Process for preparing maytansinoid antibody conjugates

ES 2 533 992 T3

DESCRIPTION

Procedure for preparing maytansinoid antibody conjugates

Field of the invention

This invention relates to a process for preparing conjugates of substantially high purity and stability, wherein the conjugates comprise a cell binding agent chemically coupled to a drug, as defined in the claims.

Background of the invention

Cancer treatment has progressed significantly with the development of pharmaceuticals that more effectively access and kill cancer cells. To this end, researchers have harnessed cell surface receptors and antigens selectively expressed by cancer cells to develop antibody-based drugs that bind to tumor-specific or tumor-associated antigens. In this regard, cytotoxic molecules such as bacterial and plant toxins, radionuclides, and certain chemotherapeutic drugs have been chemically linked to monoclonal antibodies that bind to tumor-specific or tumor-associated cell surface antigens (see, eg, applications International Patent Nos. WO 00/02587, WO 02/060955 and WO 02/092127, US Patents 5,475,092, 6,340,701, and 6,171,586, US Patent Application Publication. No. 2003/004210 A1, and Ghetie et al., J. Immunol. Methods, 112: 267-277 (1988)). Such compounds are typically referred to as "conjugates" of toxins, radionuclides, and drugs, respectively. They are also often referred to as immunoconjugates, radioimmunoconjugates, and immunotoxins. Tumor cell death occurs upon binding of the drug conjugate to a tumor cell and the release and / or activation of the drug's cytotoxic activity. The selectivity provided by the drug conjugates minimizes toxicity towards normal cells, thereby increasing the tolerability of the drug in the patient.

Procedures for conjugating antibodies to sulfhydryl-containing cytotoxic agents such as maytansinoids have been previously described (see, eg, US Patents 5,208,020, 5,416,064 and 6,441,163). For example, US Patents 5,208,020 and 5,416,064 describe a process for making antibody-maytansinoid conjugates in which the antibody is first modified with a heterobifunctional reagent as described in US Pat. 4,149,003, 4,563,304 and US Patent Application Publication No. 2004/0241174 A1. US Patents 5,208,020 and 5,416,064 further describe conjugation of a modified antibody with an excess of a sulfhydryl-containing cytotoxic agent at pH 7, followed by purification on Sephadex ™ G25 chromatography columns. Purification of antibody-drug conjugates by size exclusion chromatography (SEC) has also been described (see, eg, Liu et al, Proc. Natl. Acad. Sci. (USA), 93: 8618-8623 (1996) and Chari et al., Cancer Research, 52: 127-131 (1992)).

Procedures that have been previously described for the preparation of antibody-drug conjugates are complex, because they are hampered with steps that are cumbersome to perform or produce immunoconjugates that are less pure or less stable than is optimally desired. For example, conjugation at a pH between 6.0 and 6.5 is not optimal to produce pure and stable conjugates. Furthermore, conjugation reactions under these conditions are generally slow and inefficient, leading to a requirement for excessive time and material utilization.

It would be desirable to modify or eliminate one or more preparation steps without compromising the quality of the product, such as purity and / or stability. It would further be desirable to have additional purification options than those described heretofore, as some options will be more effective with certain combinations of cell binding agents, linkers and drugs, than with others.

In view of the foregoing, there is a need in the art to develop improved methods of preparing cell-binding agent-drug conjugate compositions that are of substantially high purity and at the same time have greater stability. Such a method is described.

Brief summary of the invention

A process is described for preparing a conjugate of substantially high purity and stability comprising a cell binding agent chemically coupled to a drug. The method comprises (a) contacting a cell binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell binding agent and thereby preparing a first mixture comprising cell binding agents having linkers attached thereto, (b) subjecting the first mixture to adsorptive chromatography, and thereby preparing a first purified mixture of cell binding agents having linkers attached thereto, (c) conjugating a drug with the cell binding agents having linkers attached thereto in the first purified mixture by reacting the cell binding agents having linkers attached thereto with a drug in a solution having a pH from about 4 to about 9 to prepare a second mixture comprising (i) cell binding agent chemically coupled via the linker to the drug, (ii) free drug and (iii) reaction by-products,

ES 2 533 992 T3 and (d) subjecting the second mixture to adsorptive chromatography to purify the cell binding agents chemically coupled by the drug linkers of the other components of the second mixture and thereby prepare a second purified mixture, as is described in the claims.

Detailed description of the invention

A process for preparing drug-cell binding agent conjugates of substantially high purity and stability is described. Such compositions can be used to treat diseases, due to the high purity and stability of the conjugates. Compositions comprising a cell binding agent, such as an antibody, chemically coupled to a drug, such as a maytansinoid, are described in, for example, US Patent Application Publication No. 2004/0241174 A1 . In this context, substantially high purity is considered to be: (a) more than 90%, preferably more than 95% of the conjugated species are monomeric, and / or (b) the level of free drug in the conjugate preparation is less than 2% (relative to total drug).

In this regard, the described method comprises (a) modifying the cell binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell binding agent and thereby preparing a first mixture comprising cell binding agents having linkers attached thereto, (b) subjecting the first mixture to adsorptive chromatography, to purify cell binding agents having linkers attached thereto from other components of the first mixture and thereby prepare a first purified mixture of cell binding agents having linkers attached thereto, (c) conjugating a drug with the cell binding agents having linkers attached thereto in the first purified mixture by reacting the cell binding agents having linkers attached thereto with the drug in a solution having a pH from about 4 to about 9 to prepare a second mixture comprising (i) cell binding agent chemically coupled via the linker to the drug, (ii) free drug and (iii) reaction by-products, and (d) subjecting the second mixture to adsorptive chromatography to remove unconjugated drugs, reactants, and by-products, as well as to obtain substantially cell-binding agent-drug conjugates. purified according to the claims.

Preferably, adsorptive chromatography resins are used in the purification steps. In a further description, adsorptive chromatography resins are used in both purification steps.

Any suitable adsorptive chromatography resin can be used. Preferred adsorptive chromatography resins include hydroxyapatite chromatography resins, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reverse phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT Type I and Type II, Bio-Rad Laboratories, Hercules, CA), hydroxyapatite HA Ultrogel (Pall Corp., East Hills, NY), and ceramic fluoroapatite (CFT Type I and Type II, Bio-Rad Laboratories, Hercules, CA). An example of a suitable HCIC resin is MEP Hypercel resin (Pall Corp., East Hills, NY). Examples of suitable HIC resins include Butyl-Sepharose, Hexyl-Sepharose, Phenyl-Sepharose, and Octyl-Sepharose resins (all from GE Healthcare, Piscataway, NJ), as well as Macroprep Methyl and Macro-prep t-Butyl (Bio- Rad Laboratories, Hercules, CA). Examples of suitable ion exchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharose resins (all from GE Healthcare, Piscataway, NJ), and Unosphere S resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable mixed mode ion exchangers include Bakerbond ABx resin (JT Baker, Phillipsburg, NJ). Examples of suitable IMAC resins include Chelating Sepharose resin (GE Healthcare, Piscataway, NJ) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable dye ligand resins include Sepharose Blue resin (GE Healthcare, Piscataway, NJ) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, CA). Examples of suitable affinity resins include Protein A Sepharose resin (eg, MabSelect, GE Healthcare, Piscataway, NJ), where the cell binding agent is an antibody, and lectin affinity resins, eg Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, NJ) in which the cell binding agent carries appropriate lectin binding sites. Alternatively, an antibody specific for the cell binding agent can be used. Such an antibody can be immobilized, for example, to Sepharose 4 Fast Flow resin (GE Healthcare, Piscataway, NJ). Examples of suitable reverse phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, CA).

