S) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1- c] [1,4] Benzodiazepine -5-ketone]

￭ 8,8 '-[(1- (4-methyl-4-methyldithio) -pentylamino) -benzene-3,5-dimethyl) -dioxy] -bis [(S ) -2-ethylene- (E) -yl-7-dimethoxy-1,2,3,11a-tetrahydro-pyrrole [2,1-c] [1,4] benzodiazepines -5-ketone]

And the corresponding mercapto derivatives or their pharmaceutically acceptable salts, hydrates or hydrated salts or the polymorphic crystal structures of these compounds or their optical isomers, racemates, diastereomers and enantiomers isomer.

Specific compounds are those represented by the following formula (XIV) or (XV):

or

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

The compound of formula (XII) can be prepared according to many methods known to those skilled in the art. The compound can be synthesized by adopting or adapting the method described below or a change that can be understood by those skilled in the art. Appropriate modifications and substitutions are easily understood by those skilled in the art, well known, or readily available through scientific literature. Specifically, these methods can be found in RC Larock, Comprehensive Organic Transformations , Wiley-VCH Publishers, 1999.

Methods for synthesizing tomycin derivatives that can be used in the present invention are described in International Application No. WO2007 / 085930. The compounds of the present invention can be prepared by various synthetic routes. Reagents and raw materials are commercially available or can be easily synthesized by those of ordinary skill in the art through well-known techniques (see, for example, WO 00/12508, WO 00/12507, WO 2005/040170, WO 2005/085260, FR1516743, Mori, etc. , (1986) Tetrahedron , 42 : 3793-3806).

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

According to the present invention, "finemycin derivative " refers to a member of the finemycin family defined in Kalesse et al. (2002) Synthesis 8 : 981-1003, and includes: finemycins such as finemycin A and fine B, Callystatins, Ratjadone such as Ratjadone A and Ratjadone B, Anguinomycin such as Anguinomynomycin A, B, C, D, kasusamycin, Leptolstatin, Leptofuranin such as Leptofuranin A, B, C, D. Derivatives of finemycin A and B are preferred.

More specifically, the finemycin derivative of the present invention may have the formula (XVI):

Where Ra and Ra 'are H or -AIk; preferably, Ra is -AIk, preferably methyl and Ra' is H; R17 is an alkyl group, the alkyl group is OR, CN, NRR ', perfluoroalkyl Optional substitution; preferably R17 is alkyl, more preferably methyl or ethyl; R9 is alkyl, the alkyl is 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-, -OU-, -NR-CO-U-, -U-NR -CO-, -U-CO-, -CO-U-; preferably when X is -O-, Y is -U-, -NR-U-, -U-NR-CO-; wherein U is selected from straight Chain or branched chain -Alk-, -Alk (OCH ₂ CH ₂ ) _m -,-(OCH ₂ CH ₂ ) _m -Alk-, -Alk (OCH ₂ CH ₂ ) _m -Alk-,-(OCH ₂ CH ₂ ) _m- , -cycloalkyl-, -heterocycle-, -cycloalkyl-Alk-, -AIk-cycloalkyl-, -heterocycle-Alk-, -Alk-heterocycle-; wherein m is selected from Integer of 1-2000; preferably, U is linear or branched-Alk-, Z is -Alk-; n is 0 or 1; preferably, n is 0; T represents H, thiol protecting group such as Ac , R ₁ or SR ₁ , wherein R ₁ represents H, methyl, Alk, cycloalkyl, optionally substituted aryl or heterocycle, or T represents

Where: Ra, Ra ', R17, R9, X, Y, Z, n are as defined above; preferably, T is H or SR ₁ , where R ₁ represents Alk, more preferably methyl; R and R' are the same or Different, H or alkyl; Alk stands for straight-chain or branched-chain alkyl; preferably Alk stands for (-(CH _2-q (CH ₃ ) _q ) _p- , where p stands for an integer from 1 to 10, and q stands for 0 to Integer of 2; preferably, Alk represents-(CH ₂ )-or -C (CH ₃ ) ₂ -or its pharmaceutically acceptable salts, hydrates or hydrated salts, or the polymorphic crystal structure of these compounds or their optical Isomers, racemates, diastereomers and enantiomers.

Specific compounds can be selected from:

￭ (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-nadeca-2,10,12,16,18-pentenoic (2 -Methylthio-ethyl) -amide

￭ (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-nineteen carbon-2,10,12,16,18-pentenoic acid) -[(2-Mercaptoethyl) -amide

￭ (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-nadeca-2,10,12,16,18-pentenoic (2 -Mercaptoethyl) -amide

￭ (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-nadeca-2,10,12,16,18-pentenoic (2 -Methyldithio-ethyl) -amide

￭ (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-nadeca-2,10,12,16,18-pentenoic (2 -Methyl-2-methyldithio-propyl) -amide

￭ (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-nadeca-2,10,12,16,18-pentenoic (2 -Mercapto-2-methyl-propyl) -amide

Or a pharmaceutically acceptable salt, hydrate or hydrated salt thereof, or the polymorphic crystal structure of these compounds or their optical isomers, racemates, diastereomers and enantiomers.

In order to link the derivative to the cell binding agent as defined in the " Cell Binding Agent" section above, the derivative must contain a moiety (linking group) that allows the derivative to be linked to the cell binding agent via a linking group, wherein the linking Groups such as disulfide bonds, sulfides (or referred to herein as thioethers), acid labile groups, photolabile groups, peptidase labile groups or esterase labile groups. The derivative is prepared so that it contains the necessary parts to connect the finemycin derivative to the cell binding agent, such as via disulfide bond, thioether bond, acid labile group, photolabile group, peptidase labile group or Esterase instability group. To further enhance the solubility in aqueous solution, the linking group may contain a polyethylene glycol spacer. In one embodiment, a sulfur or disulfide linker is used, because the reducing environment of the target cell can cause sulfide or disulfide cleavage, and release derivatives accompanied by increased cytotoxicity.

The compounds of the present invention can be prepared by various synthetic routes. Reagents and raw materials are commercially available or can be easily synthesized by a person of ordinary skill in the art through well-known techniques. A method for synthesizing a finemycin derivative that can be used in the cytotoxic conjugate of the present invention and a method for conjugating the finemycin derivative to a cell binding agent such as an antibody are described in detail in European Patent Application No. EP1864682 in.

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

CC-1065 is an effective anti-tumor antibiotic, which is isolated from the culture broth of Streptomyces zelensis . CC-1065 is about 1,000 times more potent in vitro than commonly used anticancer drugs such as doxorubicin, methotrexate, and vincristine (BKBhuyan et al., Cancer Res. 42: 3532-3537). CC-1065 and its analogs are disclosed in US Patent Nos. 6,372,738, 6,340,701, 5,846,545, and 5,585,499.

The cytotoxic efficacy of CC-1065 is related to its alkylation activity and its DNA-binding or DNA-insertion activity. These two activities are located in different parts of the molecule. Therefore, the alkylation activity is contained in the cyclopropapyrroloindole (CPI) subunit, and the DNA-binding activity is in the two pyrrolidine indole subunits.

Although CC-1065 has certain attractive features as a cytotoxic agent, it has limitations in therapeutic use. Administration of CC-1065 to mice resulted in delayed hepatotoxicity, leading to death on the 50th day after a single intravenous dose of 12.5 μg / kg (Reynolds et al. (1986) J. Antibiotics XXIX: 319-334)). This has stimulated efforts to develop analogues that do not cause delayed toxicity, and the synthesis of simpler analogues using CC1065 as a model has been described (MA. Warpehoski et al., 1988, J. Med. Chem. 31 590- 603.

In another series of analogues, the CPI portion was replaced with a cyclopropyl phenylindole (CBI) portion (DL Boger et al., 1990, J. Org. Chem. , 55: 5823-5833; DL Boger et al . , 1991, BioOrg .Med . Chem . Lett . 1: 115-120 ). These compounds maintain the high in vitro potency of the parent drug without causing delayed toxicity in mice. Similar to CC-1065, these compounds are alkylating agents that bind to the minor groove of DNA in a covalent mode to cause cell death. However, clinical evaluations of the most promising analogues, Adozelesin and Carzelesin, have yielded disappointing results (BFFoster et al., 1996, Investigational New Drugs , 13: 321-326; I. Wolff et al., 1996, Clin. Cancer Res., 2: 1717-1723). These drugs show poor therapeutic efficacy because of their high systemic toxicity.

By changing the distribution in vivo through targeted delivery to the tumor site, low toxicity to non-targeted tissues is obtained, and thus lower system toxicity is obtained, which can greatly improve the therapeutic efficacy of CC-1065 analogs. In order to achieve this goal, conjugates of CC-1065 analogs and derivatives with cell binding agents that specifically target tumor cells have been described (US Pat. Nos. 5,475,092; 5,585,499; 5,846,545). Generally these conjugates show high target-specific cytotoxicity in vitro and exhibit excellent antitumor activity in mouse human tumor xenograft models (Chari et al., 1995, Cancer Res. , 55: 4079-4084 ).

Recently, prodrugs of CC-1065 analogues with enhanced solubility in aqueous media have been described (European Patent Application EP1832577). In these prodrugs, the phenol group of the alkylated part of the molecule is protected with a functional group, which makes the drug stable when stored in an acidic aqueous solution, and provides the drug with improved water solubility compared to unprotected analogs. The protecting group is easily cleaved at physiological pH in vivo to provide corresponding active drugs. In the prodrugs described in EP1832577, the phenol substituent is protected as sulfonic acid-containing phenyl carbamate, which has a charge at physiological pH, and thus has enhanced water solubility. To further enhance water solubility, an optional polyethylene glycol spacer can be introduced into the linker between the indole subunit and the cleavable linker such as a disulfide group. The introduction of this spacer does not change the efficacy of the drug.

Methods for synthesizing CC-1065 analogs that can be used in the cytotoxic conjugates of the present invention and methods for conjugating these analogs to cell binding agents such as antibodies are described in detail in EP1832577 and U.S. Patent Nos. 5,475,092, 5,846,545, 5,585,499, 6,534,660 and 6,586,618 and US applications US2003 / 0199519 and US2003 / 0195365.

Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C ), Chlorambucil, calicheamicin, tubulysin and tubulysin analogues, duocarmycin and dokamycin analogues, dolastatin and dorastatin Statin analogs are also suitable for the preparation of the conjugates of the invention. The drug molecule can also be linked to the antibody molecule through an intermediary carrier molecule such as hemoglobin. Doxarubicin and Danorubicin compounds as described in, for example, US Patent No. 6,630,579 can also be effective cytotoxic agents.

In a specific embodiment of the invention, the at least one cytotoxic agent is maytansine DM1 of formula (I). In another specific embodiment of the invention, the at least one cytotoxic agent is maytansine DM4 of formula (II).

These cytotoxic agents are conjugated to cell binding agents, antibodies, and epitope binding fragments of antibodies as disclosed herein.

Connector

As used herein, " linker " means a chemical moiety that contains a covalent bond or chain of atoms that covalently connects a polypeptide to a drug moiety.

Conjugates can be prepared by in vitro methods. To connect drugs or prodrugs to cell binding agents, especially antibodies, linking groups are 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 Group.

Conjugation of the cell binding agent as defined in the " Cell Binding Agent " section above, particularly the antibody of the present invention, with the cytotoxic agent as defined in the " Cytotoxic Agent" section above can be made using a variety of bifunctional protein conjugating agents. Functional protein coupling agents include but are not limited to N -succinimidopyridyl dithiobutyrate (SPDB), butyric acid 4-[(5-nitro-2-pyridyl) disulfide] -2, 5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4- (pyridin-2-yldithio) -2-sulfo-butyric acid (sulfo-SPDB), (2-pyridine Dithio) propionic acid N -succinimidyl ester (SPDP), ( N -maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), Amino sulfane (IT), bifunctional derivatives of imino esters (e.g. dimethyl adipimidate HCL), active esters (e.g. disuccinimide suberate ( disuccinimidyl suberate)), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzyl) hexamethylenediamine), bis-diazo derivatives such as bis- (p- (Diazobenzyl) -ethylenediamine, diisocyanate (such as toluene 2,6-diisocyanate) and di-active Fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

In a specific embodiment, the linker is selected from the group consisting of: N-succinimidopyridyl dithiobutyrate (SPDB), 4- (pyridin-2-yldithio)- 2-sulfo-butyric acid (sulfo-SPDB) and (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester (SMCC).

The cell binding agent of the conjugate of the present invention can be covalently linked to at least one cytotoxic agent via a cleavable or non-cleavable linker.

The linker may be a " cleavable linker " that promotes the release of cytotoxic agents in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase labile linker, a photolabile linker, or a disulfide-containing linker can be used (for example, see US Patent No. 5,208,020). The linker may also be a " non-cleavable linker " (such as an SMCC linker) that may cause better tolerance in some cases.

Alternatively, a fusion protein comprising a cell binding agent as defined above in the " Cell Binding Agent" section of the present invention, especially an antibody and a cytotoxic polypeptide, can be produced by recombinant technology or peptide synthesis. The length of the DNA may include the respective regions encoding the two parts of the conjugate, which are adjacent to each other or separated by a region encoding a linker peptide that does not destroy the expected properties of the conjugate.

The cell-binding agents of the present invention, particularly antibodies, can also be used in dependent enzyme-mediated prodrug therapy by conjugating polypeptides with enzymes that activate prodrugs, which enzymes prodrugs (eg, peptide-based chemotherapeutic agents, (See WO81 / 01145) to active anticancer drugs (for example, see WO 88/07378 and US Patent No. 4,975,278). The enzyme component of the immunoconjugate that can be used in ADEPT contains any enzyme that can act on the prodrug in a manner that converts the prodrug into its active, more cytotoxic form. Enzymes that can be used in the method of the present invention include, but are not limited to: alkaline phosphatase, which can be used to convert phosphate-containing prodrugs to free drugs; arylsulfatase, which can be used to convert sulfate-containing prodrugs It is a free drug; cytosine deaminase, which can be used to convert non-toxic fluorocytosine into the anticancer drug 5-fluorouracil; proteases, such as serratia protease, thermophilic protease, subtilisin, carboxyl Peptidases and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs into free drugs; D-propylamine carboxypeptidase, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrates Compound lyases, such as O-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs to free drugs; P-endoamidase, which can be used to convert P-endoamidamide Derivative drugs are converted to free drugs; and penicillin (a penicillin) amidase, such as penicillin V amidase or penicillin G amidase, which can be used to place the amine nitrogen via phenoxyacetamide or phenylacetamide, respectively The radical-derived drugs are converted into free drugs. The enzyme can be covalently bound to the polypeptide of the present invention by techniques well known in the art, such as using the heterobifunctional cross-linking reagents discussed above.

According to a specific embodiment, in the conjugate of the present invention, the cytotoxic agent is maytansinoid, specifically DM1 or DM4.

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

In a specific embodiment, the linker is N-succinimidopyridyl dithiobutyrate (SPDB) and the cytotoxic agent is DM4. In another specific embodiment, the linker is 4- (pyridin-2-yldithio) -2-sulfo-butyric acid (sulfo-SPDB) and the cytotoxic agent is DM4.

More specifically, the conjugate is selected from the group consisting of:

i) Antibody-SPDB-DM4 conjugate of formula (XVIII)

ii) Antibody-sulfo-SPDB-DM4 conjugate of formula (XIX)

And

iii) Antibody-SMCC-DM1 conjugate of formula (XX)

Generally, the conjugate can be obtained by a method including the following steps: (i) contacting an optionally buffered aqueous solution of a cell binding agent (eg, an antibody according to the invention) with a solution of a linker and a cytotoxic compound; (ii) then , Optionally separating the conjugate formed in (i) from the unreacted cell binding agent.

The aqueous solution of the cell binding agent may be buffered with a buffer such as, for example, potassium phosphate, acetate, citrate or N-2-hydroxyethylpyrazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer depends on the nature of the cell binding agent. The cytotoxic compound is in a solution in an organic polar solvent, such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA).

The reaction temperature is usually 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 refractometry and / or UV detector. If the yield of the conjugate is too low, the reaction time can be extended.

A person skilled in the art can use a variety of different chromatography methods to perform the separation of step (ii): for example, by SEC, adsorption chromatography (such as ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography , Mixed support chromatography such as hydroxyapatite chromatography or high performance liquid chromatography (HPLC) to purify the conjugate. Purification by dialysis or diafiltration can also be used.