According to the described method, a first mixture is produced comprising the cell binding agent having linkers attached thereto, as well as reactants and other by-products. Purification of the modified cell binding agent from reactants and by-products is carried out by subjecting the first mixture to a purification procedure. In this regard, the first mixture is purified using adsorptive chromatography. This first purification step provides a first purified mixture, that is, an increased concentration of the cell binding agents having linkers attached thereto, and a decreased amount of unbound bifunctional crosslinking reagent, compared to the first mixture. before purification according to the description.

After purification of the first mixture to obtain a first purified mixture of cell binding agents having linkers attached thereto, a drug is conjugated to the cell binding agents that

ES 2 533 992 T3 have linkers attached to them in the first purified mixture by reacting the cell binding agents having linkers attached thereto with a drug in a solution having a pH of from about 4 to about 9, after which produces a second mixture comprising (i) the cell binding agent chemically coupled via the linker to the drug, (ii) free drug, and (iii) reaction by-products. Although the conjugation reaction is carried out at a pH of about 4 to about pH 9, the reaction is preferably carried out at a pH of about 6 or less or at a pH of about 6.5 or greater, most preferably at a pH of about 4 at about 6 or at a pH of about 6.5 to about 9, and especially at a pH of 4 to less than 6 or at a pH of more than 6.5 to 9. When the conjugation step is performed at a pH of about 6.5 or higher, some sulfhydryl-containing drugs may be prone to dimerize by disulfide bond formation. The removal of trace metals and / or oxygen from the reaction mixture, as well as the optional addition of antioxidants or the use of linkers with more reactive leaving groups, or the addition of drug in more than one aliquot, may be required to allow a effective reaction in such situation.

The method described may optionally include the addition of sucrose to the conjugation step used in the inventive method to increase the solubility and recovery of the cell-binding agent-drug conjugates. Desirably, sucrose is added at a concentration of from about 0.1% (w / v) to about 20% (w / v) (eg, about 0.1% (w / v), 1% (p / v), 5% (p / v), 10% (p / v), 15% (p / v) or 20% (p / v)). Preferably, sucrose is added at a concentration of from about 1% (w / v) to 10% (w / v) (eg, about 2% (w / v), about 4% (w / v), about 6% (w / v) or about 8% (w / v)). Furthermore, the conjugation reaction may also comprise the addition of a buffering agent. Any suitable buffering agent known in the art can be used. Suitable buffering agents include, for example, a citrate buffer, an acetate buffer, a succinate buffer, and a phosphate buffer.

After the conjugation step, the second mixture is subjected to a purification step. In this regard, the second mixture is purified using adsorptive chromatography. This second purification step provides a second purified mixture, that is, an increased concentration of the cell binding agents chemically coupled by the drug linkers and a decreased amount of one or more other components of the second mixture, compared to the second mixing before purification according to the description.

The cell binding agent is an antibody (eg, monoclonal antibodies and fragments thereof).

The term "antibody" as used herein refers to any immunoglobulin, any immunoglobulin fragment, such as Fab, F (ab ') 2, dsFv, sFv, diabodies and tribodies, or immunoglobulin chimeras, that can bind to an antigen on the surface of a cell (eg, containing a complementarity determining region (CDR)). Any suitable antibody can be used. One of ordinary skill in the art will appreciate that the selection of an appropriate antibody will depend on the population of cells being targeted. In this regard, the type and number of cell surface molecules (i.e., antigens) that are selectively expressed in a particular cell population (typically and preferably a diseased cell population) will govern the selection of an appropriate antibody for use in the composition. Cell surface expression profiles are known for a wide variety of cell types, including tumor cell types, or, if unknown, can be determined using routine molecular biology and histochemical techniques.

The antibody can be polyclonal or monoclonal, but is most preferably a monoclonal antibody. As used herein, "polyclonal" antibodies refer to heterogeneous populations of antibody molecules, typically contained in the sera of immunized animals. "Monoclonal" antibodies refer to homogeneous populations of antibody molecules that are specific for a particular antigen. Monoclonal antibodies are typically produced by a single clone of B lymphocytes ("B cells"). Monoclonal antibodies can be obtained using various techniques known to those of skill in the art, including standard hybridoma technology (see, eg, Kohler and Milstein, Eur. J. Immunol., 5: 511519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, cSh Press (1988), and CA Janeway et al. (Eds.), Immunobiology, 5^to Ed., Garland Publishing, New York, NY (2001)). Briefly, the hybridoma method of producing monoclonal antibodies typically involves injecting any suitable animal, typically and preferably a mouse, with an antigen (ie, an "immunogen"). The animal is subsequently sacrificed, and B cells isolated from its spleen are condensed with human myeloma cells. A hybrid cell (ie, a "hybridoma") is produced, which proliferates indefinitely and continuously secretes high titers of an antibody with the desired specificity in vitro. Any appropriate method known in the art can be used to identify hybridoma cells that produce an antibody with the desired specificity. Such methods include, for example, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and radioimmunoassay. The hybridoma population is screened to isolate individual clones, each of which secretes a single species of antibody to the antigen. As each hybridoma is a clone derived from fusion with a single B cell, all the antibody molecules it produces are identical in structure, including their antigen-binding site and isotype. Monoclonal antibodies can also be generated using other suitable techniques, including EBV-hybridoma technology (see, eg, Haskard and Archer, J. Immunol. Methods, 74 (2): 361-67 (1984), and Roder et al., Methods Enzymol., 121: 140-67 (1986)), Bacteriophage vector expression systems (see, eg, Huse et al., Science, 246: 1275-81 (1989)), or phage display libraries comprising fragments of

ES 2 533 992 T3 antibodies, such as Fab and scFv (single chain variable region) (see, eg, US patents 5,885,793 and 5,969,108, and international patent applications WO 92 / 01047 and WO 99/06587).

The monoclonal antibody can be isolated from or produced in any suitable animal, but is preferably produced in a mammal, more preferably a mouse or human, and most preferably a human. Methods for producing an antibody in mice are well known to those of skill in the art and are described herein. With respect to human antibodies, one of skill in the art will appreciate that polyclonal antibodies can be isolated from the sera of human subjects vaccinated or immunized with an appropriate antigen. Alternatively, human antibodies can be generated by adapting known techniques to produce human antibodies in non-human animals such as mice (see, eg, US Patents 5,545,806, 5,569,825, and 5,714,352, and US Patent Application Publication 2002/0197266 A1).

Although they are the ideal choice for therapeutic applications in humans, human antibodies, particularly human monoclonal antibodies, are typically more difficult to generate than mouse monoclonal antibodies. Mouse monoclonal antibodies, however, induce a rapid host antibody response when administered to humans, which can reduce the therapeutic or diagnostic potential of the antibody-drug conjugate. To circumvent these complications, a monoclonal antibody is preferably not recognized as "foreign" by the human immune system.

For this purpose, phage display can be used to generate the antibody. In this regard, phage libraries encoding antibody antigen-binding variable (V) domains can be generated using standard molecular biology and recombinant DNA techniques (see, eg, Sambrook et al. (Eds.) , Molecular Cloning, A Laboratory Manual, 3^to Edition, Cold Spring Harbor Laboratory Press, New York (2001)). Phage encoding a variable region with the desired specificity are selected for specific binding to the desired antigen, and a complete human antibody comprising the selected variable domain is reconstituted. Nucleic acid sequences encoding the reconstituted antibody are introduced into a suitable cell line, such as a myeloma cell used for the production of hybridomas, such that human antibodies having the characteristics of monoclonal antibodies are secreted by the cell (see , eg, Janeway et al., cited above, Huse et al., cited above, and US Patent 6,265,150). Alternatively, monoclonal antibodies can be generated from mice that are transgenic for specific human heavy and light chain immunoglobulin genes. Such methods are known in the art, and are described in, for example, US Patents 5,545,806 and 5,569,825, and Janeway et al., Cited above.