As used herein, the term " aggregate " means an association that can be formed between two or more cell-binding agents, the agent being modified by conjugation or otherwise. Aggregates can be formed under the influence of many parameters, such as high concentration of cell binder in solution, pH of solution, high shear force, number of bonded dimers and their hydrophobic characteristics, and temperature (see Wang and Gosh, 2008, J. Membrane Sci., 318: 311-316, and references cited therein); note that the relative influence of some of these parameters has not been established. In the case of proteins and antibodies, those skilled in the art will refer to Cromwell et al. (2006, AAPS Jounal, 8 (3): E572-E579). The aggregate content can be determined using techniques well known to the skilled person, such as SEC (see Walter et al., 1993, Anal. Biochem., 212 (2): 469-480).

After step (i) or (ii), the solution containing the conjugate can be subjected to additional chromatography, ultrafiltration and / or diafiltration steps (iii).

At the end of these steps the conjugate is recovered in the aqueous solution.

In the embodiment of the present invention where the cytotoxic agent is a maytansinoid, in order to link the maytansinoid to a cell binding agent as defined in the " Cell Binding Agent " section above, such as a heavy chain comprising the sequence SEQ ID NO: 9 and The humanized huDS6 antibody of the light chain of sequence SEQ ID NO: 10 is linked, and maytansinoid may comprise a linking portion. The linking part contains chemical bonds that allow the release of fully active maytansinoids at specific sites. 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. It is preferably a disulfide bond.

The connecting part also contains reactive chemical groups. In an embodiment, the reactive chemical group may be covalently linked to the maytansinoid via a disulfide bond linking moiety.

Specific reactive chemical groups are N -succinimide and N -sulfosuccinimide.

A special maytansinoid biotin containing a linking part containing a reactive chemical group is C-3 ester of maytansinol and its analogs, where the linking part contains a disulfide bond, and the chemically reactive group contains N -amber Amide imino or N -sulfosuccinimide ester.

Many positions on maytansinoid can function as the position of chemical connection with the linking group. For example, the C-3 position having a hydroxy group, the C-14 position having a hydroxymethyl modification, the C-15 position having a hydroxy modification, 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 particularly preferred.

Although in terms of the linking moiety containing a disulfide bond, the ester synthesis of maytansinol having a linking moiety has been described, those skilled in the art will understand that linking moieties with other chemical bonds (as described above) can also be used Inventions are just like other maytansine. Specific examples of other chemical bonds include acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds. The content of US Patent No. 5,208,020 teaches the production of maytansinoids having such bonds.

The synthesis of maytansinoids and maytansinoid derivatives having a disulfide moiety carrying reactive groups is described in US Patent Nos. 6,441,163 and 6,333,410 and US Application No. 2003/0055226.

Maytansinoids such as DM1 containing reactive groups and cell binding agents as defined above in the section " Cell binding agents ", especially antibodies (such as the heavy chain comprising the sequence SEQ ID NO: 9 and the sequence SEQ ID NO: Humanized huDS6 antibody of the light chain of 10) reacted to produce a cytotoxic conjugate. These conjugates can be purified by HPLC or by gel filtration.

Several superiorities for producing such cell-binding agents-maytansinoid, especially antibody-maytansinoid conjugates are provided in U.S. Patent Nos. 6,333,410 and 6,441,163 and U.S. Application Nos. 2003/0055226 and 2004/0001838 Scheme.

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

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

Maytansinoids and cell binding agents can be linked using PEG linking groups, as described in US Patent Application No. 2004/0001838. These PEG linking groups are soluble in both water and non-aqueous solvents, and can be used to join one or more cytotoxic agents to cell binding agents. Exemplary PEG linking groups include heterobifunctional PEG linkers that are bonded at one end through a functional sulfhydryl or disulfide group and at the other end through an active ester with a cytotoxic agent and a cell binding agent at the opposite end of the linker.

As a general example of the synthesis of cytotoxic conjugates using PEG linking groups, specific details refer again to US Application No. 2004/0001838. Synthesis begins with the reaction of one or more cytotoxic agents with reactive PEG moieties and cell binding agents, resulting in the passage of cell binding agents (eg, the heavy chain comprising sequence SEQ ID NO: 9 and the light chain comprising sequence SEQ ID NO: 10 Humanized huDS6 antibody) amino acid residues to replace the terminal active ester of each reactive PEG moiety to obtain a cytotoxic conjugate comprising one or more cytotoxic agents covalently bonded to the cell binding agent via a PEG linking group Compound.

Any technique can be used to form the conjugate molecules of the invention. Specifically, the tomycin derivative of the present invention can be connected to an antibody or other cell binding agent as defined in the " Cell Binding Agent " section above via an acid-labile linker, or through a photolabile linker. The derivative can be condensed with a peptide having a suitable sequence and then linked to a cell binding agent to generate a peptidase-labile linker. The conjugate can be prepared to contain a primary hydroxyl group, which can be succinated, and attached to a cell binding agent to produce a conjugate that can release a free derivative by intracellular esterase cleavage. Preferably, the derivative is synthesized to contain a free or protected thiol group, and then one or more disulfide- or thiol-containing derivatives are combined with the cell binding agent via disulfide bonds or thioether linkages, respectively.价 连接。 Price connection.

Various conjugation methods are taught in US Patent Nos. 5,416,064 and 5,475,092. The tomycin derivative can be modified to obtain a free amino group, and then attached to an antibody or other cell binding agent via an acid-labile linker or a photolabile linker. A tomycin derivative having a free amino or carboxyl group can be condensed with a peptide and then attached to a cell binding agent to generate a peptidase-labile linker. The tomycin derivative having a free hydroxyl group on the linker can be succinated and connected to a cell binding agent to produce a conjugate that can release the free drug by intracellular esterase cleavage. Most preferably, the tomycin derivative is treated to generate free or protected thiol groups, and then the disulfide- or thiol-containing tomycin dimer is connected to the cell binding agent via a disulfide bond.

In one embodiment, the monoclonal antibody or cell-binding agent-tomycin derivative conjugates are those conjugates linked via disulfide bonds, which are capable of delivering tomycin derivatives as discussed above. Such cell binding is prepared by known methods such as by modifying monoclonal antibodies with succinimidopyridyl-dithiopropionate (SPDP) (Carlsson et al., 1978, Biochem. J., 173: 723-737) Conjugate. The resulting thiopyridyl group is then replaced by treatment with a thiol-containing toyomycin derivative to generate a disulfide-linked conjugate. Alternatively, in the case of an aryl dithiomyrophyllin derivative, the cell binding conjugate is affected by directly replacing the aryl-thiol of the tomycin derivative with a thiol group previously introduced into the antibody molecule Formation. Conjugates containing 1 to 10 toyamycin derivative drugs linked via disulfide bridges can be easily prepared by either method.

More specifically, 2.5 mg / ml concentration in a 0.05M potassium phosphate buffer (pH 7.5) containing 2mM EDTA was treated with a tomycin derivative containing thiols (1.3 molar equivalents / dithiopyridyl group) Solution of the antibody modified with dithio-nitropyridyl. The thiol nitropyridine released from the modified antibody was monitored spectrophotometrically at 325 nm, and was completed in about 16 hours. The antibody-toyamycin derivative conjugate is purified by gel filtration through a Sephadex G-25 or Sephacryl S300 column, and unreacted drugs and other low molecular weight materials are removed. The number of tomycin derivative moieties bound to each antibody molecule can be determined by measuring the absorbance ratio at 230 nm and 275 nm. By this method, an average of 1-10 tomycin derivative molecules / antibody molecules can be connected via disulfide bonds.

The method previously described by Liu et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 8618-8623 can be used to determine the effect of conjugation on the binding affinity against antigen-expressing cells. The cytotoxicity of tomycin derivatives and their antibody conjugates to cell lines can be measured by the backstepping of cell proliferation curves, as described by Goldmacher et al., 1985, J Immunol., 135 3648-3651. The cytotoxicity of these compounds on adherent cell lines can be determined by a cloning method, as described by Goldmacher et al., 1986, J. Cell Biol., 102: 1312-1319.

Drug to antibody ratio

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

This DAR value can vary depending on the nature of the cell binding agent used, especially antibodies and drugs (i.e. cytotoxic agents) and the experimental conditions used for conjugation (e.g. cytotoxic agent / cell binding agent ratio, reaction time, solvent and co-solvent ( If there is a) nature) and change. Therefore, the contact between the cell binding agent and the cytotoxic agent results in a conjugate comprising several drug to antibody ratios different from each other; optionally naked cell binding agent; optionally aggregates. Therefore, the measured DAR is an average value.