Most preferably, the antibody is a humanized antibody. As used herein, a "humanized" antibody is one in which the complementarity determining regions (CDRs) of a mouse monoclonal antibody, which form the antigen-binding loops of the antibody, are grafted onto the framework of a human antibody molecule. Due to the similarity of the frameworks of mouse and human antibodies, it is generally accepted in the art that this method produces a monoclonal antibody that is antigenically identical to a human antibody but that binds to the same antigen as the mouse monoclonal antibody of the that the CDR sequences were derived. Methods for generating humanized antibodies are well known in the art, and are described in detail in, for example, Janeway et al., Cited above, US Patents 5,225,539, 5,585,089 and 5,693,761. European Patent No. 0239400 B1, and UK Patent No. 2188638. Humanized antibodies can also be generated using the antibody surface reconstruction technology described in US Patent 5,639,641 and Pedersen et al., J. Mol. Biol., 235: 959-973 (1994). Although the antibody employed in the conjugate of the described composition is most preferably a humanized monoclonal antibody, a human monoclonal antibody and a mouse monoclonal antibody, described above, are also described.

Antibody fragments that have at least one antigen-binding site, and thus recognize and bind at least one antigen or receptor present on the surface of a target cell, are also described. In this regard, proteolytic cleavage of an intact antibody molecule can produce various antibody fragments that retain the ability to recognize and bind antigens. For example, limited digestion of an antibody molecule with papain protease typically produces three fragments, two of which are identical and are called Fab fragments, since they retain the antigen-binding activity of the parent antibody molecule. Cleavage of an antibody molecule with the enzyme pepsin normally produces two antibody fragments, one of which retains both antigen-binding arms of the antibody molecule, and is therefore called the F (ab ') 2 fragment. Reduction of an F (ab ') 2 fragment with dithiothreitol or mercaptoethylamine produces a fragment called the Fab' fragment. A single chain variable region (sFv) antibody fragment, consisting of a truncated Fab fragment comprising the variable domain (V) of an antibody heavy chain linked to a V domain of an antibody light chain by means of a synthetic peptide, can be generated using recombinant DNA technology techniques (see, eg, Janeway et al., cited above). Similarly, disulfide stabilized variable region (dsFv) fragments can be prepared by recombinant DNA technology (see, eg, Reiter et al., Protein Engineering, 7: 697-704 (1994)). Any antibody fragment that recognizes and binds to a desired cell surface receptor or antigen can be employed. Antibody fragments are further described in, for example, Parham, J. Immunol., 131: 2895-2902 (1983), Spring et al., J. Immunol., 113: 470-478 (1974), and Nisonoff et al., Arch. Biochem. Biophys., 89: 230-244 (1960)). Antibody-antigen binding can be assayed using any method

Suitable ES 2 533 992 T3 known in the art, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, eg, Janeway et al., Cited above, and US Patent Application Publication No. 2002/0197266 A1).

Furthermore, the antibody can be a chimeric antibody or an antigen-binding fragment thereof. By "chimeric" is meant that the antibody comprises at least two immunoglobulins, or fragments thereof, obtained or derived from at least two different species (eg, two different immunoglobulins, such as a combined human immunoglobulin constant region. with a murine immunoglobulin variable region). The antibody may also be a domain antibody (dAb) or an antigen-binding fragment thereof, such as, for example, a camelid antibody (see, eg, Desmyter et al., Nature Struct. Biol., 3: 752, (1996)), or a shark antibody, such as, for example, a novel antigen receptor (IgNAR) (see, eg, Greenberg et al., Nature, 374: 168 (1995) , and Stanfield et al., Science, 305: 1770-1773 (2004)).

Any suitable antibody can be used in the context of the description. For example, monoclonal antibody J5 is a murine IgG2a antibody that is specific for Common Acute Lymphoblastic Leukemia Antigen (CALLA) (Ritz et al., Nature, 283: 583-585 (1980)) , and can be used to access cells that express CALLA (eg, acute lymphoblastic leukemia cells). Monoclonal antibody MY9 is a murine IgG1 antibody that specifically binds to the CD33 antigen (Griffin et al., Leukemia Res., 8: 521 (1984)), and can be used to access cells that express CD33 (eg. , acute myelogenous leukemia (AML) cells).

Similarly, the anti-B4 monoclonal antibody (also called B4) is a murine IgG1 antibody that binds to the CD19 antigen on B cells (Nadler et al., J. Immunol., 131: 244-250 (1983)), and can be used to access B cells or diseased cells that express CD19 (eg, non-Hodgkin lymphoma cells and chronic lymphoblastic leukemia cells). N901 is a murine monoclonal antibody that binds to the CD56 (neural cell adhesion molecule) antigen found on cells of neuroendocrine origin, including small cell lung tumors, which can be used in the conjugate to target drugs to cells of neuroendocrine origin. Antibodies J5, MY9 and B4 are preferably surface reconstructed or humanized prior to use as part of the conjugate. Surface reconstruction or humanization of antibodies is described in, for example, Roguska et al., Proc. Natl. Acad. Sci. USA, 91: 969-73 (1994).

In addition, monoclonal antibody C242 binds to the CanAg antigen (see, eg, US Patent 5,552,293), and can be used to target the conjugate to CanAg expressing tumors, such as colorectal tumors, pancreatic, non-small cell lung, and gastric cancers. HuC242 is a humanized form of the C242 monoclonal antibody (see, eg, US Patent 5,552,293). The hybridoma from which HuC242 is produced is deposited under the ECACC identification number 90012601. HuC242 can be prepared using CDR grafting methodology (see, eg, US Patents 5,585,089, 5,693 .761 and 5,693,762) or surface reconstruction technology (see, eg, US Patent 5,639,641). HuC242 can be used to target the conjugate to tumor cells expressing the CanAg antigen, eg, colorectal, pancreatic, non-small cell lung and gastric cancer cells.

To access cell ovarian cancer and prostate cancer cells, an anti-MUC1 antibody can be used as a cell binding agent in the conjugate. Anti-MUC1 antibodies include, for example, anti-HMFG-2 (see, eg, Taylor-Papadimitriou et al., Int. J. Cancer, 28: 17-21 (1981)), hCTM01 (see, eg, van Hof et al., CancerRes., 56: 5179-5185 (1996)), and DS6. Prostate cancer cells can also be accessed with the conjugate using a specific anti-prostate membrane antigen (PSMA) as a cell binding agent, such as J591 (see, eg, Liu et al., Cancer Res., 57: 3629-3634 (1997)). In addition, cancer cells expressing the Her2 antigen, such as breast, prostate and ovarian cancers, can be accessed using the trastuzumab antibody. Anti-IGF-IR antibodies that bind to the insulin-like growth factor receptor can also be used in the conjugate.