A method that can be used to determine DAR (herein DAR UV) includes measuring the ratio of the absorbance of the substantially purified conjugate solution at λ _D to 280 nm spectrophotometrically. 280 nm is a wavelength commonly used to measure protein concentration, such as antibody concentration. The wavelength λ _{D is} selected to allow discrimination between the drug and the antibody, that is, as known to those skilled in the art, λ _D is a wavelength at which the drug has a high absorbance and the distance between λ _D and 280 nrm is sufficiently far to avoid a large overlap of absorption peaks between the drug and the antibody. In the case of maytansinoid molecules, λ _D may be selected to be 252 nm. The method of DAR calculation can be derived from Antony S. Dimitrov (ed.), LLC, 2009, Therapeutic Antibodies and Protocols, Vol. 525, 445, Springer Science: Conjugates at λ _D (A _λD ) and 280 nm (A ₂₈₀ ) The absorbance is measured using a typical spectrophotometer device (to allow calculation of "DAR" parameters). The absorbance can be expressed as follows: A _λD = ( c _D x ε _{D λD} ) + ( c _A x ε _{A λD} )

A ₂₈₀ = ( c _D x ε _{D 280} ) + ( c _A x ε _{A 280} )

Where: c _D and c _A are the concentration of drug and antibody in the solution

ε _DλD and ε _D280 are the molar extinction coefficients of the drug at λ _D and 280 nm, respectively

ε _AλD and ε _A280 are the molar extinction coefficients of the antibody at λ _D and 280 nm, respectively.

Analysis of these two equations with two unknowns yields the following equation: c _D = [( ε _{A 280} x A _λD )-( ε _{A λD} x A ₂₈₀ )] / [( ε _{D λD} x ε _{A 280} )-( ε _{A λD} x ε _{D 280} ))

c _A = [ A _280- ( c _D x ε _{D 280} )] / ε _{A 280}

The average DAR is then calculated from the ratio of drug concentration to antibody concentration: DAR = c _D / c _A.

In a specific embodiment, λ _D is 252 nm.

Therefore, in a specific embodiment, the conjugate is characterized by a drug-to-antibody ratio (DAR) of 3-4, specifically 3.5, from the concentration of cytotoxic agent (c _D ) to the concentration of cell-binding agent (c _A ) ratio calculation DAR; DAR = c _D / c _A

Where c _D = [( ε _{A 280} × A ₂₅₂ )-( ε _{A 252} × A ₂₈₀ )] / [( ε _{D 252} × ε _{A 280} )-( ε _{A 252} × ε _{D 280} )] c _A- [ A _280- ( c _D × ε _{D 280} )) / ε _{A 280}

And ε _D252 and ε _D280 are the molar extinction coefficients of cytotoxic agents at ₂₅₂ nm and 280 nm, ε _A252 and ε _A280 are the molar extinction coefficients of cell binding agents at ₂₅₂ nm and 280 nm, and A ₂₅₂ and A ₂₈₀ are conjugates, respectively. The absorbance at ₂₅₂ nm (A ₂₅₂ ) and 280 nm (A ₂₈₀ ) was measured using classic spectrophotometric equipment.

treatment

The present inventors proved that patients with cancer, especially breast cancer, lung cancer, or bladder cancer patients, more specifically breast cancer patients, when huDS6-DM4 is administered to him / her as follows, show that the tumor does not progress or the tumor subsides but has a comparative Low ocular toxicity: within at least two cycles of administration, weekly doses corresponding to repeated doses of at least 60 mg / m ² as a new cycle of administration.

The present invention therefore relates to a conjugate for use in a method of treating cancer, comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, as defined in the section " Cell binding agent " above, which Linked to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent" section above ), wherein the method comprises administering the conjugate as follows: within at least two cycles, during each cycle, the The conjugate is administered at least twice during each cycle at a planned dose of at least 60 mg / m ² per week corresponding to the cycle.

The present invention also relates to the use of conjugates for the manufacture of pharmaceuticals aimed at treating cancer, the conjugates comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, as described above for " cell binding" Definition of " agent ", which is linked to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above), wherein the conjugate is administered as follows: within at least two cycles, where in each cycle The conjugate was administered at least twice during each cycle at a planned dose corresponding to a weekly dose of at least 60 mg / m ² of the cycle.

The present invention also relates to a method of treating cancer in a patient in need thereof, the method comprising administering a conjugate to a patient in need thereof, the conjugate comprising (i) and human mucin-1 (MUC1) glycoprotein-bound cell-binding agent (as defined above in the " Cell-Binder " section), which is linked to (ii) at least one cytotoxic agent (as defined above in the " Cytotoxic Agent " section): in at least two cycles Within, where in each cycle, the conjugate is administered at least twice during each cycle at a planned dose corresponding to a weekly dose of at least 60 mg / m ² of the cycle.

In the context of the present invention, as used herein, the term " treating " or " treating " means reversing, alleviating the condition or condition to which the term is applied, inhibiting the progress of the condition or condition, or preventing the condition or condition, Or one or more symptoms of these conditions or conditions.

The term " treating cancer " as used herein means inhibiting the growth of tumor malignant cells and / or the process of metastasis from the tumor. Therefore, this treatment can make the tumor not progress, especially for more than 12 weeks. This treatment also allows tumor growth to subside, which is a measurable decrease in tumor size. In specific embodiments, this treatment causes the tumor or metastasis to partially regress. In another specific embodiment, this treatment completely regresses the tumor or metastasis.

According to the present invention, the terms " patient " or " patient in need thereof " are intended to target human or non-human mammals affected or likely to be affected by malignant tumors.

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

The conjugate of the present invention is administered in a therapeutically effective amount, that is, an amount of conjugate that is applicable to any medical treatment at a reasonable benefit / risk ratio and is sufficient to treat the cancer disease. However, it can be understood that the total daily dosage of the conjugate of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the condition to be treated and the severity of the condition; the activity of the specific conjugate used; the specific composition used, the patient's age, weight, General health, gender and diet; time of administration, route of administration and excretion rate of the specific conjugate used; duration of treatment; drugs used in combination or concurrent use with the specific conjugate used and well known in the medical field factor.

In a specific embodiment, the therapeutically effective amount of the conjugate administered to the patient is the dose planned for the administration of a dose of 60 to 80 mg / m ² per week corresponding to each cycle of the treatment method. In a specific embodiment, the therapeutically effective amount of the conjugate administered to the patient is the dose planned for the weekly dose of 60 mg / m ² corresponding to each cycle of the treatment method. In one embodiment, the cycle is a three-week period, and the therapeutically effective amount of the conjugate administered to the patient is a dose of 90 mg / m ² on the first day of each cycle of the treatment method, And another dose of 90 mg / m ² on the 8th day of each cycle of the treatment method.

It should be further noted that the therapeutically effective amount is also a safe dose with regard to eye disorders in particular.

In another specific embodiment, the cycle is a three-week period, and the therapeutically effective amount of the conjugate administered to the patient is 120 mg / m ^{2 on} the first day of each cycle of the treatment method Dose, and another dose of 120 mg / m ² on the 8th day of each cycle of the treatment method.

According to the present invention, the conjugate is administered repeatedly during the treatment method according to a protocol that includes at least two cycles, and the conjugate is administered at least twice during each cycle.

In a specific embodiment, the number of cycles is two. In a more specific embodiment, the number of cycles is 3, 4 or 6.

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

In a more specific embodiment, the cycle is a two-week period, and in each cycle, the conjugate is administered at a dose of 120 mg / m ² on the first day of the cycle.

The conjugates of the present invention can be administered in the form of a pharmaceutical composition comprising a pharmaceutically acceptable excipient, and optionally a sustained release matrix such as a biodegradable polymer to form a therapeutic composition.

" Pharmaceutically " or " pharmaceutically acceptable " refers to molecular entities and compositions that do not produce adverse, allergic, or other adverse reactions when administered to mammals, especially humans (if appropriate). Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of formulation aids.

The form and administration route of the pharmaceutical composition containing the conjugate of the present invention naturally depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient.

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

In particular, the pharmaceutical composition comprising the conjugate of the present invention may contain a vehicle that is pharmaceutically acceptable for formulations that can be injected. These may specifically be isotonic, sterile, saline solution (monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or a mixture of said salts), or sterile water may be added as appropriate Or physiological saline is allowed to constitute a dry, especially lyophilized composition of the injectable solution.

For preparing pharmaceutical compositions, an effective amount of the conjugate of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

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

The carrier can also be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (such as glycerin, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions and by using surfactants, stabilizers, cryoprotectants or antioxidants. The prevention of microbial effects can be brought about by antibacterial and antifungal agents. In many cases, it is preferable to include isotonic agents such as sugar or sodium chloride.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle which contains a 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 preparation methods are vacuum drying and freeze-drying techniques, which can produce a powder consisting of the active ingredient plus any desired additional ingredients from a solution previously sterile filtered.

After formulation, the solution will be administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulation can be easily administered in various dosage forms such as the injectable solution types described above, but drug release capsules and the like can also be used.

For example, for parenteral administration of aqueous solutions, the solution (if necessary) should be properly buffered and the liquid diluent is first made isotonic with the aid of sufficient saline or glucose. These specific aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, under the inspiration of the present invention, those skilled in the art will know the sterile aqueous medium that can be used. For example, a dose can be dissolved in 1 ml of NaCl isotonic solution and added to 1000 ml of subcutaneous injection or injected at the proposed infusion site (for example, see "Remington's Pharmaceutical Sciences" 15th edition, page 1035 1038 and 1570-1580). Depending on the condition of the subject being treated, the dosage will inevitably vary. The person responsible for administration will in any case determine the appropriate dose for the individual subject.

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

The cancer to be treated according to the present invention includes any type of malignant cancer, specifically solid tumors such as breast cancer, lung cancer, more specifically non-small cell 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, when the intensity of at least 30% of the cells of the sample measured by immunohistochemistry (IHC) is 2 + / 3 +, the tumor is considered positive for CA 6. More specifically, the immunohistochemistry (IHC) assay includes: a murine monoclonal antibody produced by the hybridoma cell line DS6 deposited with the American Type Culture Collection (ATCC) under deposit number PTA-4449.

In a further embodiment, the cancer to be treated is breast cancer, more specifically triple negative breast cancer (TNBC), which is non-positive for estrogen, progesterone, or HER2 receptors.

The conjugate of the present invention can be administered in combination with a drug to prevent or control keratitis, especially in combination with a prophylactic or curative ophthalmic composition for keratitis.

In a specific embodiment, prophylactic treatment to prevent ocular disorders, prophylactic treatment to prevent keratitis is administered before, simultaneously or after each administration of the conjugate.

The present invention further relates to a conjugate for use in a method of treating cancer, comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, as defined in the section " Cell binding agent " above, which Connected to (ii) at least one cytotoxic agent (as defined in the section " Cytotoxic Agent " above), wherein the method includes at least two cycles, one cycle is a three-week period, and in each cycle:

- conjugate on day cycle at a dose of 120mg / m ² is administered, a dose of Bing and 120mg / m ² is administered on day 8 of the cycle, Bing

-Before, at the same time or immediately after each administration of the conjugate, administration to prevent ocular disorders, in particular prophylactic treatment for the prevention of keratitis.

The present invention also relates to the use of the conjugate for the manufacture of a medicament for the treatment of cancer, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above in " Cell binding agent " Definition), which is linked to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above), wherein the conjugate is administered in at least two cycles, one cycle is a period of three weeks, Among them, each cycle:

- conjugate on day cycle at a dose of 90mg / m ² is administered, Bing at a dose of 90mg / m ² is administered on day 8 of the cycle, Bing

-Before, at the same time or immediately after each administration of the conjugate, administration to prevent ocular disorders, in particular prophylactic treatment for the prevention of keratitis.

The present invention also relates to the use of the conjugate for the manufacture of a medicament for the treatment of cancer, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above in " Cell binding agent " Definition), which is linked to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent" section above ), wherein the conjugate is administered in at least two cycles, one cycle is a period of three weeks, Among them, each cycle:

- conjugate on day cycle at a dose of 120mg / m ² is administered, Bing at a dose of 120mg / m ² is administered on day 8 of the cycle, Bing

-Before, at the same time or immediately after each administration of the conjugate, administration to prevent ocular disorders, in particular prophylactic treatment for the prevention of keratitis.

The present invention also relates to the use of the conjugate for the manufacture of a medicament for the treatment of cancer, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above in " Cell binding agent " Definition), which is linked to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above), wherein the conjugate is administered in at least two cycles, one cycle is a period of two weeks, Among them, each cycle:

-The conjugate is administered at a dose of 120 mg / m ² on the first day of the cycle, and

-Before, at the same time or immediately after each administration of the conjugate, administration to prevent ocular disorders, in particular prophylactic treatment for the prevention of keratitis.

The present invention also relates to a method of treating cancer in a patient in need thereof, the method comprising at least two cycles, one cycle is a period of three weeks, and in each cycle:-to the patient in need The conjugate is administered, comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as defined in the " Cell Binding Agent " section above), and (ii) at least one cytotoxic agent (such as Defined in the " cytotoxic agent " section above) linked, the conjugate was administered at a dose of 90 mg / m ² on the first day of the cycle and at a dose of 90 mg / m ² on the eighth day of the cycle,- Before, at the same time, or immediately after each administration of the conjugate, the patient is administered prophylactic treatment for the prevention of ocular disorders, more specifically for the prevention of keratitis.

The present invention also relates to a method of treating cancer in a patient in need thereof, the method comprising at least two cycles, one cycle is a period of three weeks, and in each cycle:-to the patient in need The conjugate is administered, comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as defined in the " Cell Binding Agent " section above), and (ii) at least one cytotoxic agent (such as Defined in the " cytotoxic agent " section above) linked, the conjugate was administered at a dose of 120 mg / m ² on the first day of the cycle and at a dose of 120 mg / m ² on the eighth day of the cycle,- Before, at the same time, or immediately after each administration of the conjugate, the patient is administered prophylactic treatment for the prevention of ocular disorders, more specifically for the prevention of keratitis.

The invention also relates to a method of treating cancer in a patient in need thereof, said method comprising at least two cycles, one cycle is between two cycles, and in each cycle:

-Administering the conjugate to the patient in need, which comprises: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as defined in the section " Cell binding agent " above), and (ii ) At least one cytotoxic agent (as defined in the " cytotoxic agent " section above) linked, the conjugate is administered at a dose of 120 mg / m ² on the first day of the cycle and

-Before, at the same time or immediately after each administration of the conjugate, administer prophylactic treatment for the prevention of ocular disorders to the patient, more specifically for the prevention of keratitis.

As used herein, " prophylactic treatment for the prevention of ocular disorders " means herein any treatment that prevents or reduces ocular adverse events caused by anti-tumor therapy administered to a subject with cancer. The ocular adverse events caused by antitumor therapy, specifically caused by tubulin adhesive cytotoxic drugs, are well known to those skilled in the art. These ocular adverse events or eye conditions include keratitis, blurred vision, abnormal eye sensation, dry eyes, increased tearing, photophobia, corneal deposition, blepharitis, eye pain, astigmatism, and conjunctival hyperemia.

In specific embodiments, the prophylactic treatment is used to prevent keratitis.

Prophylactic treatment for the prevention of ocular disorders, specifically for the prevention of keratitis, is well known to those skilled in the art.

In a specific embodiment, the prophylactic treatment for the prevention of ocular disorders, particularly for the prevention of keratitis, includes the administration of vasoconstrictors and / or corticosteroids, and / or the use of cold eye masks.

" Vasoconstrictor " means any compound that induces vasoconstriction at the corneal level, ie, narrows blood vessels caused by the contraction of the muscular wall of the blood vessel. Examples of vasoconstrictors are well known to those skilled in the art, and include: antihistamines, caffeine, methylphenidate, oxymetazoline, phenylephrine (or neofolin), propylhexenamine, pseudo Ephedrine and tetrahydrozoline. In a specific embodiment, the vasoconstrictor is neofolin, more specifically neofolin in the form of eye drops.

The present inventors proved that by administering a vasoconstrictor, specifically neofolin, when the conjugate is administered, a particularly effective prevention of ocular adverse events such as keratitis can be obtained.

Correspondingly, in a specific embodiment, the vasoconstrictor is administered at the time of administration of the conjugate, specifically neofolin, more specifically in the form of less than 2.5%, especially in the form of drops, specifically a dose of 3 drops .