Particularly preferred antibodies are humanized monoclonal antibodies, examples of which include huN901, huMy9-6, huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, and rituximab (see, eg, US Patents 5,639,641 and 5,665,357, US Provisional Patent Application No. 60 / 424,332 (which is related to US Patent Application Publication No. No. 2005/0118183 A1), international patent application WO 02/16401, Pedersen et al., Cited above, Roguska et al., Cited above, Liu et al., Cited above, Nadler et al., Cited above, Colomer et al., Cancer Invest., 19: 49-56 (2001), Heider et al., Eur. J. Cancer, 31A: 2385-2391 (1995), Welt et al., J. Clin. Oncol., 12: 1193-1203 (1994), and Maloney et al., Blood, 90: 2188-2195 (1997)). Most preferably, the antibody is humanized monoclonal antibody huN901 or humanized monoclonal antibody huMy9-6. Other preferred antibodies include CNTO95, huDS6, huB4, and huC242. Other humanized monoclonal antibodies are known in the art, and can be used in conjunction with the disclosure.

The conjugate comprises a maytansinoid. A maytansinoid, which includes maytansinol, is a compound that inhibits microtubule formation and is highly toxic to mammalian cells. Examples of suitable maytansinol analogs include those with a modified aromatic ring and those with modifications at other positions. Such maytansinoids are described in, for example, US Patents 4,256,746, 4,294,757,

ES 2 533 992 T3

4,307,016, 4,313,946, 4,315,929, 4,322,348, 4,331,598, 4,361,650, 4,362,663, 4,364,866, 4,424,219, 4,371,533, 4,450,254, 5,475,092, 5,585. 499, 5,846,545 and 6,333,410.

Examples of maytansinol analogs having a modified aromatic ring include: (1) C-19-dechloro (US Patent 4,256,746) (prepared by LAH reduction of ansamitocin P2), (2) C-20 -hydroxy (p C-20demethyl) +/- C-19-dechloro (US patents 4,361,650 and 4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH), and (3) C -20-demethoxy, C-20-acyloxy (-OCOR), +/- dechlor (US Pat. 4,294,757) (prepared by acylation using acyl chlorides).

Examples of maytansinol analogs having modifications of positions other than an aromatic ring include: (1) C-9-SH (US Patent 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5), (2) C-14-alkoxymethyl (demethoxy / CH2OR) (US Patent 4,331,598), (3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH₂OAc) (US patent 4,450,254) (prepared from Nocardia), (4) C-15-hydroxy / acyloxy (US patent 4,364,866) (prepared by the conversion of maytansinol by Streptomyces), (5) C-15-methoxy (US patents 4,313,946 and 4,315,929) (isolated from Trewia nudiflora), (6) C-18-N-demethyl (US patents). 4,362,663 and 4,322,348) (prepared by the demethylation of maytansinol by Streptomyces), and (7) 4,5-deoxy (US Pat. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

In a preferred embodiment of the invention, the conjugate uses the thiol-containing maytansinoid DM1, also known as N²'-desacetyl-N²'- (3-mercapto-1-oxopropyl) -maitansine, as a cytotoxic agent. The structure of DM1 is represented by formula (I):

<img file="ES2533992T3_D0001.tif" />

In another preferred embodiment of the invention, the conjugate uses the thiol-containing maytansinoid DM4, also known as N²'-desacetyl-N²'- (4-methyl-4-mercapto-1-oxopentyl) -maitansine, as a cytotoxic agent. The structure of DM4 is represented by the formula (II):

<img file="ES2533992T3_D0002.tif" />

Other maytansines may be used in the context of the invention, including, for example, thiol- and disulfide-containing maytansines bearing a mono- or dialkyl substitution on the carbon atom bearing the sulfur atom. Particularly preferred is a maytansinoid having at the C-3 position (a) C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyl functionality, and (b) an acylated amino acid side chain with an acyl group bearing a hindered sulfhydryl group, wherein the carbon atom of the acyl group bearing the thiol functionality has

ES 2 533 992 T3 one or two substituents, said substituents being CH3, C2H5, linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, phenyl substituted, or heterocyclic aromatic or heterocycloalkyl radical, and in addition where one of the substituents can be H, and wherein the acyl group has a straight chain length of at least three carbon atoms between the carbonyl functionality and the sulfur atom.

Additional maytansins for use in the context of the invention include compounds represented by formula (III):

<img file="ES2533992T3_D0003.tif" />

where Y 'represents (CR7R8) l (CR9 = CR10) pCECqAr (CR5R6) mDu (CR11 = CR12) r (CEC) sBt (CR3R4) n-CR1R2SZ, where each of R1 and R2 are independently CH3, C2H5 , linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, and where R2 can also be H , where A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or substituted aryl, or aromatic heterocyclic radical, or heterocycloalkyl, where each of R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are independently H, CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical, where each of l, m, n, o, p, q, r, s and t are independently zero or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not zero at any time, and where Z is H, SR or COR, where R is linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (III) include compounds of formula (III) in which (a) R1 is H, R2 is methyl and Z is H, (b) R1 and R2 are methyl and Z is H, (c) R1 is H, R2 is methyl, and Z is -SCH3, and (d) R1 and R2 are methyl, and Z is -SCH3.

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

<img file="ES2533992T3_D0004.tif" />

<img file="ES2533992T3_D0005.tif" />

(IV-D) (ÍV-DJL) in which Y represents (CR7R8) l (CR5R6) m (CR3R4) nCR1R2SZ,

ES 2 533 992 T3 wherein each of R1 and R2 are independently CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl , substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and wherein R2 can also be H, wherein each of R3, R4, R5, R6, R7 and R8 are independently H, CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, wherein each of l, m and n are independently an integer from 1 to 5, and further n may be zero, where Z is H, SR or COR, where R is linear or branched alkyl or alkenyl having 1 to 10 carbon atoms, cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical, and wherein May represents a maytansinoid carrying the side chain at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C20 desmethyl.

Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D, L) include compounds of formulas (IV-L), (IV-D) and (IV-D, L) in the that (a) R1 is H, R2 is methyl, each of R5, R6, R7, and R8 are H, each of l and m are 1, n is 0, and Z is H, (b) R1 and R2 are methyl, each of R5, R6, R7, and R8 are H, each of l and m are 1, n is 0, and Z is H, (c) R1 is H, R2 is methyl, each of R5, R6, R7, and R8 are H, l and m are each 1, n is 0, and Z is -SCH3 ,, or (d) R1 and R2 are methyl, each of R5, R6, R7, and R8 are H, each of l and m are 1, n is 0, and Z is -SCH3.

Preferably, the maytansinoid is represented by formula (IV-L).

Additional preferred maytansins also include compounds represented by formula (V):

<img file="ES2533992T3_D0006.tif" />

where Y represents (CR7R8) l (CR5R6) m (CR3R4) nCR1R2SZ, where each of R1 and R2 are independently CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched alkyl or alkenyl or cyclic having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and wherein R2 can also be H, where each of R3, R4, R5, R6, R7 and R8 are independently H, CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, wherein each of l, m and n are independently an integer from 1 to 5, and further n may be zero, where Z is H, SR or COR, where R is linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (V) include compounds of formula (V) wherein (a) R1 is H, R2 is methyl, each of R5, R6, R7, and R8 are H; each of l and m are 1; n is 0, and Z is H, (b) R1 and R2 are methyl; each of R5, R6, R7, and R8 are H; l and m are 1; n is 0; and Z is H, (c) R1 is H, R2 is methyl, each of R5, R6, R7 and R8 are

ES 2 533 992 T3

H, each of l and m are 1, n is 0, and Z is -SCH3, or (d) R1 and R2 are methyl, each of R5, Re, R7, and R8 are H, l and m are 1, n is 0, and Z is -SCH3.