" Corticosteroid " means herein any compound of the corticosteroid class which is a steroid hormone. Examples of corticosteroids are well known to those skilled in the art, and include: corticosteroids such as hydrocortisone, hydrocortisone, hydrocortisone acetate, cortisone acetate, pivaloyl hydrosulfide Cortisone (tixocortol pivalate), prednisolone, methylprednisolone and prednisolone; acetonides such as triamcinolone acetonide, triamcinolone alcohol, mometasone (mometasone), Ansi Amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, and halcinonide; betamethasone cortex Steroids such as betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, and fluocortolone; halogenated esters of corticosteroids such as hydrocortisone-17-valerate, halomethasone ( halometasone), beclomethasone dipropionate (alclometasone dipropionate), betamethasone valerate, betamethasone dipropionate, betamethasone dipropionate Prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate And fluprednidene acetate; and unstable prodrug esters of corticosteroids such as hydrocortisone-17-butyrate, hydrocortisone-17-propyl acetate, hydrocortisone-17- Butyric acid propionate, cyclosonide and prednicarbate. In a specific embodiment, the corticosteroid is dexamethasone, more specifically dexamethasone in the form of an ocular gel.

The inventors have demonstrated that by administering corticosteroids, specifically dexamethasone, more specifically in the form of an ocular gel, three times a day for two days from the day of conjugate administration, a particularly effective ocular adverse event such as cornea can be obtained Prevention of inflammation.

Therefore, in a specific embodiment, the corticosteroid, specifically dexamethasone, more specifically 0.16%, and more specifically the ocular gel form, is administered three times a day for 2 days from the day of conjugate administration.

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

Therefore, in a specific embodiment, a cold eye mask is used throughout the administration of the conjugate.

The inventors have also shown that when vasoconstrictors, corticosteroids and cold eye masks are used, particularly effective prevention of ocular adverse events such as keratitis can be obtained.

Therefore, in a specific embodiment, (i) the vasoconstrictor is administered when the conjugate is administered, specifically neofolin, more specifically less than 2.5%, and more specifically in the form of drops, specifically A dose of 3 drops, (ii) corticosteroids, specifically dexamethasone, more specifically 0.16%, and more specifically ocular gels, three times a day for 2 days from the day of conjugate administration , And (iii) use a cold eye mask throughout the administration of the conjugate.

Therefore, the present invention also relates to the simultaneous, separate or sequential use of a vasoconstrictor as defined above and a corticosteroid as defined above specifically in combination with the conjugate of the present invention, optionally in combination with a cold eye mask, for the prevention of cancer and Adverse ocular events (specifically keratitis) in patients under anticancer treatment.

The present invention also relates to vasoconstrictors as defined above for use in combination with the corticosteroids defined above and optional cold eye masks, specifically in combination with the conjugates of the present invention, for the prevention of cancer and anti-cancer treatment Methods of ocular adverse events (specifically keratitis) in patients.

The present invention also relates to corticosteroids as defined above for use in combination with the vasoconstrictors defined above and optional cold eye masks, specifically in combination with the conjugates of the present invention, for the prevention of cancer and anti-cancer treatment Methods of ocular adverse events (specifically keratitis) in patients.

The present invention also relates to a method for preventing ocular adverse events (specifically keratitis) in patients suffering from cancer and anti-cancer therapy, in particular using the conjugates of the present invention, which includes simultaneous, separate or sequential administration The patient provides a vasoconstrictor as defined above and a corticosteroid as defined above, and it is further preferred that the patient wears a cold eye mask.

In a specific embodiment, the vasoconstrictor is administered when the anticancer treatment is specifically the conjugate of the present invention, specifically neofolin, more specifically less than 2.5%, and also in the form of drops, specifically 3 doses.

In another specific embodiment, the administration of a corticosteroid, specifically dexamethasone, more specifically 0.16%, and also specifically in the form of an ocular gel from the day when the anticancer treatment is specifically the conjugate of the present invention, Three times a day and 2 days.

In a specific embodiment, a cold eye mask is used during the entire administration of the anticancer treatment specifically to the conjugate of the present invention.

In a more specific embodiment, (i) when the anticancer treatment is specifically a conjugate of the present invention, a vasoconstrictor is administered, specifically neofolin, more specifically less than 2.5%, and also specifically drops Form, specifically a dose of 3 drops, (ii) Corticosteroids, specifically dexamethasone, more specifically 0.16%, and specifically eye Part of the gel form, and carried out for two days, and (iii) throughout the period of administration of anticancer treatment specifically the conjugate of the present invention, using a cold eye mask.

The present invention also relates to a conjugate used in a method for treating cancer, comprising: (i) a cell binding agent that specifically binds to tumor cells, which is connected to (ii) at least one cytotoxic agent, wherein The cytotoxic agent is a tubulin-binding agent; and wherein the method includes administering the conjugate in a patient in need thereof, and each time the conjugate is administered, administering Prophylactic treatment for the prevention of ocular disorders, which includes two or more of the following:-administration of an ocular vasoconstrictor,-administration of an ocular corticosteroid, and-use of a cold eye mask.

In specific embodiments, the cell-binding agent binds to an antigen that is also naturally present on the surface of tumor cells in the eye, for example in corneal epithelium, conjunctival basal cells and / or lacrimal gland cells.

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

In a specific embodiment, the prophylactic treatment includes two or more of the following:-three drops of 2.5% neofolin administered in each eye,-the dexamethas is administered from the day of administration of the conjugate Misong (0.1%) eye gel is performed three times a day for two days, and-a cold eye mask is used during the administration of the conjugate.

More specifically, the prophylactic treatment includes a combination: three drops of 2.5% neofolin administered to each eye; an eye gel of dexamethasone (0.16%) administered from the date of administration of the conjugate Perform three times a day for two days; and use a cold eye mask during conjugate administration.

The present invention also relates to an article of manufacture comprising: a) packaging materials; b) a conjugate comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above for " cell binding agent " Part of the definition), connected to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above); and c) the label or package insert included in the packaging material, indicating at least two In each cycle, the conjugate is administered at least twice during each cycle, and in each cycle at a dose corresponding to the weekly scheduled dose of at least 60 mg / m ² of the cycle.

In an embodiment, the label or package insert indicates that in at least two three-week cycles, during each cycle, the conjugate is at 120 mg / m ² on day 1 of the cycle and on day 8 It is administered at a dose of 120 mg / m ² .

In an alternative embodiment, the label or package insert indicates that in at least two three-week cycles, during each cycle, the conjugate at 120 mg / m ² on the first day of the cycle and on the eighth It is administered at a dose of 120 mg / m ² per day. The invention also relates to an article of manufacture comprising: a) packaging material; b) a conjugate comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above for " cell binding agent " Defined in), linked to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above); and c) the label or package insert included in the packaging material, indicating at least two three-week cycles, each cycle, the conjugate on day 1 cycle at 120mg / m ² dose administered, and the period of 8 days at a dose of 120mg / m ² is administered in Before, at the same time or immediately after each administration of the conjugate, prophylactic treatment to prevent ocular disorders is administered.

The invention also relates to an article of manufacture comprising: a) packaging material; b) a conjugate comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above for " cell binding agent " Part of the definition), connected to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above); and c) the label or package insert included in the packaging material, indicating at least two a three-week cycles, each cycle, the conjugate at a dose of 90mg / m ² is administered on day 1 of the cycle, and the 8 day cycle at a dose of 90mg / m ² is administered in each Before, at the same time, or immediately after the second administration of the conjugate, prophylactic treatment to prevent ocular disorders is administered.

The present invention also relates to an article of manufacture comprising: a) packaging materials; b) a conjugate comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein (as described above for " cell binding agent" Part of the definition ), connected to (ii) at least one cytotoxic agent (as defined in the " cytotoxic agent " section above); and c) the label or package insert included in the packaging material, indicating at least two In a two-week cycle, in each cycle, the conjugate is administered at a dose of 120 mg / m ² on the first day of the cycle, optionally before, simultaneously, or immediately after each administration of the conjugate. Preventive treatment to prevent eye diseases.

Brief description of the sequence

【Example】

After every 3 weeks (10-240mg / m ² ), every 2 weeks (120mg / m ² ), every 3 weeks D1, D8 (90mg / m ² ) after intravenous huDS6-DM4 intravenous administration, the first time in human (FIH) studies to assess safety, dose-limiting toxicity (DLT) / recommended dose (RD) and pharmacokinetics (PK)).

1067 patients were pre-screened for CA6 expression: 60 patients were positive for CA6 ( 1%), 24 cases of CA6 positive ( The intensity of 30% of tumor cells is 2 + / 3 +). The effect of CA6 antigen expression on paraffin-embedded tumor tissue was evaluated by immunohistochemistry (IHC). The IHC assay uses the murine monoclonal antibody DS6 produced by the hybridoma cell line deposited with the American Type Culture Collection (ATCC) under deposit number PTA-4449.

Including those with most of the expression of CA6, 30% of tumor cells have 114 patients with solid tumors that are heavily pre-treated by immunohistochemistry with a strength of 2 + / 3 +.

During the dose escalation phase, 34 patients who were heavily pre-treated with CA6 positive solid tumors including pancreatic, ovarian, and breast tumors were included. In ² of 8 patients treated with the highest dose of 240 mg / m ² every 3 weeks (q3w), dose-limiting toxicity (DLT) [grade 3 diarrhea (cycle 1) and grade 3 cornea were observed Inflammation (cycle 2)]. Overall, huDS6-DM4 was observed to be well tolerated from a dose of 150 mg / m ² every 3 weeks, with reversible corneal adverse events that occurred late (known from previous maytansine-ADC), and hematology is rare Events and some peripheral neuropathy.

The initial recommended dose (RD) of 190 mg / m ² q3w was used during the expansion period (breast, ovary, and pancreas). However, a high incidence of corneal lesions was mainly observed in the second cycle. Decided to reduce the dose to 150 mg / m ² q3w: corneal toxicity is reduced, but unfortunately the activity is affected.

The inventors found that there was a highly significant correlation between ocular toxicity and huDS6-DM4 exposure (AUCs and C _max ) in the first cycle. However, it is impossible to distinguish whether ocular toxicity is most related to C _max or AUC because they are strongly related to each other. The inventors decided to reduce C _max by approximately 30% in an attempt to limit the occurrence of ocular toxicity, but in order to maintain the activity of huDS6-DM4, drug exposure was maintained.

Therefore, two new alternative plans were selected and tested in the ongoing phase I study: 90 mg / m ² (D1D8 q3w) on days 1 and 8 every 3 weeks, and 120 mg / m ² (q2w) every two weeks. Choosing these alternatives, the benefit / risk ratio is increased by maintaining a lower C _max but a similar AUC for more than 12 weeks and a higher C _min compared to the 190 mg / m ² plan.

Define Go No Go criteria based on efficacy and safety parameters for selecting RD: Yes 25% of eye events cause dose changes (delay and / or dose reduction) or drug discontinuation, and overall response rate (ORR) 15% or no progress at 12 weeks 50%.

A total of 33 patients were included in the two alternative plans (17 and 16 patients in 90 mg / m ² D1D8 q3w and 120 mg / m ² q2w, respectively).

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

Table 1     

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

All the results presented below report on patients treated at the recommended doses (150 mg / m ² Q3w and 190 mg / m ² Q3w and the two alternatives) in the expanded section.

-lab testing

No abnormalities in level 4 laboratory tests were observed. Hematological toxicity was limited, and no differences were observed between the q3w tables except platelets. No severe liver or kidney function test abnormalities related to treatment were observed.

-Treatment-emergent adverse events

Almost all patients experienced at least one adverse event. Of these, 48% had Grade 3 adverse events and 12% had related Grade 3 adverse events. 7% had adverse events that caused permanent treatment discontinuation (including 2 patients with keratitis).

Ocular corneal events are the main adverse events and globally are dose / plan dependent. The main ocular corneal event was keratitis, with a higher incidence (65%) at 190mg / m ² q3w, similar incidence at 150mg / m ² q3w and 90mg / m ² D1D8 q3w (about 35%), at 120mg / m ² q2w has a low incidence (13%).

As shown in Table 4 below, ocular disorders that caused dose changes accounted for 68.2% (28/41) of all TEAEs that caused dose changes.

Eye disorders causing dose changes were observed in 31% of patients treated at high doses:

-The highest incidence (61%) is 190mg / m ² q3w

-Intermediate (~ 24-29%) at 150mg / m ² q3w and 90mg / m ² D1D8 q3w

-The lowest incidence rate (6%) is 120mg / m ² q2w

Keratitis represents 93% of eye disorders that cause dose changes.

The ocular adverse events are summarized in Table 5 below.

Eye disorders were observed in 55% of treated patients. More specifically, Grade 3 ocular disorders were observed in 11% (18% in 90 mg / m ² D1D8 q3w).

Keratitis was observed in 38% of patients, with a higher incidence (> 60%) at 190 mg / m ² q3w and a lower incidence at 120 mg / m ² q2w.

Blurred vision was observed in 17% of patients.

A total of 10 patients (6 in the 90 mg / m ² D1D8 q3w plan and 4 in the 120 mg / m ² q2w plan) received preliminary ophthalmic prophylaxis, and no ocular disorders leading to dose changes were observed. Only one patient had keratitis at 90 mg / m ² D1D8 q3w. In this latter alternative plan, the incidence of ocular corneal events (mainly keratitis) as the main AE is less than or similar to 150 mg / m ² q3w and less than 190 mg / m ² q3w.

This prophylactic treatment is a prophylactic (eg, preliminary) prevention of keratitis performed within the time of each infusion:

-Vasoconstrictor ( 2.5%; 3 drops) (ie new forint)

-From the start of the infusion, the corticosteroid eye gel (Sterdex / dexamethasone 0.16%) is taken three times a day (morning, noon and evening) for 2 days.

-From the beginning to the end of the infusion, a cold eye mask (unless the patient cannot tolerate it).

Other overall TEAEs are: fatigue (32.6%), peripheral neuropathy (31.6%), GI disorders [(nausea (29%), abdominal pain (26%), diarrhea (25%)] and neutropenia (2.6% ) .Noticing lower liver and kidney abnormalities.

ORR was evaluated in 109 of 114 patients. As shown in Table 6, similar ORRs were obtained at 90 mg / m ² D1D8 q3w (2 local reactions / 15 cases) and at 190 mg / m ² q3w (1 complete reaction and 2 local reactions / 23 cases).

Tumor regression was noted in approximately 60% of patients in 190 mg / m ² q3w and 90 mg / m ² D1D8 q3w, and in approximately 35% of patients in 150 mg / m ² q3w and 120 mg / m ² q2w.

Considering all the tested doses, one complete response (ovary), 8 local responses (3 breasts, 2 ovaries, 1 NSCLC, 1 cervix, and 1 bladder) and 39 cases were stable. Similar ORRs were observed in 13.3% (2/15) of 90 mg / m ² D1D8 q3w and 13% (3/23) in 190 mg / m ² q3w.

Table 7 below summarizes the results obtained by the different plans tested.

Conclusion :

huDS6-DM4 is an active drug with a remarkably visible response in breast cancer, lung cancer and bladder cancer. Both the 90 mg / m ² D1D8 q3w and 120 mg / m ² q2w plans resulted in acceptable safety for ocular disorders, better than the 190 mg / m ² q3w plan.

A similar ORR was observed at 90 mg / m ² D1D8 q3w compared to 190 mg / m3q3w. The initial prevention of eye disorders may be beneficial, and it leads to a more improved eye safety profile for huDS6-DM4.