Other additionally preferred maytansines include compounds represented by the formula (VI-L), (VI-D), or (VID, L):

<img file="ES2533992T3_D0007.tif" />

(VI-L) (VI-D) (VI-D, L), where Y2 represents (CR7R8) l (CR5R6) m (CR3R4) nCR1R2SZ2, where each of R1 and R2 are independently CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and wherein R2 can also be H , wherein each of R3, R4, R5, R6, R7, and R8 are independently H, CH3, C2H5, linear or cyclic alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, wherein each of l , m and n are independently an integer from 1 to 5, and further n can be zero, where Z2 is SR or COR, where R is linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical, and wherein May is a maytansinoid.

Additional preferred maytansins include compounds represented by formula (VII):

<img file="ES2533992T3_D0008.tif" />

where Y2 - represents (CR7R8) l (CR9 = CR10) p (CEC) qAr (CR5R6) mDu (CR11 = CR12) r (CEC) sBt (CR3R4) nCR1R2SZ2, where each of R1 and R2 are independently CH3 , C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, and wherein R2 also can be H, where each of A, B and D is independently cycloalkyl or cycloalkenyl having 3 to 10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical, wherein each of R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are independently H, CH3, C2H5, linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10

ES 2 533 992 T3 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical, wherein each of 1, m, n, o, p, q, r, s and t are independently zero or an integer from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and t are not zero at any time, and where Z2 is SR or -COR, where R is linear alkyl or alkenyl having 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.

Preferred embodiments of formula (VII) include compounds of formula (VII) wherein R1 is H and R2 is methyl.

Drug conjugates can be prepared by in vitro methods. To link a drug or prodrug to the antibody, a linking group is used. Suitable linking groups are well known in the art, and include disulfide groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Preferred linking groups are disulfide groups. For example, conjugates can be constructed using a disulfide exchange reaction between the antibody and the drug or prodrug.

In accordance with the disclosure, the antibody is modified by reacting a bifunctional crosslinking reagent with the antibody, thereby resulting in the covalent attachment of a linker molecule to the antibody. As used herein, a "bifunctional crosslinking reagent" is any chemical moiety that covalently links a cell-binding agent to a drug, such as the drugs described herein. In a preferred embodiment of the invention, a part of the binding moiety is provided by the drug. In this regard, the drug comprises a linker moiety that is part of a larger linker molecule that is used to bind the cell-binding agent to the drug. For example, to form the maytansinoid DM1, the side chain at the C-3 hydroxyl group of maytansine is modified to have a free sulfhydryl group (SH). This thiolated form of maytansine can react with a modified cell binding agent to form a conjugate. Therefore, the final linker is assembled from two components, one of which is provided by the cross-linking reagent, while the other is provided by the DM1 side chain.

Any suitable bifunctional crosslinking reagent can be used in connection with the present method, as long as the linker reagent provides preservation of the therapeutic characteristics, eg, cytotoxicity, and accessibility of the drug and cell binding agent, respectively. Preferably, the linker molecule binds the drug to the cell-binding agent via chemical bonds (as described above), such that the drug and the cell-binding agent are chemically coupled (eg, covalently linked) each other. Preferably, the binding reagent is a cleavable linker. More preferably, the linker is cleaved under mild conditions, that is, conditions within a cell under which the activity of the drug is unaffected. Examples of suitable cleavable linkers include disulfide linkers, acid-labile linkers, photo-labile linkers, peptidase-labile linkers, and esterase-labile linkers. Disulfide-containing linkers are linkers cleavable by disulfide exchange, which can occur under physiological conditions. Acid labile linkers are cleavable linkers at acidic pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide suitable conditions for cleaving acid-labile linkers. Photolabile linkers are useful on the surface of the body and in many cavities of the body that are accessible to light. Also, infrared light can penetrate tissues. Peptidase-labile linkers can be used to cleave certain peptides inside or outside cells (see eg, Trouet et al., Proc. Natl. Acad. Sci. USA, 79: 626-629 (1982), and Umemoto et al., Int. J. Cancer, 43: 677-684 (1989)).

Preferably, the drug is linked to a cell binding agent via a disulfide bond. The linker molecule comprises a reactive chemical group that can react with the cell-binding agent. The preferred reactive chemical groups for reaction with the cell binding agent are Nsuccinimidyl esters and N-sulfosuccinimidyl esters. Additionally, the linker molecule comprises a reactive chemical group, preferably a dithiopyridyl group, that can react with the drug to form a disulfide bond. Particularly preferred linker molecules include, for example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (see, eg, Carlsson et al., Biochem. J., 173: 723-737 (1978 )), N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB) (see, eg, US Pat. 4,563,304), N-succinimidyl 4- (2-pyridyldithio) pentanoate (SPP) (see, eg, CAS registry number 341498-08-6), and other reactive cross-linkers that are described in the US patent. USA 6,913,748.

Although cleavable linkers are preferably used in the inventive method, a non-cleavable linker can also be used to generate the conjugate described above. A non-cleavable linker is any chemical moiety that is capable of linking a maytansinoid to a cell binding agent in a stable, covalent manner. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions under which the drug or the cell binding agent remains active.

ES 2 533 992 T3

Suitable crosslinking reagents that form non-cleavable linkers between a drug and the cell binding agent are well known in the art. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the cell-binding agent, as well as a maleimido- or haloacetyl-based moiety for reaction with the cell. drug. Crosslinking reagents comprising a maleimido-based moiety include N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl 4- (maleimidomethyl) -cyclohexane-1-carboxy- (6-amidocaproate), which is a “long chain” analog of SMCC (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), εmaleimidocaproic acid hydroxysuccinimidyl ester ( EMCS), m-Maleidobenzoyl-N-hydroxysuccinimide (MBS) ester, N- (amaleimidoacetoxy) -succinimide ester (AMAS), N-succinimidyl 6- (e-maleimidopropionamido) hexanoate (SMPH), 4- (pmaleimidophenyl) -butyrate N-succinimidyl (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI). Crosslinking reagents comprising a haloacetyl-based moiety include Nsuccinimidyl 4- (iodoacetyl) -aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-3 (bromoacetamido) propionate. -succinimidyl (SBAP).

Other crosslinking reagents which lack a sulfur atom and which form non-cleavable linkers can also be used in the inventive method. Such linkers can be derived from dicarboxylic acid-based moieties. Suitable dicarboxylic acid-based moieties include, but are not limited to, α, ωdicarboxylic acids of the general formula (IX):

HOOC-X1-Yn-Zm-COOH (IX), where X is a linear or branched alkyl, alkenyl or alkynyl group having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl group bearing 3 to 10 atoms of carbon, Z is a substituted or unsubstituted aromatic group bearing 6 to 10 carbon atoms, or a substituted or unsubstituted heterocyclic group in which the heteroatom is selected from N, O or S, and wherein each of the , m and n are zero or 1, provided that l, m and n are not all zero at the same time.

Many of the non-cleavable linkers described herein are described in detail in US Patent Application No. 10 / 960,602, which corresponds to US Patent Application Publication No. 2005/0169933 A1.

Additional information regarding maytansinoids, cytotoxic agents comprising the same, drug conjugates, and related methods of preparation is described in US Patent Application No. 11 / 352,121 and US Patent Application No. 10 / 849,136, which corresponds to US Patent Application Publication No. 2004/0235840 A1.

The following examples further illustrate the invention.

Comparative Example 1

This example demonstrates the purification of a modified antibody with a heterobifunctional modification reagent using TFF.

The monoclonal antibody huN901 (final concentration 8 mg / ml) was incubated with Nsuccinimidyl 4- (2-pyridyldithio) pentanoate (SPP, 5.6-fold molar excess) for approximately 180 minutes at 20 ° C in potassium phosphate buffer. 50 mM (pH 7.5) containing 50 mM NaCl, 2 mM EDTA and 5% ethanol. In a first group, the reaction mixture was purified using a Sephadex ™ G25F resin column equilibrated and eluted in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl and 2 mM EDTA. In a second group, the reaction mixture was purified using a Pellicon XL TFF system (Millipore, Billerica, MA), and the antibody was diafiltered (5 volumes) in 50 mM potassium phosphate, 50 mM NaCl (pH 6.5) and 2 mM EDTA using a 10,000 molecular weight cutoff membrane (Ultracel ™ regenerated cellulose membrane, Millipore, Billerica, MA). Both samples were conjugated with DM1 (1.7-fold molar excess over unbound linker) for 18 hours at pH 6.5 in potassium phosphate buffer containing 50 mM NaCl and a final concentration of 3% DMA.

In both groups, the yields were determined spectrophotometrically (wavelength 280 nm) for the combined modification and purification step. Linker / antibody ratios were also determined by treatment with dithiothreitol to release pyridine-2-thione, which has an extinction coefficient of 8,080 M^-1cm^-1 at 343 nm. Drug / antibody ratios were determined spectrophotometrically (280 nm and 252 nm wavelengths) for the conjugation step. Furthermore, the removal of small molecular species related to SPP was measured by HPLC Hisep.

The resulting data are shown in Table 1.

ES 2 533 992 T3

Table 1: Purification methods for modified huN901 using G-25F versus TFF

<td></td><td></td><td>Sephadex ™ G25F Resin</td><td>TFF</td>

<td rowspan="3">Modification stage</td><td>Stage performance</td><td> 94 %</td><td> 98 %</td>

<td>Linker / antibody ratio</td><td> 4,9</td><td> 4,9</td>

<td>Small molecules related to SPP</td><td> 0,2 %</td><td> 0,2 %</td>

<td>Conjugation stage</td><td>Drug / antibody ratio</td><td> 3,7</td><td> 3,7</td>

As shown in Table 1, the use of TFF provides a quality drug conjugate at least equivalent to the non-adsorptive chromatography procedure (G25), while being more convenient and scalable.

Example 2

This example demonstrates the purification of a modified antibody with a heterobifunctional modification reagent using adsorptive chromatography.

The huB4 antibody was modified with N-succinimidyl 4- (2-pyridyldithio) butanoate (SPDB, 5.4-fold molar excess) for 120 minutes at room temperature in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl, 2 mM EDTA and 5% ethanol. In a first group, the reaction mixture was purified using the Sephadex resin column.^TM G25F described in Example 1. In a second group, the reaction mixture was loaded onto a column of ceramic hydroxyapatite (CHT, Bio-Rad Laboratories, Hercules, CA), which was equilibrated in potassium phosphate buffer.

12.5 mM (pH 6.5) and eluted with 80 mM potassium phosphate buffer (pH 6.5).

In both groups, the yields and linker / antibody ratios were determined as described in Example 1. The first group had a 91% yield and a linker / antibody ratio of 4.2. The second group had an 89% yield and a linker / antibody ratio of 4.2.

Antibody CNTO95 (final concentration 10 mg / ml) was modified with Nsuccinimidyl 4- (2-pyridyldithio) butanoate (SPDB, 4.5-fold molar excess) for 120 minutes at 20 ° C in 10 mM sodium phosphate buffer (pH 7.5) containing 2.7% sucrose and 5% ethanol. In a first group, the reaction mixture was purified using a Sephadex ™ G25F resin column in 12.5 mM potassium phosphate buffer (pH 6.6) containing 12.5 mM NaCl and 0.5 mM EDTA. In a second group, the reaction mixture was loaded onto a SP Sepharose Fast Flow column (GE Healthcare, Piscataway, NJ), which was equilibrated in 10 mM sodium phosphate buffer (pH 7.5) and eluted with phosphate buffer. 50 mM potassium (pH 7.5), containing 50 mM NaCl.

In both groups, the yields and linker / antibody ratios were determined as described in Example 1. The first group had a 96% yield and a linker / antibody ratio of 4.0. The second group had a 97% yield and a linker / antibody ratio of 4.1.

The data obtained in this example demonstrate that adsorptive chromatography can be used to purify an antibody modified with a heterobifunctional modification reagent.

Comparative example 3

This example demonstrates the beneficial effects of conjugating a modified antibody to a drug at a pH above 6.5.

In a first experiment, the CNTO95 antibody was modified and purified as described in Example 2. Then, the modified antibody was divided into two groups. In the first group, conjugation was carried out in potassium phosphate

12.5 mM at pH 6.5 containing 12.5 mM NaCl, 0.5 mM EDTA, 3% DMA and a 1.7-fold molar excess of drug per linker at 20 ° C. In the second group, the conjugation reaction was at pH 7.5. The conjugated antibody was purified on NAP-10 columns.

The drug / antibody ratio was measured for both groups. The resulting data are shown in Table 2.

ES 2 533 992 T3

Table 2: Drug / Antibody Ratio in a pH 6.5 vs. 7.5 Conjugation Reaction

<td>Reaction time (hours)</td><td>Drug / Antibody Ratio in pH 6.5 Conjugation Reaction</td><td>Drug / Antibody Ratio in pH 7.5 Conjugation Reaction</td>

<td> 0,5</td><td> --</td><td> 3,0</td>

<td> 1</td><td> 2,3</td><td> 3,4</td>

<td> 1,5</td><td> --</td><td> 3,5</td>

<td> 2</td><td> 2,8</td><td> 3,5</td>

<td> 2,75</td><td> --</td><td> 3,6</td>

<td> 3,5</td><td> 3,2</td><td> 3,6</td>

<td> 5</td><td> 3,4</td><td> 3,7</td>

As the data set out in Table 2 shows, conjugation occurs faster at pH 7.5 than at pH 6.5.

In a second experiment, the humanized monoclonal antibody huB4 was modified with either (a) a 4.9-fold molar excess of SPDB relative to antibody, or (b) a 4.8-fold molar excess of SPDB relative to antibody. In both situations, the reaction was in 50 mM potassium phosphate, 50 mM potassium chloride, and 2 mM EDTA (pH 6.5) in 5% ethanol for a total of 120 minutes at room temperature. Sample (a) was purified on a Sephadex ™ G25F resin column equilibrated in 50 mM potassium phosphate, 50 mM sodium chloride and 2 mM EDTA at pH 6.5. Sample (b) was purified in an equivalent manner, except that the chromatography buffer was adjusted to pH 7.5. Both samples were conjugated with DM4 (1.7-fold molar excess over bound linker) for 18 hours at room temperature at a final dimethylacetamide (DMA) concentration of 3%.

Thus, sample (a) was conjugated to pH 6.5, and sample (b) was conjugated to pH 7.5. The samples were then purified on a Sephadex ™ G25F resin column equilibrated in 9.6 mM potassium phosphate and 4.2 mM sodium chloride at pH 6.5. Both samples were incubated at 4 ° C for up to 7 months and subjected to analysis for free drug released at intervals. The resulting data are shown in Table 3.