<110> Sanofi (SANOFI)

<120> Therapeutic plan for tumor treatment with anti-Muc1 class maytansine immunoconjugate antibody

<130> FR2016 / 006

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CDR1-H of huDS6

<400> 1

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CDR2-H of huDS6

<400> 2

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> CDR3-H of huDS6

<400> 3

<210> 4

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CDR1-L of huDS6

<400> 4

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CDR2-L of huDS6

<400> 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CDR3-L of huDS6

<400> 6

<210> 7

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain variable region of HuDS6

<400> 7

<210> 8

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> HuDS6 light chain variable region

<400> 8

<210> 9

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> The heavy chain of HuDS6

<400> 9

<210> 10

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> HuDS6 light chain

<400> 10

Claims (41)

A conjugate used in a method for treating cancer, comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, which is linked to (ii) at least one cytotoxic agent, wherein The method includes at least two cycles, wherein, for each cycle, the conjugate is administered at a dose corresponding to a weekly planned dose of at least 60 mg / m ² of the cycle, and wherein the conjugate The substance is administered at least twice during each cycle. The conjugate for its use as claimed in claim 1, wherein in each cycle, the conjugate is administered at a dose corresponding to the weekly planned administration of 60 to 80 mg / m ² of the cycle.     The conjugate as claimed in claim 1 or 2 for its use, wherein the cycle is a period of two weeks or three weeks.     The conjugate for 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 as follows: day 1 at a dose of 90mg / m ² on day 8 of said cycle at 90mg / m ² dose.     The conjugate for use thereof according to any one of claims 1 to 4, wherein, before, at the same time, or immediately after each administration of the conjugate, prophylactic treatment for preventing ocular disorders is administered.     The conjugate for its use according to claim 5, wherein the cycle is a three-week period, and, for each cycle, the conjugate is administered as follows: on the first day of the cycle, at 120 mg / m ² at the dose of 120 mg / m ² on the 8th day of the cycle.     The conjugate for its use according to claim 5 or 6, wherein the prophylactic treatment for the prevention of ocular disorders includes: administration of vasoconstrictors and / or corticosteroids, and / or use of cold eye masks.         The conjugate for its use according to claim 7, wherein the corticosteroid is administered three times a day for 2 days starting from the day the conjugate is administered.         The conjugate for its use according to claim 7, wherein a cold eye mask is used throughout the administration of the conjugate.         The conjugate for its use according to any one of claims 1 to 9, wherein the cell binding agent binds to the extracellular domain of the MUCl glycoprotein.         The conjugate for its use according to any one of claims 1 to 10, wherein the cell-binding agent recognizes and binds to the CA6 glycotope on the MUCl glycoprotein.         The conjugate for its use according to any one of claims 1 to 11, wherein the cell-binding agent is an antibody or an epitope-binding fragment thereof.         The conjugate for its use according to claim 12, wherein the antibody or epitope binding fragment thereof comprises one or more complementarity determining regions (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.         The conjugate for its use according to claim 13, wherein the antibody or epitope binding fragment thereof comprises the sequence CDR1-H of sequence SEQ ID NO: 1, the sequence CDR2-H of sequence SEQ ID NO: 2, the sequence SEQ ID NO : 3 CDR3-H, sequence SEQ ID NO: 4 CDR1-L, sequence SEQ ID NO: 5 CDR2-L and sequence SEQ ID NO: 6 CDR3-L.         The conjugate as claimed in claim 13 or 14 for its use, wherein the antibody or epitope binding fragment thereof comprises the heavy chain variable region of the sequence SEQ ID NO: 7 or a sequence at least 85% identical thereto.         The conjugate for its use according to any one of claims 13 to 15, wherein the antibody or epitope binding fragment thereof comprises the light chain variable region of the sequence SEQ ID NO: 8 or a sequence at least 85% identical thereto.     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.     The conjugate for its use according to any one of claims 1 to 16, wherein the cell binding agent is a heavy chain comprising SEQ ID NO: 9 and a light chain of the sequence SEQ ID NO: 10 or at least 85% identical thereto Monoclonal antibody.         The conjugate for its use according to any one of claims 1 to 18, wherein the at least one cytotoxic agent is selected from the group consisting of maytansinoid, small drug, tomycin (tomayamycin) derivatives, finemycin derivatives, prodrugs, taxanes, CC-1065 and CC-1065 analogs.     The conjugate for its use according to claim 19, wherein the at least one cytotoxic agent is maytansine DM1 of formula (I) The conjugate for its use according to claim 19, wherein the at least one cytotoxic agent is maytansine DM4 of formula (II)     The conjugate for its use according to any one of claims 1 to 21, wherein the cell binding agent is covalently linked to the at least one cytotoxic agent via a cleavable or non-cleavable linker.         The conjugate for its use according to claim 22, wherein the linker is selected from the group consisting of: N-succinimidopyridyl dithiobutyrate (SPDB), 4- (pyridine-2 -Yldithio) -2-sulfo-butyric acid (sulfo SPDB) and succinimido (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).         The conjugate for its use according to claim 22, wherein the linker is N-succinimidopyridyl dithiobutyrate (SPDB) and the cytotoxic agent is DM4.         The conjugate for its use according to claim 22, wherein the linker is 4- (pyridin-2-yl-dithio) -2-sulfo-butyric acid (sulfo SPDB) and the cytotoxic agent For DM4.     The conjugate for its use according to any one of claims 1 to 25, wherein the conjugate is characterized by a drug-to-antibody ratio (DAR) of 3-4, the DAR from the cytotoxic agent concentration (cD) Calculate the ratio of cell binding agent concentration (c _A ): DAR = c _D / c _A where c _D = [( ε _{A 280} × A ₂₅₂ )-( ε _{A 252} × A ₂₈₀ )] / [( ε _{D 252} × _{ε A 280) - (ε A} 252 × ε D 280)] c A - [A 280 - (c D × ε D 280)] / ε A 280 and ε _D252 and ε _D280 are the cytotoxic agent at 252nm And 280 nm molar extinction coefficients, ε _A252 and ε _A280 are the molar extinction coefficients of the cell binding agent at ₂₅₂ nm and 280 nm, respectively, and A ₂₅₂ and A ₂₈₀ are the conjugate measured using a classic spectrophotometric device at ₂₅₂ nm (A ₂₅₂ ) and 280 nm (A ₂₈₀ ) absorbance.     The conjugate for its use according to any one of claims 1 to 26, wherein the number of cycles is 2.         The conjugate for its use according to any one of claims 1 to 27, wherein the conjugate is administered intravenously.         The conjugate for its use as claimed in claim 28, wherein the conjugate is administered at a rate of 1 mL / min for 30 minutes, and then increased to a maximum rate of 2 mL / min in the absence of hypersensitivity.         The conjugate for its use according to any one of claims 1 to 29, wherein the cancer is a solid tumor.         The conjugate for its use according to any one of claims 1 to 30, wherein the cancer is a CA6 positive tumor.         The conjugate for its use according to claim 31, wherein the tumor is identified as CA6 when at least 30% of the cells of the sample exhibit an intensity level of 2 + / 3 + as determined by immunohistochemistry (IHC) Positive.         The conjugate for its use according to claim 32, wherein the immunohistochemistry (IHC) assay includes: a hybridoma cell line DS6 deposited by the American Type Culture Collection (ATCC) deposited with the accession number PTA-4449 Mouse monoclonal antibodies produced.         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.         The conjugate for its use according to claim 34, wherein the cancer is breast cancer.         The conjugate for its use according to claim 35, wherein the breast cancer is triple negative breast cancer and is not positive for estrogen, progesterone or HER2 receptors.         A conjugate comprising: (i) a cell binding agent that specifically binds to tumor cells, which is connected to (ii) at least one cytotoxic agent, and is used in a method of treating cancer, wherein the cytotoxic agent is micro Tubulin binding agent; and wherein, the method comprises administering the conjugate in a patient in need thereof, and each time the conjugate is administered, prophylactic treatment to prevent ocular disorders, The prophylactic treatment includes two or more of the following:-administration of an ocular vasoconstrictor,-administration of an ocular corticosteroid, and-use of a cold eye mask.         The conjugate of claim 37, wherein the prophylactic treatment includes two or more of the following:-three drops of 2.5% neofolin administered in each eye,-from the day of administration of the conjugate, Eye gel administered with dexamethasone (0.16%) three times a day for two days, and-use cold eye mask during the administration of the conjugate.     An article comprising: a) packaging materials; b) a conjugate, comprising: (i) a cell binding agent that binds to human mucin-1 (MUC1) glycoprotein, and (ii) at least one cytotoxic agent Connection; and c) a label or package insert contained in the packaging material indicating that the conjugate is administered at least twice per cycle in at least two cycles, and for each cycle to correspond to the The conjugate is administered at a planned dose of at least 60 mg / m ² per week of the cycle. The article of claim 39, wherein the label or package insert also indicates that the cycle is a period of three weeks, and for each cycle the conjugate is administered as follows: (i) at the first of the cycle days at 90mg / m ² dose and dose on day 8 of the cycle at 90mg / m ^2; or (ii) on day 1 of the cycle at a dose of 120mg / m ² and 8 of the cycle At a dose of 120 mg / m ² per day.     The article of claim 39 or 40, wherein the label or package insert also indicates that prophylactic treatment for the prevention of ocular disorders is administered before, at the same time, or immediately after each administration of the conjugate.