Table 3: Free Drug Release Over Time from Conjugated Samples at pH 6.5 and 7.5

<td>Time (months)</td><td>Conjugation at pH 6.5</td><td>Conjugation at pH 7.5</td>

<td> 0</td><td> 1,0</td><td> 0,8</td>

<td> 1,5</td><td> 1,8</td><td> 1,0</td>

<td> 2,5</td><td> 3,2</td><td> 1,9</td>

<td> 7</td><td> 4,0</td><td> 2,8</td>

As the data shown in Table 3 show, the release of free drug is substantially slower from sample (b), which had been conjugated at pH 7.5, relative to sample (a), which had been conjugated to pH 6.5. Accordingly, the conjugated drug product prepared at pH 7.5 is shown to be more stable with respect to free drug release over time compared to the conjugated drug product prepared at pH 6.5. Conjugation at pH 7.5 also shows better drug uptake than at pH 6.5, thus requiring less drug use.

Comparative Example 4

This example demonstrates the beneficial effects of conjugating a modified antibody to a drug at a pH below 6.0.

The monoclonal antibody huN901 (final concentration 8 mg / ml) was incubated with Nsuccinimidyl 4- (2-pyridyldithio) pentanoate (SPP, 5.6-fold molar excess) for approximately 180 minutes at 20 ° C in potassium phosphate buffer. 50 mM (pH 7.5) containing 50 mM NaCl, 2 mM EDTA and 5% ethanol. In a first group, the reaction mixture was purified using a column of Sephadex ™ G25F resin equilibrated and eluted in 50 mM sodium citrate buffer (pH 5.0) containing 50 mM NaCl and 2 mM EDTA. In a second group, the reaction mixture was purified using a Sephadex resin column.^TM G25F equilibrated and eluted in 50 mM potassium phosphate buffer

ES 2 533 992 T3 (pH 6.5) containing 50 mM NaCl and 2 mM EDTA. Both samples were conjugated with DM4 (molar excess of

1.7 times on bound linker) for 3, 19, 25, 48 and 120 hours at room temperature in a final dimethylacetamide (DMA) concentration of 3%.

Thus, the first group of samples was conjugated in 50 mM sodium citrate buffer (pH 5.0) containing 50 mM NaCl and 2 mM EDTA, and the second group of samples was conjugated in 50 mM potassium phosphate buffer (pH 6 , 5) containing 50 mM NaCl and 2 mM EDTA. The samples were then purified using a balanced Sephadex ™ G25F resin column eluted in 50 mM potassium phosphate buffer (pH 6.5) containing 50 mM NaCl.

In both groups, the linker / antibody ratios were determined by treatment with dithiothreitol to release pyridine-2-thione, which has an extinction coefficient of 8,080 M '¹cm '¹ at 343 nm. Drug / antibody ratios were determined spectrophotometrically (280 nm and 252 nm wavelengths) for the conjugation step.

The first group had a linker / antibody ratio of 4.3. The second group had a linker / antibody ratio of 4.2.

The drug / antibody ratios over time for the two groups are set forth in Table 4.

Table 4: Rate of incorporation of DM1 into SPP modified huN901 as a function of conjugation pH

<td rowspan="2">Reaction time (hours)</td><td colspan="2">Drug / antibody ratio (mol / mol)</td>

<td>Conjugation at pH 5.0</td><td>Conjugation at pH 6.5</td>

<td> 3</td><td> 2,43</td><td> 2,97</td>

<td> 19</td><td> 3,38</td><td> 3,28</td>

<td> 25</td><td> 3,41</td><td>NT</td>

<td> 48</td><td> 3,46</td><td> 3,17</td>

<td> 120</td><td> 3,44</td><td> 2,85</td>

As is evident from the data set forth in Table 4, the conjugate that is prepared by conjugating the modified antibody to the drug at a pH of 5.0 achieves a higher and more stable level of bound drug during the course of the reaction. of conjugation than the conjugate prepared at a conjugation pH of 6.5. In addition to increased stability, the results indicate that a higher drug / antibody level is achieved after conjugation at pH 5.0 than when the same amount of drug is used at a conjugation pH of 6.5, indicating this for more efficient use of the drug at pH 5.0.

In both groups, the amounts of conjugated monomer were determined over time. The resulting data are shown in Table 5.

Table 5: Effect of conjugation pH on the level of conjugated monomer during conjugation of SPP-modified huN901 with DM1

<td rowspan="2">Reaction time (hours)</td><td colspan="2">Conjugated monomer (%)</td>

<td>Conjugation at pH 5.0</td><td>Conjugation at pH 6.5</td>

<td> 3</td><td> 98,5</td><td> 98,0</td>

<td> 19</td><td> 98,8</td><td> 98,2</td>

<td> 25</td><td> 99,1</td><td>NT</td>

<td> 48</td><td> 99,2</td><td> 98,6</td>

<td> 120</td><td> 99,2</td><td> 97,8</td>

As is evident from the data set forth in Table 5, the conjugate that is prepared by conjugating the modified antibody to the drug at a pH of 5.0 has a higher level of conjugated monomer than the conjugate prepared at a conjugating pH. of 6.5.

ES 2 533 992 T3

Comparative Example 5

This example further demonstrates the benefits of conjugating a drug to a modified antibody at a pH less than 6.

The BIWA 4 antibody was modified with SPP (molar excess of SPP shown in Table 6) for 120-140 minutes at room temperature in 50 mM potassium phosphate buffer (pH 6.5), 50 mM NaCl, 2 mM EDTA and ethanol at 5%. Aliquots of modified antibody were purified on independent NAP 25 columns equilibrated in buffers having various pH values ​​(pH 4.6-6.5). Buffers pH 4.6-5.9 were composed of 35 mM sodium citrate, 150 mM sodium chloride, and 2 mM EDTA. The pH 6.5 buffer was PBS with 2 mM EDTA.

The modified antibody was conjugated at each pH with DM1 (1.7-fold molar excess over linker) in dimethylacetamide (DMA, 3% final concentration). After incubation for 17-18 hours at room temperature, the conjugated antibody samples were purified by chromatography on equilibrated NAP 25 columns in PBS (pH 6.5). Linker / antibody ratios (E / A in Table 6) were determined by treatment with dithiothreitol to release pyridine-2-thione, which has an extinction coefficient of 8,080 M '¹cm '¹ at 343 nm. Drug / antibody ratios were determined spectrophotometrically (280 nm and 252 nm wavelengths) for the conjugation step. The conjugated monomer, high molecular weight species and low molecular weight species were determined by SEC-HPLC using a balanced TSKG3000SWXL column developed in 0.2M potassium phosphate buffer (pH 7.0) containing potassium chloride. 0.2 M and 20% isopropanol.

The results of this analysis are shown in Table 6.

Table 6: Characteristics of the drug conjugate product in relation to pH

<td>Tampon</td><td>Molar excess of SPP</td><td>E / A</td><td>FA</td><td>Monomer (%)</td><td>High PM (%)</td><td>Low PM (%)</td><td>Conjugation step yield (%)</td>

<td>pH 4.6</td><td> 4,7</td><td> 3,8</td><td> 3,6</td><td> 97,5</td><td> 2,2</td><td> 0,4</td><td> 74</td>

<td>pH 5.1</td><td> 4,4</td><td> 4,7</td><td> 3,6</td><td> 97,6</td><td> 1,9</td><td> 0,6</td><td> 75</td>

<td>pH 5.6</td><td> 5,0</td><td> 4,9</td><td> 3,6</td><td> 97,7</td><td> 1,5</td><td> 0,8</td><td> 85</td>

<td>pH 5.9</td><td> 5,5</td><td> 5,3</td><td> 3,7</td><td> 96,4</td><td> 2,3</td><td> 1,4</td><td> 76</td>

<td>pH 6.5</td><td> 6,6</td><td> 6,4</td><td> 3,7</td><td> 95,1</td><td> 2,8</td><td> 1,9</td><td> 71</td>

The data set forth in Table 6 demonstrates that conjugation of SPP-modified BIWA 4 with DMI was effective at a pH below 6.0, compared to conjugation at pH 6.5. The amounts of linker and drug, specifically SPP and DM1 linker, required to achieve a particular final drug / antibody ratio were reduced at lower pH. Furthermore, the levels of conjugated monomer, the high molecular weight species and the low molecular weight species were more optimal, and the yields were improved, at lower pH.

Comparative Example 6

This example demonstrates that the step to purify the modified antibody can optionally be eliminated. The drug can be added simultaneously with the bifunctional modifying reagent or at some point afterwards.

In an example of drug addition after modifying reagent, the humanized monoclonal antibody CNTO95 was modified at a concentration of 20 mg / ml with the bifunctional modifying reagent SPDB to a molar excess of SPBD over the antibody of 4.6 for 120 min at 20 ° C. The modification buffer was 44 mM phosphate buffer (pH 7.5) containing 5.3% sucrose and 5% ethanol. An aliquot of the modified antibody was purified on Sephadex ™ G25F resin (standard four-step procedure), equilibrated and eluted in 12.5 mM potassium phosphate buffer (pH 7.5) containing 12.5 mM NaCl, and was subsequently conjugated with DM4 (1.7-fold molar excess of drug over bound linker) to a final modified antibody concentration of 10 mg / ml in 12.5 mM potassium phosphate buffer (pH 7.5) containing NaCl 12.5 mM and 10% DMA for 20 hours at room temperature. A second aliquot of the modified antibody was conjugated immediately at the end of the 120 minute modification reaction (three step procedure), without being further purified.

The protein and buffer concentrations of the modification reaction mixture were adjusted to give a modified protein concentration of 10 mg / ml and a 28 mM potassium phosphate buffer composition (pH 7.5) containing 5.9 mM NaCl. and 2.7% sucrose. DM4 (1.7-fold molar excess over the starting SPDB) was then added and the DMA was adjusted to a final concentration of 10%. After 20 hours of incubation at room temperature, both conjugated antibody aliquots were purified on Sephadex ™ G25F resin equilibrated in 10 mM histidine and 10% sucrose at pH 5.5.

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Linker / antibody (E / A) ratios were determined by treatment with dithiothreitol to release pyridine-2thiona, which has an extinction coefficient of 8,080 M '¹cm '¹ at 343 nm. Drug / antibody (D / A) ratios and yield were determined spectrophotometrically (280 nm and 252 nm wavelengths) for the conjugation step. Monomer percentages were assayed by SEC-HPLC. The percentages of free drug were assayed by HPLC on a Hisep column. The results of these analyzes are shown in Table 7.

Table 7: Optional Elimination of the Purification Step for the Modified Antibody

<td>Parameters</td><td>4-stage procedure</td><td>3-stage procedure</td>

<td>Starting SPDB</td><td>4.6 x</td><td>4.6 x</td>

<td>E / A</td><td> 4,1</td><td>Undetermined</td>

<td>GIVES</td><td> 3,9</td><td> 4,0</td>

<td>Performance</td><td> 79%</td><td> 91%</td>

<td>% of Monomer</td><td> 95,8%</td><td> 96,1%</td>

<td>% of free drug</td><td> 2,4%</td><td> 1,1%</td>

As the results set forth in Table 7 demonstrate, the step to purify the modified antibody can be eliminated in the context of the invention.

Comparative Example 7

This example demonstrates an improved means of purifying an antibody that has been modified with a heterobifunctional modifying reagent and then conjugated to a maytansinoid.

The huN901 antibody modified with SPP (7-fold molar excess) and purified on Sephadex ™ G25F resin, as described in Example 1, was conjugated to the maytansinoid DM1 (1.7-fold molar excess over the linker, dissolved in dimethylacetamide (DMA), final concentration 3%).

A first conjugate sample was purified by standard chromatography on Sephadex ™ G25F resin in phosphate buffered saline (PBS, pH 6.5).

A second conjugate sample was purified by a Pellicon XL TFF system (Millipore, Billerica, MA), as described in Example 1.

A third conjugate sample was purified using a MEP Hypercell resin column equilibrated in 50 mM Tris (pH 8.0), and eluted with 50 mM sodium acetate (pH 4.0).

A fourth conjugate sample was purified using a UNOsphere S resin column equilibrated in 50 mM sodium phosphate (pH 6.5) and eluted with 0.2 M NaCl and 50 mM sodium phosphate (pH 6.5).

A fifth conjugate sample was purified using a CHT resin column (Bio-Rad Laboratories, Hercules, CA) equilibrated in 50 mM sodium phosphate (pH 6.5) and eluted with 0.3 M NaCl and 50 mM sodium phosphate. (pH 6.5).

A sixth conjugate sample was purified using a SP Sepharose resin column equilibrated in 35 mM sodium citrate, 10 mM sodium chloride (pH 5.0), and eluted with 0.25 M NaCl, 35 mM sodium citrate (pH 5.0).

The conjugated monomer was determined by SEC-HPLC using a column of TSKG3000SWxl resin balanced and developed in 0.2M potassium phosphate buffer at pH 7.0, containing 0.2M potassium chloride and 20% isopropanol. The yield of the conjugation step was determined by dividing the yield of conjugated antibody by the amount of modified antibody that was conjugated (determined spectrophotometrically at a wavelength of 280 nm).

The results of these analyzes are shown in Table 8.

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Table 8: Comparison of conjugation and purification steps

<td>Conjugate sample</td><td>Conjugation and purification stage</td><td>Conjugated monomer%</td><td>Stage performance%</td>

<td>1 (control)</td><td>G25F resin</td><td> 93,2</td><td> 86</td>

<td>2 (invention)</td><td>TFF</td><td> 92,8</td><td> 85</td>

<td>3 (invention)</td><td>MEP Hypercell resin</td><td> 94,5</td><td> 74</td>

<td>4 (invention)</td><td>UNOsphere resin</td><td> 96,3</td><td> 81</td>

<td>5 (invention)</td><td>CHT resin</td><td> 97,9</td><td> 72</td>

<td>6 (invention)</td><td>SP Sepharose resin</td><td> 95,1</td><td> 81</td>

The results in Table 8 show that all the inventive purification methods (groups 2-6) gave similar yields to those obtained with the control procedure (group 1). Each inventive chromatographic method provided an improvement in the level of conjugated monomer and can be easily scaled up.

Besides CHT (ceramic hydroxyapatite), CFT (ceramic fluoroapatite) can also be used under similar chromatographic conditions. Alternatively, both CHT and CFT can be used in non-adsorptive mode, such that the desired product (substantially monomeric conjugate) is not retained by the resins, while the high molecular weight species are retained and thus separated. of the desired product.

Although a standard solvent / buffer composition for conjugation comprises 3% DMA, 50 mM potassium phosphate, 50 mM NaCl, and 2 mM EDTA at pH 6.5 (as used in Example 1), other compositions are more compatible with some of the chromatographic steps described herein and provide other benefits over the standard procedure. For example, conjugation can be performed in 3% DMA, 12.5 mM potassium phosphate, 12.5 mM NaCl, and 0.5 mM EDTA at pH 6.5. Under these conditions, the amount of DM4 incorporated relative to the amount of the linker incorporated into the huB4 antibody was approximately 10% higher than for standard conditions. Furthermore, these conditions are more compatible with loading on resins such as cation exchange resins and CHT resins.

All references, including publications, patent applications, and patents cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and set forth in its entirety in the present report.